This employed a loose patch clamp technique which examined ionic currents in response to superimposed 10-ms V1 steps to varying degrees of depolarisation, followed by V2 steps to a fixed, +100 mV depolarisation relative to resting membrane potential following 40 mV hyperpolarising prepulses of 50 ms duration. The activation and inactivation properties of the resulting Na+ membrane current densities revealed reduced maximum currents and steepnesses in their voltage dependences after addition of the Epac activator 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (1 µM) to the bathing Krebs-Henseleit solutions. Contrastingly, voltages at half-maximal current and timecourses of currents obtained in response to the V1 depolarising steps were unchanged. These effects were abolished by further addition of the RyR-inhibitor dantrolene (10 µM). In contrast, challenge by dantrolene alone left both currents and their parameters intact. These effects of Epac activation in inhibiting skeletal muscle, Nav1.4, currents, complement similar effects previously reported in the homologous Nav1.5 in murine cardiomyocytes. They are discussed in terms of a hypothesis implicating Epac actions in increasing RyR-mediated SR Ca2+ release resulting in a Ca2+-mediated inhibition of Nav1.4. The latter effect may form the basis for Ca2+-dependent Na+ channel dysregulation in SCN4A channelopathies associated with cold- and K+-aggravated myotonias.We investigated effects of pharmacological triggering of  However, recent intense interest has concerned alternative or co-existent PKA-independent activation mechanisms particularly involving exchange proteins directly activated by cAMP (Epac) in distinct critical cell physiological processes. Yet few studies report on Epac-mediated regulation of excitable membrane, as opposed to metabolic, signalling2, in particular involving ryanodine receptor (RyR) function or its downstream effects on Na+ channel (Nav)-mediated signalling. Those studies available were restricted to cardiac myocytes. Thus, in intact in situ murine ventricular and atrial myocytes, challenge by the cAMP analog 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate (8-CPT) at concentrations specifically acting on Epac as opposed to PKA3 inhibited voltage-dependent Na+ currents under loose patch clamp recording conditions under which their intracellular Ca2+ homeostasis conditions were thereby preserved4. This accompanied pro-arrhythmic reductions in action potential conduction velocities in intact perfused hearts5. Both actions were reversed by the ryanodine receptor (RyR) blocker dantrolene which by itself contrastingly did not alter Na+ currents4.Intracellular cellular 3′-5′-cyclic adenosine monophosphate (cAMP) is known to activate protein kinase A (PKA)-mediated phosphorylation of a wide range of regulatory cellular Ca2+ release that would in turn modify Nav1.5 function. In murine cardiomyocytes, Epac is thought to cause a downstream RyR phosphorylation stimulating SR Ca2+ release thereby modifying Ca2+ homeostasis. Thus, the Epac activating agent, 8-CPT, elicits occurrences of spontaneous cytosolic Ca2+ ([Ca2+]i) transients. It also increases the amplitudes of evoked [Ca2+]i transients following action potential excitation. Finally, it results in an appearance of spontaneous propagated cytosolic Ca2+ waves in rat and mouse cardiomyocytes6. These findings were accompanied by pro-arrhythmic extrasystolic electrophysiological events in intact perfused hearts10. Both effects persisted in the presence of the PKA inhibitor H-8911. However, they were abolished by genetic ablation of Epac2, β1-adrenoreceptors or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-δ, as well as by RyR2-S2814 phosphorylation12. The resulting altered [Ca2+]i in turn could then potentially modulate voltage-gated Na+ channels (Nav) that generate propagated action potentials. The intracellular C-terminus domains of cardiac Nav1.5 possess EF hand-like motifs to which Ca2+ can bind directly. Nav1.5 also possesses an IQ-like domain to which Ca2+ can bind indirectly via calmodulin (CaM) as well as phosphorylation sites for CaMKII13. Different reports have variously implicated all three of these sites in the modified or inhibited Nav1.5 function17 observed when intracellular Ca2+ was varied in patch-clamped cardiomyocytes18.These findings were consistent with an action of Epac activation upon Nav1.5 through an increased RyR-mediated sarcoplasmic reticular (SR) Ca2+-induced Ca2+ release, by differing surface membrane Cav1.1 as opposed to Cav1.2 L-type Ca2+ channel isoforms, not involving activation of membrane Ca2+ current19. These events are initiated by depolarisation driven by Nav1.4 rather than Nav1.5 channel opening. Furthermore, abnormal skeletal muscle Nav1.4 and cardiac muscle Nav1.5 function cause distinct clinical consequences. Genetic abnormalities affecting Nav1.5 potentially cause clinical cardiac pro-arrhythmic effects. Nav1.4 dysfunction is contrastingly implicated in hyperkalaemic and hypokalaemic periodic paralysis22, paramyotonia congenita21, cold- and K+-aggravated myotonia23, and sudden infant death syndrome24. Cold- and K+-aggravated myotonias particularly are associated with compromised Ca2+-mediated regulation of Nav1.425.Skeletal myocytes represent a cell type distinct from cardiac myocytes. They express differing skeletal muscle RyR1, rather than cardiac RyR2, isoforms. These are activated by direct charge coupling as opposed to Cai on activation26. Furthermore, homologies between Nav1.4 and Nav1.5 are compatible with similarities in functional properties13. In preliminary reports, Nav1.4 function was inhibited by Ca2+ entry through neighbouring Ca2+ channels, photorelease of caged Ca2+ in transfected HEK293 cells and skeletal muscle cell lines25, and following release of mitochondrial Ca2+ in murine skeletal muscle fibres27. CaM overexpression similarly negatively shifted steady-state voltage-dependences of Nav1.4 inactivation. This was rescued by expressing mutant CaM with impaired Ca2+ binding29. However, this evidence largely derives from cultured or heterologous cell lines studied by whole-cell patch-clamp methods that themselves perturb intracellular Ca2+ homeostasis. Furthermore, other reports demonstrated inhibitory effects of Ca2+ and CaM on Nav1.4 even under conditions when they did not inhibit cardiac Nav1.528. Finally, previous explorations of Epac actions in skeletal myocytes concerned actions of Epac1 in inhibiting proteolysis, inducing mitochondrial biogenesis30 and regulating AMP-activated protein kinase31. They did not study electrophysiological effects.However, in common with cardiomyocytes, skeletal myocytes possess G-protein coupled β-adrenergic receptors which generate cAMP+ current activation and inactivation properties, through its action on RyR1-mediated release of intracellularly stored Ca2+ for the first time. They studied mammalian, murine, muscle, thought to share many genetic and physiological properties with in situ human skeletal muscle32. Transcriptome analyses has demonstrated close similarities between murine soleus and human skeletal muscle33. Mouse models are amenable to further detailed studies of recently available genetically modified murine Epac1−/− and Epac2−/− variants34. The present experiments employed the Epac activator 8-CPT and controls using the RyR inhibitor dantrolene comparable with manoeuvres previously used to explore parallel regulatory features of Nav1.5. They similarly used loose patch clamp recordings and its related pulse protocols. Such experimental configurations similarly minimised intracellular perturbations particularly those involving [Ca2+]i homeostasis associated with conventional patch clamp methods4.The present experiments explore downstream effects of Epac activation on skeletal Nav1.4 function, characterised by NaV0 voltage step from the RMP to a membrane potential of (RMP-40) mV of duration 50 ms was applied to remove any residual Na+ channel inactivation that might exist at the RMP. It would maximise and standardise the proportion of Na+ channels in the resting state relative to the inactivated state prior to imposition of the remaining V1 and V2 voltage steps. Use of the double pulse protocol allowed an analysis of the voltage-dependences of both Na+ current activation and inactivation within the same protocol  mV to (RMP + 120) mV through successive sweeps in 5 mV increments. The currents obtained in response to these were used to investigate the voltage-dependence of Na+ current activation. In each sweep, this was then followed by superimposing a further V2 voltage step to a constant strongly depolarised membrane potential of (RMP + 100) mV. This would activate the remaining Na+ channels which had not entered the inactivated state by the end of the prior depolarising V1 step. Currents from this second step accordingly allowed characterisation of the degree of Na+ current inactivation induced by the preceding varying V1 steps, and thus the voltage-dependence of Na+ current inactivation.The loose patch clamp technique alters transmembrane voltage by changing the voltage on the extracellular rather than the intracellular side of the intact membrane under study Fig. . Accordicol Fig. . Thus, dV1 step was made to a membrane potential of (RMP + 40) mV. Inward currents are represented as downward, negative, deflections and outward currents as upward, positive, deflections. Records typically began with a small upward deflection in response to the V0 pre-pulse to a membrane potential of (RMP-40) mV. The initial V1 test voltage steps yielded early inward current transients, I1, whose amplitudes enabled quantification of activation. The subsequent V2 voltage step similarly elicited inward currents, I2, whose amplitude would be expected to vary with the inactivation expected to take place with the preceding pulse V1. The amplitudes of I1 and I2 were measured in relationship to the current recorded just before the V1 and V2 voltage steps respectively. The magnitude of I1 would represent the degree of Na+ current activation in response to V1 while changes in I2 would provide measures of the degree of Na+ current inactivation produced by the preceding V1 voltage step. The Na+ current amplitudes, I1 and I2, in response to the respective V1 and V2 voltage steps thus permitted quantification of the voltage-dependences of Na+ current activation and inactivation respectively.Figure V1 voltage steps. We then repeated this procedure in the reverse order from the largest to the smallest V1 voltage steps. The mean of the two measurements obtained at each V1 step was then calculated and used for analysis. This process was repeated four times in the examination of each patch, with the resulting traces averaged to make up experimental records for display and analysis. Figure I1 and I2 shown separately for each repeat. These demonstrate the consistency of the findings obtained from a patch that was studied that gave stable results throughout the duration  to give current densities (pA/µm2). The pretreatment inward Na+ currents showed their typical activation and inactivation time courses  mV similarly elicited inward Na+ currents with typical activation and inactivation time courses. However, the resulting deflections decreased in amplitude the more depolarised the V1 excursion. This is as expected for Na+ channel inactivation that is dependent upon, and becomes more marked with, the preceding voltage level V1. In a significant number of records the initial inward currents following V1 and V2 steps were followed by a more gradual development of an outward current expected from a delayed K+ current activation. However, challenge by 8-CPT markedly decreased the inward Na+ current amplitudes whether with the V1 or V2 voltage steps at all the test voltages explored by the V1 steps ) for each value of voltage excursion in the V1 step. The rising phases of the current-voltage (I-V) relationships from each patch obtained before and following pharmacological intervention were then characterised by fits to a Boltzmann function: I1 = I = Imax/[1 + exp{(V* − V)/k}]), relating the peak current I at any given voltage excursion V1 through a maximum value of such peak current (Imax), the voltage V* at half-maximal current activation, and a steepness factor term k. The collected n values were then expressed as means ± SEM and values of the parameters compared (a) before and (b) during drug treatment by unpaired 2-tailed t-testing to a P < 0.05 significance level.The reduction in Natep Fig.  before atep Fig.  during tImax,V* and k for the activation I-V curves under each pharmacological condition were expressed as means ± SEM . 8-CPT reduced Na+ current with small changes in its voltage-dependence. Thus, although there were similar voltages at half-maximal current, V* (45.61 ± 1.66 mV (n = 6) vs. 45.09 ± 3.35 mV (n = 6); t-statistic (t) = 0.13, degrees of freedom (φ) = 10, P ≫ 0.05), there were increased values of k, reflecting decreased voltage sensitivity of current activation, following addition of 8-CPT (from 3.56 ± 0.62 mV (n = 6) to 5.75 ± 0.57 mV (n = 6); t = 2.36, φ = 10, P < 0.05), and marked reductions in the values of the maximum current Imax with 8-CPT challenge (from −99.06 ± 15.19 pA/μm2 (n = 6) to −38.04 ± 4.48 pA/μm2 (n = 6); t = 3.52, φ = 10, P < 0.01).The values of + inactivation. Figure + current inactivation produced by varying V1 voltage steps prior to the V2 test step to a fixed membrane potential (e) before and (f) following treatment with 8-CPT. Peak currents, I2, in response to the V2 step are plotted (mean of I2 ± SEM) for each value of voltage excursion in the preceding V1 step. These fell with increasing depolarisation produced by the V1 voltage step, reflecting the voltage-dependence of Na+ current inactivation. The dependences of I2 upon V1 for each patch were then characterised by fits to a Boltzmann function: I2 = I = Imax(1 − 1/[1 + exp{(V* − V)/k}]), where I is the peak current associated with given voltage excursion V1, Imax the maximum peak current, V* the voltage at half-maximal current inactivation, and k the steepness factor term. The collected n values were then again expressed as means ± SEM and values of the parameters compared during treatment and before drug treatment by unpaired 2-tailed t-testing to a P < 0.05 significance level. The values for Imax, V* and k, were expressed as means ± SEM .Concordant observations were obtained from analysis of NaV* (30.70 ± 1.23 mV (n = 6) vs. 25.78 ± 2.35 mV (n = 9); t = 1.78, φ = 13, P ≫ 0.05), but increases in k (from 8.82 ± 0.20 mV (n = 6) to 10.24 ± 0.28 mV (n = 9); t = 4.13, φ = 13, P < 0.01), demonstrating slightly decreased voltage sensitivities of peak current inactivation, following addition of 8-CPT. There were also pronounced reductions in the values of the maximum current Imax with 8-CPT challenge (from 71.86 ± 9.96 pA/μm2 (n = 6) to 34.61 ± 3.50 pA/μm2 (n = 9); t = 4.48, φ = 13, P < 0.001).Analysis of inactivation data demonstrated again that introduction of 8-CPT resulted in marked reductions in the inward current. There were similar voltages at half-maximal current, + currents. This is illustrated by comparing time courses of the inward currents following the V1 voltage step before and following 8-CPT challenge. Figure V1 steps between (RMP + 40) and (RMP + 120) mV from a single patch employing the pulse protocol (a) before and (b) following challenge by 8-CPT. Figure V1 excursions were superimposable at least in the initial stages following the depolarising steps. This was apart from small differences at deflections of 40 mV which are consistent with the shallower voltage dependence of both activation and inactivation observed in the presence of 8CPT.8-CPT did not produce major kinetic changes in the observed Na+ current magnitudes following challenge by extracellular 8-CPT, in intact cardiomyocytes whilst minimising intracellular perturbations. In view of its lipophilic nature and its intracellular Epac-dependent site of action, reversibility of these actions was tested by challenge using dantrolene in combination with 8-CPT. Dantrolene is known to exert, similarly intracellular, actions directly blocking RyR-mediated release of SR Ca2+ whether added extracellularly or injected36. The final experiments that followed applied dantrolene challenge alone in the absence of 8-CPT, to control for any intrinsic actions of dantrolene on Na+ current. This strategy thus tested the reversibility of the intracellular effects of Epac activator on RyR mediated actions on Na+ channel function through matching this with applications of a known RyR antagonist. In addition, adopting this control strategy thus additionally directly confirmed a hypothesis attributing the observed 8-CPT actions on Na+ current to actions on the RyR-Ca2+ release channel.The above experiments thus demonstrated significant effects on NaµM) and dantrolene (10 µM). Dantrolene is a known RyR blocker. It could therefore be used to test for an involvement of RyR-mediated Ca2+ release in the effects of Epac activation on Na+ channel function, by exploring whether it would abrogate the effect of 8-CPT. In these control experiments, simultaneous administration of 8-CPT and dantrolene resulted in current amplitudes similar to those observed before pharmacological challenge, indicating that the effect of 8-CPT in inhibiting Na+ current was abolished  vs. −37.60 ± 6.62 pA/μm2 (n = 13); t = 0.54, φ = 19, P ≫ 0.05), V* (49.92 ± 1.44 mV (n = 8) vs. 46.79 ± 0.80 mV (n = 13); t = 1.96, φ = 19, P ≫ 0.05), and k (7.30 ± 0.62 mV (n = 8) vs. 6.32 ± 0.36 mV (n = 13); t = 1.39, φ = 19, P ≫ 0.05). Similar fits of Boltzmann functions to inactivation data gave concordant results for which Imax was 38.77 ± 5.47 pA/μm2 (n = 8) and 32.62 ± 6.00 pA/μm2 (n = 13) ; V* was 28.50 ± 3.19 mV (n = 8) and 25.57 ± 2.16 mV (n = 13) ; and k was 11.33 ± 0.49 mV (n = 8) and 11.31 ± 0.85 mV (n = 13) . As treatment with dantrolene could reverse the effects of 8-CPT on Na+ current amplitudes, activation of RyR is likely involved in this 8-CPT-induced inhibition of Na+ currents.The subsequent experiments accordingly explored the extent to which the effects outlined above could be abrogated by challenge with a combination of 8-CPT  alone  and −53.60 ± 9.26 pA/μm2 (n = 7) ; V*, 49.43 ± 1.53 mV (n = 7) and 45.91 ± 1.78 mV (n = 7) ; and k, 5.63 ± 0.54 mV (n = 7) and 5.53 ± 0.61 mV (n = 7) . Similarly, the inactivation data  and 41.91 ± 7.24 pA/μm2 (n = 7) ; V*, 30.40 ± 1.72 mV (n = 7) and 26.75 ± 2.20 mV (n = 7) ; and k, 10.19 ± 0.66 mV (n = 7) and 9.91 ± 0.53 mV (n = 7) . Therefore, as dantrolene alone was unable to affect Na+ currents, the reversal of 8-CPT action by dantrolene is due to interference of the inhibitory action of 8-CPT and not by the direct actions of dantrolene on Na+ currents.The above results are consistent with a hypothesis implicating a RyR-dependent Caone Fig. . Analysione Fig.  demonstrves Fig.  gave actata Fig.  gave for2+. Intense recent interest has been directed at such PKA-independent activation mechanisms in distinct critical cell physiological processes. Yet there have been no studies of their actions on excitable as opposed to metabolic function2 in skeletal muscle, particularly its RyR1-mediated signalling, or further downstream effects on Nav1.4 function. Although such studies on Epac action have been made on cardiac myocytes6, skeletal muscle activation is distinct in its involving direct charge-coupled as opposed to Ca2+-induced Ca2+ release by RyR1 rather than RyR2, driven by surface membrane Cav1.1 as opposed to Cav1.2 activation following depolarisation, that does not involve Ca2+ current. Furthermore, these events are initially driven by Nav1.4 rather than Nav1.5 opening19. The present experiments demonstrated inhibitory effects on skeletal muscle Nav1.4 activation by the Epac activator 8-CPT. These were reversed by the RyR-blocker dantrolene. Yet dantrolene challenge by itself did not affect Na+ current. The observed effects on Nav1.4 kinetics, and activation and inactivation steady state properties were quantifiable in terms of maximum currents, steepness factors and half-maximal voltages.The present experiments demonstrate for the first time a downstream modulation of skeletal muscle Nav1.4 function by the recently characterised Epac signalling system, brought about by its action on RyR1-mediated release of intracellularly stored Ca37 avoiding membrane or cellular disruption thereby leaving intracellular, particularly [Ca2+]i, homeostasis unperturbed. This approach can be applied to intact cells in tissue preparations in situ and further permits pipette re-use in successive recording protocols39. Currents from the same pipette before and after drug treatment could therefore be compared at multiple recording sites within the same in situ muscle fibre. In contrast, conventional tight seal patch clamping methods often include Ca2+-sequestrating ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) in the pipette solutions that could perturb intracellular Ca2+ homeostasis, often require isolated or cultured cells and enzyme-cleaning of membranes, and involve pipette replacement between successive patches.The experimental procedures used loose patch clamp methods, apposing micropipettes on the surface membranes of intact skeletal myocytes. This formed relatively low resistance, <~2 MΩ, seals3 was applied at concentrations (~1 µM) considered 300-fold preferentially selective for Epac over PKA activated pathways42. Although 8-CPT also inhibits phosphodiesterase isoforms, it only does so at considerably higher concentrations43. Firstly, in common with the previous findings in murine cardiac, atrial and ventricular muscle4, 8-CPT reduced skeletal muscle cell Na+ current amplitudes whether quantified from peak currents, Imax, or measurements from both activation and inactivation current-voltage curves. 8-CPT also increased their corresponding steepness factors, k, reflecting decreased Na+ channel voltage sensitivities. Secondly, superimposition of scaled membrane currents before and after 8-CPT challenge suggested that 8-CPT contrastingly did not affect Na+ current kinetics, other than modest changes corresponding to the alteration in Na+ channel voltage sensitivity.The cAMP analogue and Epac agonist 8-CPT4. Dantrolene is a ryanodine receptor (RyR)-sarcoplasmic reticular (SR) Ca2+ channel blocker acting through stabilising RyR closed states by enhancing interactions between its N-terminal and the central domains particularly under conditions of increased open channel probability46. In cardiac muscle, dantrolene inhibited RyR2-mediated diastolic Ca2+ release, decreasing frequencies and durations of aberrant Ca2+ sparks in cardiomyocytes modeling catecholaminergic polymorphic ventricular tachycardia (CPVT)46 and cardiac failure models47. Its observed action thus implicated mechanisms involving RyR-mediated SR Ca2+ release in the observed 8-CPT action on Nav1.5. Furthermore, challenge with dantrolene alone did not affect either Na+ current amplitude or values of k. This excludes actions of dantrolene arising from any direct effects on Na+ current. Thus, both dantrolene and 8-CPT likely produced their respective effects upon skeletal muscle Nav1.4 through a common Ca2+-dependent mechanism. Through these pharmacological procedures, voltages of half-maximal activation, V*, for both activation and inactivation were unchanged. Thus, certain regulatory processes that act on cardiac Nav1.5 may well also do so in skeletal muscle Nav1.4.These effects of 8-CPT were abrogated by further addition of dantrolene, in parallel with the previous study in cardiac muscle cells+ channel function in skeletal muscle. First, they suggest that skeletal muscle also possesses an Epac signalling pathway capable of influencing ion channel function. Epac is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPases Ras-related protein (Rap)1 and Rap248. Of the three Epac1, Epac2 and Related to Epac (Repac) isoforms3, Epac2 has been associated with regulating Ca2+-dependent processes48, including pancreatic β-cell excitation-secretion coupling53 and activation of spontaneous transient outward currents in vascular smooth myocytes54. In cardiac muscle, pro-arrhythmic effects of Epac activators on both spontaneous SR Ca2+ release and diastolic Ca2+ transients6 were abolished by genetic ablation particularly of Epac2, as well as of β1 adrenoreceptor, Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ), and with RyR2-S2814 phosphorylation12. These effects may involve a novel pathway in which an activated Epac upregulates Rap1 action in turn stimulating phospholipase C (PLCε) mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis to diacylglycerol (DAG)55. DAG triggers a protein kinase C (PKC) mediated CaMKII activation in turn promoting a RyR2 phosphorylation associated with increased SR Ca2+ release. PLCε also exerts GEF activity that amplifies Rap1 activation initiating further, positive feedback, activation of PLCε. Conversely, CaMKII inhibition abolished such Epac-induced alterations in Ca2+ homeostasis6.The present findings extend signalling schemes involving Epac-dependent cAMP-triggered activation pathways suggested for cardiac muscle to a similar regulation of Na2+ release and cytosolic Ca2+. This is consistent with their conserved CaMKII phosphorylation sites at potentially regulatory locations in the RyR1/RyR2 loop connecting their third and fourth repetitive sequences56. Epac activation may also induce inositol 1,4,5-triphosphate (IP3) receptor (IP3R)-mediated SR Ca2+ release57, likely via IP3 generation from PIP2 by PLCε.Secondly, the findings are consistent with the CaMKII activation phosphorylating skeletal muscle RyR1 as it does cardiac muscle RyR2. These effects are associated with increased SR Cain vitro suggestions that the resulting increased [Ca2+]i may then inhibit Nav1.4 function in intact skeletal myocytes in situ27 in common with its action on cardiac Nav1.558. Nav1.4 and Nav1.5 isoforms possess similar amino acid sequences consistent with such multiple structural and functional homologies13. In common with cardiac Nav1.5, Ca2+ could then regulate Nav1.4 through a number of possible mechanisms. Thus: (a) an EF-hand motif in the Nav1.5 C-terminus domain was reported to directly bind Ca2+ with an affinity typical of Ca2+-sensor proteins14. Such EF-hand-like domains also occur in Nav1.4. (b) Ca2+ could bind at EF-hand motifs of CaM that thereby acts as a Ca2+ sensor. The CaM could then bind to an isoleucine-glutamine (IQ) domain conserved between Nav1.4 and other Nav isoforms. Thus, CaM co-expression with Nav1.4 negatively shifted the steady-state voltage-dependences of Na+ current activation, an effect abolished both by expression of a mutant CaM with impaired Ca2+ binding28 and by mutation of the Nav1.4 IQ domain29. The mutant Nav1.4 remained Ca2+-sensitive, suggesting persistent additional Ca2+-mediated mechanisms regulating Nav1.4 gating independent of Nav1.4 IQ domains. Thus: (c) Nav1.4 and Nav1.5 share sites phosphorylated by CaMKII action17 to extents dependent on [Ca2+]i. CaMKII activation downstream of the Epac2/Rap1/PLCε cascade could also phosphorylate Nav1.4 independently of Ca2+ release. Additionally, CaMKII is itself regulated by Ca2+/CaM. (d) Nav1.4 can also be phosphorylated by PKC resulting in reduced Na+ current amplitudes in vitro59. Similar activation of PKC by the Epac2/Rap1/PLCε cascade may thus also directly contribute to the inhibition of Na+ currents. (e) Ca2+ may produce longer-term inhibition of Na+ channel expression: gain of function, RyR2-P2328S, mutations appear to downregulate cardiomyocyte Nav1.5 expression60.Thirdly, the present findings directly confirm previous 2+]i-dependent inhibition of Nav1.4 could be physiologically important in regulating skeletal muscle excitability. Nav1.4 dysfunction has been reported following mutations in its encoding SCN4A gene. This may be implicated in skeletal muscle disorders including hyperkalaemic periodic paralysis, K+-aggravated myotonia61 and sudden infant death syndrome24. In particular, SCN4A abnormalities resulting in Ca2+-dependent Na+ channel dysregulation have been associated with cold- and K+-aggravated myotonias25. Thus, a Nav1.4 mutation localised to the EF-hand-like domain was observed in a patient with K+-aggravated myotonia62. Patch-clamp analysis of Na+ currents from such mutant channels have showed impaired inactivation and slowed Na+ current kinetics62. Small defects in Nav1.4 inactivation, which can also occur following weakening of Ca2+-dependent inhibition, can also predispose to myotonias61. Finally, the resulting inhibition of myocyte excitability may also contribute to weakness in dystrophic muscle, where resting [Ca2+]i is constitutively substantially raised63.Clinical evidence suggests that a [Ca2, 1.0; CaCl2, 1.8; glucose, 10; and Na-pyruvate, 2.0; pH adjusted to 7.4) during dissection. Isolated muscles were transferred intact into the Sylgard-bottomed experimental bath and secured with A1 insect pins.This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). Chemical reagents used were purchased from Sigma-Aldrich  unless otherwise stated. Muscle preparations were obtained from C67BL6 wild-type mice housed in a licensed facility at room temperature, given free access to sterile rodent chow and water, and exposed to 12 hour light/dark cycles. Mice were killed by cervical dislocation immediately before use by Home Office-licensed personnel, according to Schedule 1 of the UK Animals (Scientific Procedures) Act (1986). Gastrocnemius and soleus muscles were isolated and dissected free of connective tissue. They were bathed in Krebs–Henseleit (KH) solution  (3) KH + 8-CPT (1 μmol/L) + dantrolene (10 μmol/L) and (4) KH + dantrolene (10 μmol/L), with <0.02% DMSO vehicle in all cases. These solutions were first filtered to remove particles with a diameter greater than 10 μm using standard filtration paper . Control of the bath temperature was important as Na+ conductance is dependent on temperature64. Bath temperature was controlled at 24–26 °C by circulating heated water through a coil in the bath, a temperature range chosen to optimise Na+ currents observed as well as preparation lifespan. The bath solution was replaced with fresh KH solution every 30 min to prevent metabolite accumulation around the muscle preparation and obviate evaporation.Experiments were performed in the following variants of the basic KH solution into which were introduced dimethyl sulfoxide (DMSO) vehicle containing 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate sodium salt (8-CPT)  and/or dantrolene sodium , or neither agent. Solutions thus contained: (1) KH control solution, (2) KH + 8-CPT  was used to pull pipettes from borosilicate glass capillaries  for the loose patch clamp studies. The pipettes were visualised under a microscope at 250x magnification and the tips scored using a diamond knife to form a small groove. A force was then applied distal to the groove. This caused the pipette tip to break off perpendicular to the long axis of the pipette. The squarely-broken tips were visualised at 400x magnification and fire-polished with an electrically-heated nichrome filament to smooth the edges of the tip. The internal diameter of the pipettes was measured at 400x magnification. Only pipettes with tips which had a very smooth edge perpendicular to the long axis of the pipette and internal diameters of 25–30 For loose patch clamp studies, the pipette tip was lowered perpendicular to the membrane of the muscle preparation. A gentle suction was applied to form a seal around the patch of membrane under the pipette. The technique thus does not involve impalement of the cell membrane in an accordingly intact muscle fibre Fig. . The curThe equivalent circuit of a typical patch is shown in Fig. + current activation and inactivation properties in a single sweep. The voltage-dependent activation and inactivation properties of Na+ currents could thus be investigated before and following introduction of the various drugs. More detail on the pulse protocol is discussed in the Results section. Data was sampled at a 50 kHz digital sampling rate and filtered with a DC-10 kHz bandwidth, using a 10 kHz Bessel low pass filter. The currents obtained from the double pulse protocol were normalised to the pipette tip cross-sectional area to give current densities, using the formula:An IBM-compatible computer was used to deliver voltage clamp steps relative to the RMP. To detect the presence of ion channels in the membrane patch being studied, depolarising pulses of (RMP + 100) mV lasting 15 ms were first applied. Only patches which produced clearly resolved inward currents with kinetics characteristic of Nav channels were selected. A double pulse protocol Fig.  was thent-test to a significance level of p-value (P) < 0.05. Pre-treatment data was kept in three separate groups depending on the subsequent pharmacological treatment due to the large variation in Na+ currents observed between muscle preparations. As the same muscle preparations were used to collect data before and during treatment with the drug(s), comparisons before and during drug treatment could be made. Curve fitting procedures applied to activation and inactivation current voltage curves were carried out by the open source fitting algorithms QtiPlot (Version 0.9.8.9 svn 2288).Analysis of each pharmacological condition compared data collected during the treatment with the drug(s) with data collected before treatment, employing Student’s unpaired 2-tailed This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). All procedures were completed by Home Office-licensed personnel and fell within the scope of Schedule 1 of the UK Animals (Scientific Procedures) Act (1986).


Recently, Neuroplastin (NPTN) and basigin were identified as previously unrecognized obligatory subunits of PMCAs that dramatically increase the efficiency of PMCA-mediated Ca2+ clearance. Here, we report the cryo-EM structure of human PMCA1 (hPMCA1) in complex with NPTN at a resolution of 4.1 Å for the overall structure and 3.9 Å for the transmembrane domain. The single transmembrane helix of NPTN interacts with the TM8-9-linker and TM10 of hPMCA1. The subunits are required for the hPMCA1 functional activity. The NPTN-bound hPMCA1 closely resembles the E1-Mg2+ structure of endo(sarco)plasmic reticulum Ca2+ ATPase and the Ca2+ site is exposed through a large open cytoplasmic pathway. This structure provides insight into how the subunits bind to the PMCAs and serves as an important basis for understanding the functional mechanisms of this essential calcium pump family.Plasma membrane Ca 2+ ATPase (PMCA) is essential for maintaining Ca2+ homeostasis in eukaryotic cells, and neuroplastin (NPTN) was recently identified as an obligatory subunit of PMCA. Here the authors present the cryo-EM structure of NPTN bound to human PMCA1, which reveals that the NPTN transmembrane (TM) helix interacts with TM10 and the TM8-9-linker of PMCA1.The plasma membrane Ca The plasma membrane Ca2+ ATPase (PMCA) plays an essential role to regulate cellular Ca2+ homeostasis in all eukaryotic cells. PMCA extrudes excess Ca2+ from the cytoplasm, a process that maintains a steep gradient between intracellular (~100 nM) and extracellular Ca2+ (~2 mM)2. In nonexcitable cells where the resting-state Ca2+ concentration remains low, PMCA is generally the principal Ca2+ clearance system4; In excitable cells such as myocytes and neurons with higher demand for Ca2+ clearance, PMCA cooperates with the sodium/calcium exchanger (NCX) and endo(sarco)plasmic reticulum Ca2+ ATPase (SERCA) in the global maintenance of cellular Ca2+ homeostasis6. In addition, the importance of PMCA in the regulation of local intracellular Ca2+ dynamics has steadily increased. It generates a microdomain in its vicinity with low Ca2+ concentration, thereby negatively regulating Ca2+-dependent interaction partners by attracting them to its locale in caveolae7. Genetic deletion or loss-of-function mutations of individual PMCAs are associated with a variety of human diseases, including cardiovascular disease, cerebellar ataxia, deafness, paraplegia, and infertility10.Tight regulation of Ca2+-ATPases were identified in animal cells, the class PIIA SERCAs and golgi secretory pathway Ca2+-ATPases (SPCAs), and the class PIIB PMCAs6. Although they share essential properties of membrane topology and working mechanism, PMCAs have some unique properties distinct from the other two Ca2+-ATPases, particularly in the regulation regions. PMCAs have a unique autoinhibitory domain at N terminus (in plants) or C terminus 14. The action of this domain can be suppressed by directly binding to calmodulin (CaM) or acidic phospholipids16. In addition, an additional phospholipid-binding domain that contains about 40 predominantly basic amino acids exists in the first cytosolic loop of mammalian PMCAs, providing the second binding site for acidic phospholipids17. Recently, two immunoglobulin (Ig) superfamily proteins, neuroplastin (NPTN) and basigin (BASI), were identified as previously unrecognized obligatory subunits of PMCAs and essential for the efficient control of the PMCA-mediated Ca2+ clearance19. However, detailed structural information is unavailable for NPTN- or BASI-bound PMCAs and the molecular mechanism underlying the active effect of the subunits on PMCAs.PMCA belongs to the family of P-type ATPases. Three Ca2+ clearance by NPTN.Due to their physiological and pathological significance and identification as novel complexes with NPTN and BASI, PMCAs represent important targets for structural characterization. In this manuscript, we report the cryo-electron microscopy (cryo-EM) structure of the human PMCA1 in complex with NPTN at an average resolution of 4.1 Å, with the local resolution at the transmembrane region of the complex reaching 3.9 Å. This structure offers important insight into how the NPTN binds to the PMCA1 and serves as a molecular framework for the mechanistic understanding of the efficient control of PMCA-mediated CaTo obtain structural information on PMCAs, full-length human PMCA1d (hereinafter referred to as hPMCA1) was successfully overexpressed in mammalian HEK293F cells by transient transfection. Surprisingly, certain smear bands with molecular weights of approximately 55 kDa co-migrated with hPMCA1 on size-exclusion chromatography  0.143 criterion Fig. . The EM ent Fig. . In a loent Fig. . However17 in the first cytosolic loop of the PMCAs are not resolved, suggesting structural flexibility in these regions. The NPTN subunit resembles a gun wherein the TM and Ig-domains form the handle and barrel, respectively  and ten transmembrane helices (TM1-10) Fig. . The C-tion Fig. . These cion Fig. . The TM7+, K+-ATPase β and γ subunits21 and the H+, K+-ATPase β subunit23. The β subunits of these ATPases make direct contact with TM7 and TM10, and the γ-subunit is next to TM9. Similarly, the accessory regulatory protein FXYD10 also interacts almost exclusively with TM924 -containing buffer  ~7.5 Å) and more closely resembles the E1-Mg2+ conformation (r.m.s.d. ~3.0 Å)  Fig. . The TM1ket Fig. . When thion Fig. . These r2+ structure, the Ca2+-binding site of hPMCA1 is formed by E433 in TM4 and by D895, N891 in TM6, and this site is highly conserved with the Ca2+-binding site II. The Ca2+-binding site I is not preserved in PMCAs due to substitution of the essential acidic residue E771 in TM5 and E908 in TM8 of SERCA by A866 and Q983 in hPMCA1  are present in the funnel, thereby contributing to cation selectivity  conformation, the TM1’ of hPMCA1-NPTN occupies a significantly higher position with respect to the membrane. The distance between the Cα atoms of T110 in hPMCA1-NPTN and L61 in SERCA(E2) can be as high as 11 Å, indicating that significant movement of the TM1 sliding door in E1-NPTN occurs to expose the Ca2+-binding site  and most of the PIV subfamily ATPases form a heterocomplex with at least one additional subunit, which is essential for function30. Only recently, the class PIIB PMCAs were identified as heteromeric complexes that are assembled from two ATPases and two essential auxiliary subunits, either NPTN or BASI10, instead of being monomers or homodimers as previously envisaged31. Atomic structures of P-type ATPases have been determined for the class PIB copper-transporting ATPase32 and zinc-transporting ATPase33, the class PIIA SERCA34, the class PIIC Na+, K+-ATPase21 and H+, K+-ATPase23, and the class PIIIA H+-ATPase35. In this manuscript, the structure of a PIIB Ca2+-ATPase in complex with its obligatory subunit marks an important step towards understanding the functional mechanisms of this essential calcium pump family.P-type ATPases are fundamental in establishing and maintaining steep gradients of key cations across membranes. The P-type ATPase superfamily encompasses 11 distinct classes, covering a wide range of cationic and lipid substrates10, suggesting that the quaternary complex may be dissociated in the detergent environment. Interestingly, the hPMCA1 alone proteins were devoid of ATPase activity  and 537–544 (N domain) of hPMCA4 serve as two receptor sites for interacting with the CaM-binding sites (CaM-BS) of autoinhibitory domainway Fig. , indicat37. Acidic phospholipids and polyunsaturated fatty acids activate the pump by binding to two sites in the pump: one is the CaM-BS17, the other is the phospholipid-binding domain in the cytosolic loop that connects TM2 and TM338. Structure analysis indicates that the phospholipid-binding domain is located in the vicinity of the large cytosolic vestibule of Ca2+ permeation pathway , the most effective acidic phospholipid in stimulating PMCA activity, is modulated during Ca2+-related signaling processes. Accordingly, a possible PIP2-mediated reversible PMCA inactivation mechanism could be envisaged39. Structures of PMCAs in more conformations during the transport cycle are necessary to fully understand the regulatory mechanisms of the subunits and the autoinhibitory domain on PMCAs.However, the molecular mechanism for the transition from the E1-NPTN state to the autoinhibited state remains unknown. As the NPTN is required for the hPMCA1 functional activity Fig. , we spec7. A number of point mutations on PMCAs have been associated with phenotypes in human and mouse47. Among the identified mutations, 5 out of 7 mutations on PMCA2, 1 out of 3 on PMCA3, and one on PMCA4 can be reliably mapped to the structure  were cultured in SMM 293T-I medium  at 37 °C under 5% CO2 in a Multitron-Pro shaker . When the cell density reached 2.0 × 106 cells per ml, the pCAG-PMCA1 plasmids were transiently transfected into the cells. For one-litre cell cultures, approximately 1.5 mg of plasmid was pre-mixed with 4.0 mg 25-kDa linear polyethylenimines (PEIs) (Polysciences) in 50 ml fresh medium for 20–30 min before transfection. The 50 ml mixture was then added to the cell culture, and the culture was incubated for 30 min for transfection. The transfected cells were cultured for 48 h before harvesting.The complementary DNA of full-length N-dodecyl β-d-maltoside (DDM) and 0.2% (w/v) cholesterol hemisuccinate (CHS). After centrifugation at 25,000 х g for 40 min at 4 °C, the supernatant was passed over an anti-FLAG M2 affinity gel (Sigma) column twice. The resin was washed three times with 10 ml wash buffer A (lysis buffer A plus 0.02% DDM and 0.004% CHS). The protein was eluted with elution buffer A (wash buffer A plus 200 µg/ml FLAG peptide (Sigma)). The eluent was incubated with nickel affinity resin  at 4 °C for 40 min, the resin was washed with wash buffer B (lysis buffer A plus 0.1% (w/v) digitonin (Sigma) and 10 mM imidazole), and the protein was eluted with elution buffer B (lysis buffer A plus 0.1% digitonin and 300 mM imidazole). The eluent was concentrated with a 100-kDa cutoff Centricon (Millipore) and subjected to size-exclusion chromatography  in a buffer containing 25 mM Tris pH 8.0, 150 mM NaCl, 1.3 µg/ml aprotinin, 1 µg/ml pepstatin, 5 µg/ml leupeptin, 0.2 mM PMSF, 0.1% digitonin, 2 mM DTT, and 5 mM EDTA. For the cryo-EM analysis, the peak fractions were concentrated to ~8 mg/ml by a 100-kDa cutoff Centricon.For purification of hPMCA1, 12 l of cells were collected and resuspended in lysis buffer containing 25 mM Tris pH 8.0, 150 mM NaCl, 1.3 µg/ml aprotinin, 1 µg/ml pepstatin, 5 µg/ml leupeptin, and 0.2 mM PMSF (lysis buffer A). The membrane fraction was solubilized at 4 °C for 2 h in 1% (w/v) To obtain the hPMCA1 alone proteins, detergent screening was performed during purification. The hPMCA1-NPTN proteins used for ATPase activity assay were purified as mentioned above. The hPMCA1 alone proteins were purified similarly, except that DDM was replaced by different detergents in washing and elution steps of the first-step purification and Superose 6 column was replaced by Superdex 200 column in the last step purification. The subunit BASI was detected by the anti-BASI antibodies (R&D Systems).49 with a slit width of 20 eV on the energy filter and a defocus range from –1.5 μm to –2.5 μm. Each stack was exposed in super-resolution mode for 5.6 s with an exposure time of 0.175 s per frame, resulting in 32 frames per stack. The total dose was approximately 50 e-/Å2 for each stack. The stacks were first motion-corrected with MotionCorr50 and binned by twofold, resulting in a pixel size of 1.091 Å/pixel. The output stacks from MotionCorr were further motion-corrected with MotionCor251, and dose weighting was performed52. The defocus values were estimated using Gctf53.Vitrobot Mark IV (FEI) was used in the preparation of the cryo-EM grids. Aliquots (3 µl each) of hPMCA1-NPTN protein were placed on glow-discharged Quantifoil (1.2/1.3) 300 mesh Au grids (Zhongjingkeyi Technology Co. Ltd.). The grids were blotted for 4 s and plunged into liquid ethane cooled with liquid nitrogen. The grids were then transferred to a Titan Krios (FEI) electron microscope equipped with a Gatan GIF Quantum energy filter and operated at 300 kV with a nominal magnification of 105,000×. Zero-loss movie stacks were automatically collected using AutoEMationII54. After 2D classification, ten good 2D classes were used to generate an initial model using e2initialmodel.py55, and a total of 1,001,249 good particles were then selected and subjected to 3D auto-refinement. The particles were further subjected to several cycles of 3D classification with six classes and a local angular search step of 3.75° with the output from different global angular search iterations of the 3D auto-refinement as input. The class with fully intact particles was considered as a good class, which contains useful high-resolution information and usually has the smallest value of the accuracy of rotation and translation. A total of non-duplicated 655,998 particles were selected from the good classes of local angular search 3D classification. These particles were subjected to local angular search 3D auto-refinement with a soft mask applied, resulting in a 4.5-Å resolution map. The particles were classified into four classes using multi-reference, and the best classes were selected and combined. The final particle number for the 3D auto-refinement is 105,118, thereby resulting in a 4.1-Å resolution map after post-processing. The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion56 with the high-resolution noise substitution method57.A diagram of the procedures used in data processing is presented in Supplementary Fig. 2+ (PDB: 3W5B), which was first fitted into the EM map by Chimera, served as a reference for model building. Model building was performed in COOT58. Bulky residues, such as Phe, Tyr, Trp, and Arg, in many of the TMs and in the P domain of hPMCA1 were clearly visible in our cryo-EM structure and used as landmarks for model building. The secondary structure predicted by Phyre220 based on the sequence of the A domain (residues 193–290) was well fitted into the map, and the bulky residues F194, R198, R219, and Y220, which were clearly resolved, and several motifs that are highly conserved between SERCA and PMCAs facilitated the sequence assignment . The protein concentrations for the assays ranged from 0.05 to 0.2 mg/ml. All reactions were performed using the reaction buffer from the assay kit with final concentration of 1.83 mM CaClSupplementary InformationPeer Review File


SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first step of de novo sphingolipid synthesis. Mutations in SPT result in a change in enzyme substrate specificity, which causes the production of atypical deoxysphinganine and deoxymethylsphinganine, rather than the normal enzyme product, sphinganine. Levels of these abnormal compounds are elevated in blood of HSN-1 patients and this is thought to cause the peripheral motor and sensory nerve damage that is characteristic of the disease, by a largely unresolved mechanism. In this study, we show that exogenous application of these deoxysphingoid bases causes dose- and time-dependent neurotoxicity in primary mammalian neurons, as determined by analysis of cell survival and neurite length. Acutely, deoxysphingoid base neurotoxicity manifests in abnormal Ca2+ handling by the endoplasmic reticulum (ER) and mitochondria as well as dysregulation of cell membrane store-operated Ca2+ channels. The changes in intracellular Ca2+ handling are accompanied by an early loss of mitochondrial membrane potential in deoxysphingoid base-treated motor and sensory neurons. Thus, these results suggest that exogenous deoxysphingoid base application causes neuronal mitochondrial dysfunction and Ca2+ handling deficits, which may play a critical role in the pathogenesis of HSN-1.Hereditary sensory neuropathy type 1 (HSN-1) is a peripheral neuropathy most frequently caused by mutations in the  Graphical illustration demonstrating the proposed pathomechanism of the toxic, atypical deoxysphinganine and deoxymethylsphinganine, produced by mutant SPT in HSN-1.2+ (SOC) channels, upon stimulation. This results in elevated Ca2+ entry when SOC channels open, which is buffered by mitochondria. This increase in mitochondrial Ca2+ uptake in turn results in mitochondrial dysfunction, leading to loss of mitochondrial membrane potential and ultimately dysfunction of cellular processes and neuronal degeneration. .The toxic sphingoid bases DSp and DMSp (or downstream metabolites) are released into the extracellular space where they destabilize neuronal cell membranes, resulting in dysfunction of neuronal ion channels such as store-operated Ca •SPTLC1 or SPTLC2 are the most common cause of HSN-1.Mutation in •Abnormal sphingolipids produced by the mutant SPTLC1/2 enzyme cause neuronal death.•Early targets of abnormal sphingolipids are the mitochondria and ER.•2+ loading.Mitochondrial damage manifests in membrane potential loss and abnormal Ca•2+ through SOC channels.Abnormal sphingolipids cause cell membrane leakage of Ca It cataSPTLC1  and deoxymethylsphinganine (DMSp), respectively  receptor signalling  Act (1986), and approved by the UCL Institute of Neurology Animal Welfare Ethical Review Board. All reagents were obtained from ThermoFisher Scientific unless otherwise specified.2.1g for 5 mins to pellet cells. The supernatant was discarded and the cell pellet re-suspended in supplemented neurobasal medium containing 2% B27 supplement, 0.5 mM GlutaMAX, 25 μM 2-mercaptoethanol, 2% horse serum, 0.1 ng/ml murine glial cell line-derived neurotrophic factor , 0.5 ng/ml human or rat ciliary neurotrophic factor (CNTF by Peprotech), 0.1 ng/ml human brain-derived neurotrophic factor  and penicillin/streptomycin (50 units per ml/50 μg per ml). Cells were plated onto 24-well plates with glass coverslips at a cell density of 5 × 104 cells/well for cell survival and neurite outgrowth assays. For live-cell imaging, cells were plated onto glass bottom dishes  at a cell density of 1 × 105 cells/well or 8 well μ-slides (Ibidi) at a cell density of 3 × 104 cells/well. All culture dishes were pre-coated overnight with poly-L-ornithine  followed by laminin  for 2 h, prior to seeding cells. These mixed ventral horn cultures were maintained in supplemented neurobasal medium (as above), under standard culture conditions . MNs were considered immature after 1 day in vitro (DIV) and mature after 5 or more DIV, due to their general morphology as well as the expression of key MN characteristics, such as expression of glutamate receptors (Van Den For primary mixed motor neuron (MN) cultures, the ventral spinal cord was dissected from embryonic day 12.5–13.5 (E12.5-E13.5) mouse embryos . Ventral horns were digested in trypsin (0.025%) for 10 mins at 37 °C. The tissue was then triturated in DNAse I  in 0.4% bovine serum albumin (BSA) in Leibovitz's L15 medium. Supernatant was collected and the remaining pellet triturated a second time in DNAse I (0.02 mg/ml) in 0.4% BSA in Leibovitz's L15 medium. Supernatants from both trituration steps were combined and centrifuged through a BSA cushion (4% in Leibovitz's L15 medium) at 380 × Van Den .2.22+ and Mg2+ free) supplemented with penicillin/streptomycin (100 units per ml/100 μg per ml). Dissected ganglia were then incubated in pre-warmed HBSS (Ca2+ and Mg2+ free) containing 60 units of papain (Sigma) at 37 °C for 10 min with gentle rotation. Papain solution was then removed and ganglia re-suspended in pre-warmed HBSS containing collagenase type II (0.1%) and dispase (1.5 units/ml) at 37 °C for 45 min with gentle rotation. DRG were then gently triturated in order to dissociate them into a cell suspension. Cells were plated onto glass bottom dishes (35 mm petri dish with 14 mm microwell) or 8 well μ-slides (Ibidi) pre-coated with poly-d-lysine  and laminin (5 μg/ml). Mixed DRG cultures were cultured in neurobasal medium supplemented with B27 supplement (2%), fetal calf serum (1%), 2 mM GlutaMAX, mouse nerve growth factor  and penicillin/streptomycin (50 units per ml/50 μg per ml). DRG cells were incubated under standard culture conditions  and used for experiments after 3–5 DIV.For primary dorsal root ganglia (DRG) cultures, DRG were harvested from 3 to 8 day old mice (C57BL/6). Spinal columns were removed, the DRG exposed and dissected into sterile HBSS (Ca2.3Metabolites of SPT enzyme products were purchased from Avanti Polar Lipids and dissolved in ethanol as stock solutions (1 mM). Cells at different stages after plating were either left untreated or treated with sphinganine (Sp), deoxysphinganine (DSp), deoxymethylsphinganine (DMSp) or ethanol , at final concentrations ranging from 0.1 μM to 10 μM. The duration of treatment ranged from 5 min to 6 days, as described in 2.3.1For cell survival and neurite outgrowth assays, at the end of each treatment regime, the cells were fixed in 4% paraformaldehyde for 20 min then washed in phosphate buffered saline (PBS). Prior to primary antibody labelling, cells were permeabilised in PBS containing 0.1% Triton X-100 (Sigma) and blocked in 5% goat serum. Mouse anti-β-III tubulin (BioLegend 801201) was used as a pan-neuronal marker (1:1000), followed by Alexa Fluor® 488 labelled anti-mouse IgG (H + L) secondary antibody . DAPI  was used to stain nucleic acids (nuclei).2.3.2Cultures were immunostained for the neuronal marker anti-β-III tubulin and stained with the nuclear marker DAPI. The cells were then imaged using the 20× objective of a Leica epifluorescence light microscope and the Leica Application Suite software. Images were taken for cell and neurite counts, as well as assessment of neurite length. A minimum of five representative images measuring 655 × 491 μm were taken per condition, per experiment. Cells positive for β-III tubulin and DAPI counter-stain were counted as neurons to establish the total number of cells surviving. Neurite outgrowth was assessed by tracing from the cell body to neurite tip. When branched, the longest neurite was measured and the shorter branch counted as a separate neurite.2.3.3d-glucose, 3 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 1.25 mM KH2PO4, pH 7.35) and then loaded with 5 μM fura-2 AM (Molecular Probes by Life Technologies) in the above recording medium supplemented with pluronic F127 (0.04%), for 30 min, at room temperature. Fura-2 was then replaced with recording medium and dye accumulation optically detected by charge-coupled device (CCD) camera microscopy following excitation at wavelengths 340 nm and 380 nm using the computer programme Andor iQ 1.9 Imaging. The ratio of fura-2 emission at 510 nm following excitation at 340/380 nm wavelengths was used as a readout measure of intracellular Ca2+ concentration; thus, an increase in this ratio signifies an increase in free Ca2+ present. Relative ER Ca2+ concentration and mitochondrial Ca2+ concentration were measured in Ca2+-free recording medium containing 0.5 mM EGTA, first using thapsigargin  to estimate ER Ca2+  in Ca2+-free recording medium, followed by replacement with standard, Ca2+-containing recording medium. In some experiments, 1 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone  was introduced 2 mins prior to thapsigargin treatment and included in recording media throughout the experiment in order to render mitochondria non-functional and to test the contribution of mitochondrial Ca2+ buffering to the overall intracellular Ca2+ increase. Relative ER Ca2+, mitochondrial Ca2+, membrane-depolarization induced Ca2+ influx and store-operated Ca2+ entry were all calculated by subtracting the baseline fura-2 ratio from the relevant peak amplitude fura-2 concentration. The drugs and conditions used to manipulate intracellular Ca2+ handling are summarised in Cultures were washed in recording medium  (MNs) or 3–5 DIV (DRG neurons) were treated with the sphingoid bases for 2 h prior to live cell imaging. Cultures were loaded with tetramethylrhodamine methyl ester (TMRM) (20 nM) in recording medium (as described above) supplemented with pluronic F127 (0.04%), for 30 min, at room temperature before imaging (no washes were performed prior to imaging). Immediately before imaging, cells were additionally loaded with the live-cell labelling dye calcein blue, AM (1 μM). Z-stack images were taken on Zeiss Laser Scanning 710 (MNs) or 780 (DRG neurons) confocal microscopes using the lasers 405 nm and 561 nm and the ZEN LE Digital Imaging 2009 or 2012 software. Z-stacks (minimum 720 × 720 pixels and 12 bit depth) were compressed to maximal intensity per pixel and thresholded  using ImageJ version 1.51n. In processed images calcein blue was used to outline individual cells. TMRM intensity (as a readout measure for ∆ψm) was calculated from the cell soma and expressed as a percentage relative to the mean TMRM intensity in control cells, for each independent imaging session. Total mitochondrial area (determined using TMRM) was expressed as a percentage of the cell soma size , from thresholded images as described above.Primary cultures at 5–8 days 2.3.5t-test or Mann-Whitney test, as appropriate. * P < 0.05, ** P < 0.01 and *** P < 0.001.Statistical analyses were performed using GraphPad Prism Version 6.0. Data were assumed to be normally distributed unless evidence was provided otherwise by the D'Agostino-Pearson normality test. Thus, statistical significance was determined by one- or two-way ANOVA with Dunnett's multiple comparison tests, Kruskal-Wallis with Dunn's multiple comparison tests, unpaired 3To determine whether abnormal deoxysphingoid bases are neurotoxic to mammalian motor neurons, which together with sensory neurons are typically affected in HSN-1, primary motor neuron cultures (MNs) were treated with either the normal enzymatic product sphinganine (Sp), the atypical products, deoxysphinganine (DSp) and deoxymethylsphinganine (DMSp), or a vehicle control (ethanol), at a range of concentrations and for varying treatment durations. The concentrations chosen were based on deoxysphingoid base concentrations previously measured in HSN-1 patient plasma .3.1in vitro (DIV) by immunostaining for the neuronal marker β-III tubulin. Previous studies have shown that atypical sphingoid bases have deleterious effects on immature primary chick MNs when treated within the first 24 h of plating, causing decreased neurite outgrowth . Therefo3.2P < 0.05). Although no significant decrease in MN neurite length was observed with 0.1 μM DMSp treatment, cells treated with 1 μM DMSp had a reduced neurite length of 27.2 ± 2.0 μm (P < 0.001). In 1 μM DSp-treated cells MN neurite length was further decreased to 12.9 ± 1.1 μm (P < 0.001). Interestingly, treatment with the typical enzyme product, Sp, was also moderately toxic, with 2 μM treatment causing a small but significant decrease (to 28.5 ± 2.3 μm) in average MN neurite length (P < 0.01). A similar pattern of toxicity was observed when the longest neurite length per MN was examined  chick neurons (DRG neurons and MNs) with deoxysphingoid bases for a short period (24 h) results in neuronal toxicity, as determined by assessment of neurite outgrowth  and 37 ± 10% (P < 0.05), respectively.At 1 DIV, MN cultures were treated for 24 h with increasing concentrations of Sp, DSp and DMSp E. Follow+, β-III tubulin− cells were observed. The extent of MN survival was quantified, and a dose-dependent decrease in MN survival was observed in response to DSp and DMSp treatments (P < 0.05) and 19 ± 9% (P < 0.05) of MNs survived, respectively.As shown in the representative images in eatments F. In culP < 0.05) and 51 ± 20% (P < 0.05) of MNs survived following 1 μM DSp and DMSp treatments, respectively.Since MNs at 1 DIV are relatively immature, the effect of sphingoid base treatments on more mature MN cultures was also examined, by treating MNs at 5 DIV for 4 days  die rapidly upon exposure to deoxysphingoid bases, within 24 h E, it is 3.52+ homeostasis. Since changes in intracellular Ca2+ levels are an important early indicator of cell stress ), indicating that short-term treatment with deoxysphingoid bases does not alter resting cytosolic Ca2+ homeostasis and these MNs appear largely healthy.Mature MNs (≥5 DIV) were treated acutely for 2 h with 1 μM of sphingoid bases, and the effects on baseline cytosolic Ca3.62+ levels were not altered by acute exposure to deoxysphingoid bases, it is possible that this is the result of effective Ca2+ buffering by intracellular organelles, such as the ER and mitochondria. We therefore examined the effects of deoxysphingoid base treatments on ER Ca2+. Mature MNs (≥ 5 DIV) were first treated for 24 h with 1 μM deoxysphingoid bases and relative ER Ca2+ levels determined by measuring the change in cytosolic Ca2+ following treatment with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor, thapsigargin, which causes the ER to release Ca2+ into the cytosol. As these experiments were performed in the absence of extracellular Ca2+ the resulting change in cytosolic Ca2+ can be used to infer a relative ER Ca2+, as shown in a typical Ca2+ recording experiment shown in 2+ in MNs. Thus, in DSp treated MNs the fura-2 ratio for relative ER Ca2+ was reduced to 0.15 ± 0.02 compared to 0.22 ± 0.02 in the vehicle treated MNs (P < 0.05), indicative of ER stress  and 1.15 ± 0.14 (P < 0.001), respectively. This shift towards higher mitochondrial Ca2+ was also observed, to some extent, in Sp-treated MNs, which had an average fura-2 ratio of 0.80 ± 0.13 (P < 0.01).Since mitochondria also play an important role in the regulation of intracellular Ca be made . Howeverial Ca2+ E. In veh2+ levels , and any of the sphingoid base treatments; Sp (2.11 ± 0.05), DSp (2.11 ± 0.05), DMSp (2.24 ± 0.10).We next investigated the source of the increased mitochondrial Ca3.92+ might originate from a second class of cell membrane channels, by examining entry of Ca2+ through the ER-regulated store-operated Ca2+ (SOC) channels. These plasma membrane channels are regulated by intracellular Ca2+ levels, namely the ER . Frequently, we also observed fluctuation in fura-2 ratios in DSp-treated MNs following Ca2+ influx through SOC channels when compared to vehicle treated MNs , and therefore active mitochondrial regulation of cytosolic Ca2+, is abolished with FCCP, there is an increase in cytosolic Ca2+ following opening of SOC channels. This increased cytosolic Ca2+ after SOC channel activation was present to similar extents in both vehicle- and DSp- treated MNs, with the fura-2 ratio changing from 0.67 ± 0.05 to 1.11 ± 0.0.13 (P < 0.01) in vehicle-treated MNs and from 1.00 ± 0.06 to 1.40 ± 0.11 (P < 0.05) in DSp-treated MNs. Application of FCCP led to exacerbated cytosolic Ca2+ levels in both vehicle-treated and DSp-treated MNs, providing evidence for an active involvement of mitochondria in Ca2+ buffering following SOC channel activation in these cells.In order to further confirm that the elevated mitochondrial Caomplex V . The res2+ levels and disturbs ER Ca2+.Taken together, these results suggest that a critical effect of deoxysphingoid bases, or their downstream metabolites, may be to alter the cell membrane, which results in dysregulation of SOC channels, which in turn affects mitochondrial Ca3.102+ loading in MNs treated acutely with the deoxysphingoid bases, we next tested whether these treatments also cause a more generalized mitochondrial dysfunction. We investigated changes in mitochondrial membrane potential (∆ψm) following 2 h sphingoid base treatments using TMRM, a cationic dye which accumulates in mitochondria as a function of ∆ψm, in both MN and DRG cultures  and 74 ± 3% (P < 0.001) of the intensity measured in vehicle treated neurons, respectively . DMSp-treated DRG neurons showed a similar decrease in ∆ψm and intriguingly, even treatment with the typical enzyme product, Sp, caused a depletion in ∆ψm. In these sensory DRG cells, 2 h treatment with DSp was also sufficient to cause a decrease in the mitochondrial area per cell soma, so that DSp-treated DRG neurons showed an average mitochondrial area of 56 ± 3% compared to 70 ± 3% in vehicle-treated DRG neurons . The mPTP opens in a bid to release mitochondrial Capoptosis . To explpoptosis , we co-tpoptosis , alongsiImmature MNs were treated with CsA (1 μM) and the sphingoid bases (1 μM) for 24 h, and cell survival was expressed as a percentage of untreated cultures. There was no significant difference in MN survival in cultures treated with the sphingoid bases alone or with addition of CsA ; 47 ± 9%4In this study we set out to investigate the intracellular effects of exogenous deoxysphingoid bases, the abnormal enzyme products of mutant SPT in HSN-1, on cultured mammalian neurons. Our results clearly show that deoxysphingoid bases are toxic to primary mammalian neurons. Toxicity by these abnormal sphingoid bases manifests as reduced neurite length and complexity of treated primary MNs  as well in vitro model of HSN-1 is dramatic, with over 50% of neurons dying after just 1 day of treatment. This is in contrast to the slowly progressing neuropathy observed in HSN-1 patients. However, DSp displayed a more severe pathology than DMSp at the same concentrations tested, as measured by neurite outgrowth assays, which does reflect the higher serum levels of DSp than DMSp measured in patients. Although the primary embryonic MNs used in these experiments may be particularly vulnerable to toxicity, it is possible that in vivo, in patients, the blood plasma may act as a “dumping ground” for deoxysphingoid bases and derivatives, whereas levels in the immediate vicinity of neurons might be lower. Moreover, in blood, deoxysphingoid bases are mainly transported on low and very low density lipoproteins and may be complexed with other proteins, which could additionally dampen their toxic effects  treatment did not result in any change in resting cytosolic Ca2+ levels . This suggests that following a 2 h treatment paradigm, MNs were functioning, are able to compensate for any damaging effects of the treatment and are not yet undergoing apoptosis. This experimental model therefore offers an opportunity to probe the underlying mechanisms of deoxysphingoid base toxicity.Although an increase in cytosolic Ca2+ homeostasis and the SPT enzyme is located in the ER membrane; the ER is thus the site of deoxysphingoid base generation. We observed Ca2+ depletion in the ER, indicative of ER stress, as early as 2 h in DRG cells, yet in MNs, which intrinsically appear to store less Ca2+ in ER and mitochondria than sensory neurons, there was no measurable depletion of Ca2+ in the ER at this time point , and thus the 2 h treatment model used in these experiments is not sufficiently short to capture changes in mitochondrial Ca2+ in DRG cells, in which depletion of ER Ca2+ is evident much earlier than in MNs. However, differences in the experimental setup used to measure relative ER Ca2+ in DRGs and MNs make it difficult to draw direct comparisons between the two cell types. MNs are developmentally younger when the relative ER Ca2+ content is measured as they are derived from embryonic mouse spinal cords, whereas DRG cells are obtained from postnatal mice and this developmental shift might be responsible for the observed differences between relative ER Ca2+ measured in motor and sensory neurons. The proposal that mitochondrial Ca2+ levels may be affected in advance of ER Ca2+ levels mirrors the recent study of Alecu et al. that tracked deoxysphinagnines to the mitochondria within 5 mins of application, and at later time points, also to the ER  treatment. Thus, the most obvious source of Ca2+ is the extracellular space through specific cationic channels: voltage-gated channels, second messenger-operated channels, receptor-operated channels, store-operated channels, or under some conditions, the reversal of the sodium-Ca2+ exchanger  channels that are regulated by the ER, causing the opening of these channels upon Ca2+ store depletion. Membrane depolarization-induced influx of Ca2+ was measured after 2 h treatment with sphingoid bases, and no difference was detected between the control and any of the treatment groups, indicating that the deoxysphingoid bases do not directly interfere with this element of normal neuronal activity, at least not at this early phase  funding scheme. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Conflicts of interest: none.We would like to thank Ione F. G. Meyer, Verna Sarajarvi and James N. Sleigh for their helpful comments during the preparation of this manuscript, James Dick for his excellent technical assistance, and all members of the Linda Greensmith and Giampietro Schiavo laboratories for helpful discussions. We thank Harriet Howard for her help with Western blot procedures. ERW is in receipt of a PhD studentship from the Medical Research Council (MRC) Centre for Neuromuscular Diseases, MRC Centre Grant (G0601943). LG is The Graham Watts Senior Research Fellow supported by The Brain Research Trust. MMR is grateful to the National Institutes of Neurological Diseases and Stroke and Office of Rare Diseases (U54NS065712) for their support. BK was supported by UCLH Charities and the European Community's Seventh Framework Programme . This work was undertaken at University College London Hospitals/University College London, which received a proportion of funding from the


Intracellular Ca2+ is the most important regulator for them to open, though PIP2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PIP2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PIP2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca They share 40% homology in their amino-acid sequences, so that they are the closest homologs among eight members of the TRPM family [2+ (Ca2+i) concentration ([Ca2+]i). The Ca2+-sensitivity of TRPM4 has been reported to be maintained by interaction with PIP2, a major phosphoinositide in plasma membrane i was varied by changing the Ca2+ concentrations of bath solutions. Although the WT rTRPM4 current amplitudes were saturated at 3 mM Ca2+i, Q825A  mutant required more than 3 mM Ca2+i to show their currents  of Q825A and D856A were rightward shifted in comparison with that of WT (50) for Ca2+ of WT rTRPM4, Q825A and D856A currents were 0.81, 7.19 and 4.42 mM, respectively.Firstly, we examined the effects of mutations of the amino acid residues at the shallow position of the proposed Ca hTRPM4  seem to contact with Ca2+ (822 and Asp862 of rTRPM4 were mutated to glutamine (E822Q) and asparagine (D862N), respectively, in order to eliminate the negative charge in their side chains . At first, we tried to measure the mutant currents under the presence of 1 mM Ca2+i, which was able to evoke substantial WT rTRPM4 currents . These results suggest that all of the mutations of the amino acid residues at the deep position of the proposed Ca2+-binding site reduced Ca2+-affinities of the mutants so much that the Ca2+-sensitivities of mutants were almost lost, especially after the desensitization.Next, we examined the effects of mutations of the amino acid residues at the deep position of the proposed Caith Ca2+ C. Glu822e chains D. Asn859currents A. Howevemeasured C. From t2+-sensitivities of E822Q, N859A or D862N were unable to be precisely estimated because there were no currents after their desensitization . Asp862 was mutated to glutamate (D862E). Although the negative charge of the residues remained in E822D and D862E or was added in N862D, the Ca2+-sensitivities of the mutants were lower than that of WT rTRPM4  in the 5’ untranslated region (UTR) . However2+-sensitivity of rTRPM5 (987 is located immediately before the TRP domain and was mutated to asparagine (D987N). Glu1000 is located in the TRP domain and was mutated to glutamine (E1000Q). As TRPM5 was inhibited by Zn2+ [2+-sensitive currents were analyzed as rTRPM5 currents. Typical whole-cell currents are shown in 2+]i was evaluated by using pipette solutions which contained different free Ca2+ concentrations. Similar to our previous report concerning rTRPM4 ethanesulfonic acid) . Bath solutions contained 145 mM NaCl and appropriate concentrations of CaCl2 or 5 mM EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) for a Ca2+-free solution. Macroscopic currents under the inside-out configuration were recorded using the ramp pulses. The holding potential was −60 mV (the intracellular side is negative), and the ramp pulses from −100 to +100 mV with durations of 400 ms were applied every 2 s. The currents were filtered at 1 kHz and sampled at 5 kHz. Water soluble phosphoinositides, diC8-PIP2 and diC8-PIP3, were obtained from Cellsignals  and dissolved in the bath solution.Patch-clamp recordings were performed as described previously ,19. BrieN,N,N’,N’-tetraacetic acid), 4 mM ATP disodium salt, 10 mM HEPES  and appropriate amounts of MgCl2 and CaCl2 in order to make solutions which contained 10−3 M free Mg2+ and 0–10−4 M free Ca2+. The free Mg2+ and Ca2+ concentrations were calculated using the CaBuf software . The pipette solutions containing 1 and 3 mM CaCl2 were made without BAPTA. The extracellular solution was composed of 145 mM NaCl, 2 mM MgCl2 and 10 mM HEPES . In most experiments, the holding potential was −60 mV and the ramp pulses from −100 to +100 mV with durations of 400 ms were applied every 2 s. The currents were filtered at 1 kHz and sampled at 5 kHz. One minute after the start of the whole-cell recording, 50 μM ZnCl2 was applied through the bath solution. Current densities were calculated by dividing the current amplitudes by the cell capacitances (pA/pF). As an indication of rTRPM5 current densities, the Zn2+-sensitive current densities were calculated by subtracting the current densities in the presence of Zn2+ from the peak current densities. All experiments were conducted at room temperature.The whole-cell patch clamp recordings for rTRPM5 current measurements were performed under the conditions described below. The pipette solution was composed of 130 mM CsCl, 10 mM BAPTA ethane-2 and 0.1 mM CaCl2) and incubated 15 min with 1.0 mg/ml EZ-link sulfo-NHS-s-s-biotin (Thermo Fisher Scientific) in cold biotinylation buffer  with gentle agitation at 4 °C. The cells were washed twice with a quenching buffer (100 mM glycine in D-PBS-CM), then rinsed once with D-PBS, scraped in cold D-PBS and pelleted at 290×g for 1 min at 4 °C. The cells were lysed in a lysis buffer ). The lysates were sonicated using Bioruptor  or Ultrasonic homogenizer . The lysates were centrifuged for 10 min at 14,000× g at 4 °C. The protein concentrations of the lysates were measured by using DC Protein Assay  and adjusted to 100 μg in 150 μL with the lysis buffer. Streptavidin-agarose beads (Sigma-Aldrich) were added to the protein extracts. The mixtures were rotated at 20 rpm for 2 h at 4 °C. The beads were pelleted by brief centrifugation (650× g for 1 min). The supernatants were taken as the unbound intracellular fractions, mixed with one fourth volume of 4× Laemmli sample buffer containing 10% β-mercaptoethanol, and heated at 50 °C for 15 min. The beads were washed three times with D-PBS containing 0.5% Triton X-100. The biotinylated proteins were eluted from the beads by mixing the beads with 4× Laemmli sample buffer containing 10% β-mercaptoethanol and heating the mixtures at 50 °C for 15 min (Surface fractions).The transfected HEK293T cells were washed twice with ice cold D-PBS-CM  and the surface fractions were separated by SDS-PAGE and transferred to a PVDF membrane. After incubation in blocking buffer containing 3% skim milk, the blots were treated with the diluted anti-TRPM5 rabbit antibody  or anti-GFP rabbit antibody  and then with horseradish peroxidase-conjugated anti-rabbit IgG antibody  as the secondary antibody. The chemiluminescent signals were produced by using Immobilon Forte  and detected by a single-lens reflex camera .maxI is the maximal current amplitude or the maximal current density, C is the concentration of Ca2+, EC50 is the half maximal effective concentration and n is the Hill coefficient.Concentration-response curves were obtained by fitting the averages of current amplitudes or current densities with the Hill equation:t test, Welch’s t test or Dunnett’s test as appropriate. A value of p < 0.05 was considered significant.All data are expressed as means ± S.E. The statistical analyses were performed using Student’s


DM), increased oxidative stress may be attributable to higher xanthine oxidase activity. In this study, we examined the relationship between oxidative stress and atrial electrical and structural remodeling, and calcium handling abnormalities, and the potential beneficial effects of the xanthine oxidase inhibitor allopurinol upon these pathological changes.There are several mechanisms, including inflammation, oxidative stress and abnormal calcium homeostasis, involved in the pathogenesis of atrial fibrillation. In diabetes mellitus (CaL was measured from isolated left atrial cardiomyocytes using voltage‐clamp techniques. Confocal microscopy was used to detect intracellular calcium transients. The Ca2+ handling protein expression was analyzed by Western blotting. Mitochondrial‐related proteins were analyzed as markers of mitochondrial function. Compared with the control group, rabbits with DM showed left ventricular hypertrophy, increased atrial interstitial fibrosis, oxidative stress and fibrosis markers, ICaL and intracellular calcium transient, and atrial fibrillation inducibility. These abnormalities were alleviated by allopurinol treatment.Ninety rabbits were randomly and equally divided into 3 groups: control, DM, and allopurinol‐treated DM group. Echocardiographic and hemodynamic assessments were performed in vivo. Serum and tissue markers of oxidative stress and atrial fibrosis, including the protein expression were examined. Atrial interstitial fibrosis was evaluated by Masson trichrome staining. IAllopurinol, via its antioxidant effects, reduces atrial mechanical, structural, ion channel remodeling and mitochondrial synthesis abnormalities induced by DM‐related increases in oxidative stress. Allopurinol, a xanthine oxidase inhibitor, reduces atrial mechanical, structural, ion‐channel remodeling and mitochondrial synthesis abnormalities caused by diabetes mellitus‐related oxidative stress.Xanthine oxidase activation is related to diabetes mellitus‐induced atrial fibrillation. Allopurinol may act as a potential upstream therapy for atrial fibrillation in the setting of diabetes.2+) homeostasis, as well as oxidative stress‐mediated damage play important roles.Atrial fibrillation (AF) is one of the most common arrhythmias observed in medical practice and is responsible for significant cardiovascular morbidity and mortality worldwide.2+ handling is a critical regulator of action potential duration, and mechanical activity of cardiomyocytes via excitation‐contraction coupling.Xanthine oxidase (XO), a key enzyme of purine catabolism, is responsible for the generation of reactive oxygen species (ROS) via several pathways that include nitric oxide (NO) and calcium signaling.The authors confirm that the data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure. Material will be available from Tianjin Institute of Cardiology, Second Hospital of Tianjin Medical University, which is responsible for maintaining availability upon request to the corresponding author.This study was approved by the Experimental Animal Administration Committee of Tianjin Medical University and Tianjin Municipal Commission for Experimental Animal Control and is fully compliant with National Institutes of Health guidelines from the United States.CaL recordings, and intracellular Ca2+ imaging. For the induction of diabetes mellitus, alloxan monohydrate  was dissolved to sterile normal saline to achieve a concentration of 5% (w/v), and a dose of 120 mg/kg was immediately administered intravenously via the marginal ear vein. The presence of diabetes mellitus was confirmed 48 hours later by blood glucose levels ≥14 mmol/L (once) or ≥11 mmol/L (twice). Subsequently, blood glucose level monitoring was performed weekly using the glucometer Optium Xceed . Rabbits in the ALLO group received allopurinol (60 mg/kg per day) orally for 8 weeks.A total of 90 male and female Japanese rabbits (weighing 1.7–2.5 kg at the beginning of the study) were obtained from Beijing Medical Animals Research Institute . They were randomly and equally divided into the control group , alloxan‐induced group with DM  and allopurinol‐treated group with DM . Ten rabbits from each group were used for echocardiographic examination, hemodynamic‐, histological‐ and electrophysiological studies, oxidative stress marker measurements, Western blot analyses, IAfter 8 weeks, rabbits were anesthetized with sodium pentobarbital , and then were placed on the table on the left lateral decubitus position. Echocardiographic parameters were obtained in the parasternal long‐axis view using a GE Vingmed machine  equipped with a 7.5‐MHz standard pediatric probe. Both two‐dimensional and M‐mode echocardiography were performed in the parasternal long‐axis view. The measurements included interventricular septal thickness and left ventricular posterior wall thickness, left ventricular cavity size end‐diastolic dimensions, left ventricular cavity size end‐systolic dimensions, and left atrial anteroposterior diameter. Measurements of LV ejection fraction were recorded according to international standards. The mean of 3 measurements were used for subsequent analysis.3 24.2, CaCl2 2.2, MgCl2 0.6, NaH2PO4 1.2, and glucose 12. The solution reservoir was saturated with 95% O2 and 5% CO2 and the pH of was set at 7.4.After echocardiography, median sternotomy was performed, following which the heart was quickly removed and placed in cold perfusion fluid (4°C). The aorta was cannulated and connected to a Langendorff perfusion system filled with warmed (37°C±0.5°C) Tyrode's solution. The perfusion pressure was maintained at 65 to 75 mm Hg, resulting in an initial coronary flow of 50 to 60 mL/min. Tyrode's solution consisted of the following (mmol/L): NaCl 130, KCl 5.6, NaHCOFour sets of silver bipolar electrodes were placed on the epicardial surface of high right atrium, high left atrium, left atrial appendage, and right ventricular apex. The S2 extra‐stimulus was delivered after a drive‐train of 8 S1S1 stimuli. The S1S2 interval was decreased by an interval of 2 ms until atrial refractoriness was reached. The atrial effective refractory period (AERP), which was defined as the longest S1S2 interval that failed to elicit atrial activity. AERP dispersion (AERPD) was defined as the difference between the longest AERP and the shortest AERP from 4 different sites. The Wenckebach cycle length of atrial–ventricular conduction was measured by right atrial incremental pacing. The interatrial conduction time, the duration from the high right atrium pacing stimulus to the beginning of high left atrium stimulus, was measured during high right atrium (at a pacing cycle length of 250 ms) pacing. AF vulnerability was tested by burst pacing (cycle length of 50 ms) for 1 second, 5 times at 30 seconds interval at amplitude of 5 V. AF was defined as rapid, irregular atrial response longer than 1000 ms. All epicardial ECGs were amplified with a custom‐made amplifier and recorded with a custom‐made computer software program .After 8 weeks, rabbits were anesthetized with 3% sodium pentobarbital (30 mg/kg) (n=10 for each group). Bipolar electrodes were placed on four limbs to obtain ECG tracings. The neck was cut in the middle and right carotid artery was isolated with great care. Following blood collection from the carotid artery for serum biochemical tests, a cannula was inserted into the right carotid artery to monitor aortic systolic and diastolic blood pressure (SBP and DBP), which were recorded after a stabilization period of 5 to 10 minutes for 3 times by 20‐second interval. Subsequently, the cannula was moved slowly through the aortic valve to left ventricle to measure left ventricle end diastolic pressure (LVEDP). The ECG and BP were continuously monitored using a custom‐made computer software program .After hemodynamic studies, the hearts were rapidly excised. The wet weight (mg) of the entire heart, left atrium, and left ventricle were measured after washing with cold phosphate–buffered saline (PBS). These were expressed relative to the total body weight (g). Isolated LA tissues were divided into 2 parts, for histological study and for Western blotting. The left atrium (LA) tissue was placed in 4% paraformaldehyde and embedded in paraffin. Tissue samples were sliced at 5‐μm thickness and stained with Masson's trichrome. The microscopic images were scanned into a personal computer and quantitatively analyzed with Photoshop 7.0 . Interstitial fibrosis was quantified on the basis of a color discrimination algorithm and expressed as a percentage of the reference tissue area . Blood vessels were excluded from quantification. The mean value of the 3 fields in each section was used for the analysis. Histological examination was performed by the investigators who were blinded to treatment assignment.Blood samples were collected from the carotid artery of rabbits at the end of the experiment and then centrifuged at 1308 g for 10 minutes. Plasma was separated for later tested. Blood urea nitrogen, creatinine, cholesterol, triglyceride, low‐density lipoprotein cholesterin, high‐density lipoprotein cholesterol, and fasting insulin levels were measured using automated techniques. Serum levels of oxidative stress markers, including nitric oxide (NO), malondialdehyde (MDA) and superoxide dismutase, were assessed by commercially available kits  according to the manufacturer's instructions. Xanthine oxidase (XO) activity in serum was determined using a xanthine oxidase activity quantitative colorimetric/fluorimetric assay kit . The principle for measurement of XO activity is that hypoxanthine can transform into xanthine under the catalysis of XO, during which superoxide anion free radicals were generated. The free radicals were combined with electron acceptor and developer to form conjugate, based on which XO activity can be calculated.g for 15 minutes before the 5× SDS‐PAGE sample loading buffer was added to each lysate, which was subsequently boiled for 3 minutes and then electrophoresed in SDS‐polyacrylamide gels. After transferred into the Polyvinylidene fluoride (PVDF) sheets , the proteins were separately incubated with primary antibodies, followed by incubation with appropriate peroxidase‐conjugated secondary antibodies. Equal protein loading of the samples was verified by β‐actin. The reactions were visualized with Western LightningTM Chemiluminescence Reagent (Millipore). The blots were exposed to autoradiographic film  according to the manufacturer's instructions. Results were approved by repeating the reactions 3 times. Fibrosis‐related factors included transforming growth factor‐β1 (TGF‐β1), p38 mitogen‐activated protein kinases (p38), phosphorylated p38 mitogen‐activated protein kinases (P‐p38), c‐Jun N‐terminal kinase (JNK), phosphorylated c‐Jun N‐termial kinase (P‐JNK), extracellular signal‐regulated kinase (ERK), phosphorylated extracellular signal‐regulated kinase (P‐ERK) as well as nuclear factor kappa‐B (NF‐κB) were measured. Xanthine oxidase (XO) and manganese superoxide dismutase (MnSOD) were measured to evaluate oxidative stress in cardiomyocytes. We analyzed alpha 1C subunit of L‐type calcium channel (Cav1.2), ryanodine Receptor 2 (RyR2), sarcoplasmic reticulum Ca2+‐ATPase2a (SERCA2a), Phospholamban (PLB), phosphorylated form of phospholamban (P‐PLB) as well as FK506‐binding protein 12.6 (FKBP12.6) for purpose of observing the influence of diabetes mellitus on remodeling of proteins involved in calcium homeostasis. Furthermore, we analyzed nuclear respiratory factor‐1, mitochondrial transcription factor A, dynamin‐related protein 1 (Drp‐1) as well as mitofusin 1 (mfn1) to observe the impact of diabetes mellitus on mitochondrial function. Detailed information of primary antibodies are detailed in Table LA tissue samples of all groups were pulverized in liquid nitrogen and extracted by 1 mL RIPA lysis buffer. The lysates were centrifuged at 12 0002+‐free Tyrode's solution at 30 mL/min for 15 minutes, followed by a perfusion containing collagenase  and 0.2% bovine serum albumin  for another 20 minutes. The atria were cut into small pieces and maintained at a 37°C high‐K+ storage solution (KB solution). Atrial cardiomyocytes were filtered on gauze and allowed to sediment by gravity for 5 minutes, followed by superfusion with Tyrode's solution (3 mL/min) for 5 minutes, after which cells were suspended in Tyrode's solution with 1 mmol/L CaCl2. Freshly isolated atrial cardiomyocytes were plated in culture dishes and stored at room temperature until use.Atrial cardiomyocytes were isolated enzymatically as previously described.CaL at room temperature (24–26°C). The patch pipette resistance was 1.0 to 2.0 MΩ when filled with pipette solution . ICaL and action potentials (APs) were recorded and filtered at 0.5 kHz using a low pass filter. The sampling frequency was set at 0.2 Hz for ICaL. After seal formation and membrane rupture, a single 10 mV hyperpolarizing pulse (from −50 mV) in the extracellular solution over a voltage range of −40 to +60 mV was applied to determine the current‐voltage relation for ICaL.The whole‐cell configuration of the patch‐clamp technique was used to record I2+]i transients were recorded in intact myocytes previously loaded with the fluorescent Ca2+ dye Fluo‐4AM  for 15 minutes and then rinsed twice in Tyrode's solution to wash away residual dye. Cardiomyocytes were electrically stimulated at 1 and 2 Hz by field stimulation using two parallel platinum electrodes. Confocal microscopy  was used to obtain the images of cardiomyocytes exhibiting Ca2+ by scanning the cell with anargon laser. Fluo‐4AM was excited at 488 nm, and emitted fluorescence was collected at >505 nm. The fluorescence values (F) were normalized by the basal fluorescence (F0) to obtain the fluorescence ratio (F/F0). Data analysis was performed with GraphPad Prism (Version 6.0).i transients were shown in Figures 2+]i transients were assessed at different pacing frequencies. At 1 Hz, significantly higher transients were observed in the DM group compared with the control group , and reduced by allopurinol . By contrast, these differences were not observed when the pacing frequency was increased to 2 Hz .Representative images of flat scanning and line scanning as well as curve graph of [Ca2+‐regulating proteins involved in excitation–contraction coupling was probed by western blot of LA tissue homogenates. As shown in Figure CaL density and [Ca2+]i transients. By contrast, no significant difference in FKBP12.6, the regulatory protein of RyR2, was observed  structural remodeling of the left ventricle; (2) a higher incidence of AF associated with prolonged IACT, AERPD and cycle length of atrial–ventricular conduction; (3) increased left atrial interstitial fibrosis; (4) significantly increased expression of NO, MDA, and XO levels; (5) higher protein expression levels of oxidative stress and atrial fibrosis, including XO, manganese superoxide dismutase, NF‐κB, TGF‐β, P‐p38, P‐JNK, ERK and P‐ERK (6) higher expression of mitochondrial‐related protein; (7) higher ISeveral pathophysiological mechanisms underlying the association between AF and DM have been identified. These include autonomic modulation,2O2 emission during oxidation of carbohydrate‐ and lipid‐based substrates is increased, corresponding to increased local oxidative stress in the tissue, demonstrating mitochondria to be the main source of ROS.Several oxidative stress mechanisms have been proposed to explain the development and perpetuation of AF in the context of diabetes mellitus.The transcription factor nuclear factor‐kappa B (NF‐κB), via alterations in angiotensin and redox signaling, enhances heterogeneity in conduction, thereby promoting intra‐atrial reentry.Ang II mediates atrial fibrosis by provoking mitogen activated protein kinases (MAPKs), promoting endogenous synthesis of TGF‐β1 and connective tissue growth factor (CTGF).2+ channels (ICa) or activation of the Na+‐Ca2+ exchanger (INCX) secondary to spontaneous Ca2+ release from the sarcoplasmic reticulum. Delayed afterdepolarizations are associated with Ca2+ overload, which activates several Ca2+‐sensitive currents, which together constitute the transient in ward current (ITI).2+ alternans can be induced in cardiac cells by making sarcoplasmic release of calcium strongly dependent upon the SR Ca2+ content. Compelling evidence suggests that action potential duration alternans, whether in the atria or the ventricles, are driven by Ca2+ alternans, which are attributable to ryanodine receptor refractoriness.2+ release will increase the local Ca2+ concentration to a high level within the mitochondria. This acts as an important activator signal for different mitochondrial enzymes leading to increased ATP generation.CaL and Ca2+ transients were increased in DM group with an enhanced expression of Ca2+ handling proteins including Cav1.2 and RyR2, leading to a rise of [Ca2+]i from the extracellular space and the SR. Furthermore, the fact that SERCA2a was slightly decreased in DM group with the reduce of P‐PLB expression, which may attenuate its activity, would impair Ca2+ uptake of SR and lead to intracellular Ca2+ overload.Calcium homeostasis is an important determinant of action potential duration, excitation–contraction coupling and the generation of contractile activity. Early and delayed afterdepolarizations, which are secondary depolarization events occurring before the subsequent AP, can initiate arrhythmias. Early after depolarizations are typically generated when the repolarization phase of the cardiac AP is prolonged, leading to reactivation of the L‐type Ca2–induced atrial fibrillation rats partly by increasing the expression of antioxidants such as GSH‐Px and manganese superoxide dismutase to reduce ROS levels in mitochondria.The association between hyperuricemia and AF is well‐established. Accumulation of uric acid inside atrial cardiomyocytes can induce remodeling by elevating ROS levels. Several previous studies have confirmed that reduction of oxidative stress through inhibition of xanthine oxidase using allopurinol or other similar drug, of NADPH‐oxidase using apocynin,Some limitations of this study should be noted. As the main aim of this study was to examine the relationship between oxidative stress, structural remodeling, and calcium homeostasis; activities of non‐calcium–related ion channels were not examined. Further studies are required to elucidate their roles in AF development of our model. Moreover, the potential beneficial effects of allopurinol for preventing AF development in humans should be examined in future studies.Our study demonstrates that allopurinol attenuated atrial structural and electrical remodeling and suppresses AF vulnerability in alloxan‐induced rabbits with diabetes mellitus. These protective effects were associated with reductions in ROS formation and atrial fibrosis‐related factors and abnormal calcium homeostasis. These findings suggest that allopurinol may be useful for treating diabetes mellitus‐related AF.This work was supported by grants  from the National Natural Science Foundation of China, Tianjin Natural Science Foundation (16JCZDJC34900 to Liu). Tse received support through a clinical assistant professorship from the Croucher Foundation of Hong Kong.None.Table S1. Primary Antibodies of ProteinsClick here for additional data file.