Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM’s N-lobe binding outside the NaV C-terminal while CaM’s C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.Skeletal muscle voltage-gated Na + channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2 +-dependent inactivation while cardiac NaV1.5 is not. Here authors use structural biology, binding and electrophysiology to parse the Ca2 +-dependent changes of CaM when bound to the NaV1.4.Skeletal muscle voltage-gated Na Of the nine human isoforms of NaV, the two isoforms studied here are predominantly expressed in skeletal muscle (NaV1.4) and cardiac tissue (NaV1.5).Voltage-gated NaVs are large (~2000 residues) transmembrane proteins composed of four homologous domains  that form a highly selective Na+ pore.2 DIV is followed by a C-terminal tail which contains a structural domain  consisting of a five-helix EF hand-like motif  followed by a long α-helix (helix αVI). Helix αVI contains an IQ (isoleucine-glutamine) motif , a high-affinity binding domain for calmodulin (CaM) in both its Ca2+-free (apo) and Ca2+-saturated form3Structurally, Na2+-sensing regulatory protein. CaM has two homologous lobes  connected by a flexible linker; each lobe is capable of binding 2 Ca2+ ions cooperatively. Strong intra-lobe cooperativity means that CaM exists primarily in four states of Ca2+-saturation: apo, (Ca2+)2-N-CaM, (Ca2+)2-C-CaM and (Ca2+)4−CaM. Each lobe may be closed, semi-open, or open depending on Ca2+-saturation. A lobe in the semi-open or open state has a solvent-exposed hydrophobic cleft that may bind target peptides, such as the NaV IQ motif, with high affinity.CaM is a small (148 residue) ubiquitous eukaryotic CaV1.5 CTerm/CaM complexes have been reported, each representing a distinct biological state: (1) NaV1.5 CTerm in complex with apo CaM (PDB ID: 4OVN4), (2) NaV1.5 CTerm in complex with apoCaM plus fibroblast growth factor homologous factor , and (3) NaV1.5 CTerm in complex with (Ca2+)4-CaM and FHF (PDB ID: 4JQ06). These structures provide snapshots of the interactions of NaV1.5 CTerm and CaM as well as clues to possible CaM binding domains in NaV1.4 CTerm; all show the C-lobe bound to the IQ-motif, with the N-lobe in varied configurations. Notably, structure 3 identifies a Ca2+-N-lobe binding motif (NLBM) in helix αVI, i.e., a post IQ-motif located past where the C-lobe is bound.Three crystal structures of NaVs has been provided by three recent cryo-EM structures of eukaryotic voltage-gated sodium channels: cockroach NavPaS 7, eel EeNaV1.4 8 and H. Sapiens NaV1.4 (PDB ID: 6AGF)9. One cytosolic region, the linker between domain DIII and DVI (DIII–DIV linker), has already been implicated in channel regulation. In NavPaS the DIII–DIV linker passes between the CTerm of the channel and the membrane, while in the other two full NaV structures the DIII–DIV linker is visible in a different orientation and no CTerm structure is observed. Previous studies have suggested that an IFM (Ile-Phe-Met) motif in the DIII–DIV linker is the putative fast inactivation gate. Studies have also suggested that the DIII–DIV linker interacts with CaM11 or NaV CTerm12 and the possibility of DIII–DIV linker involvement in NaV 1.4 Ca2+ regulation merits consideration.Information regarding the global topology of NaV1.4 and NaV1.5 have distinct responses to increases in the Ca2+ concentration. NaV1.4 exhibits a unique CaM mediated Ca2+-dependent inactivation (CDI) which results in a reduction of bulk Na+ current by approximately 30% after the elevation of cellular Ca2+ levels to ~10 µM.13 In contrast, NaV1.5 exhibits no CDI even at elevated Ca2+ levels. It is still not known what are the structural and thermodynamic mechanisms responsible for this difference in behavior are, given the high overall homology between the two NaVs , especially in the regulatory and CaM-binding CTerm .NaV1.4 CTerm, one bound to apo CaM and the other bound to (Ca2+)4-CaM, as well as binding data between the CTerm of each isoform and CaM in four states of Ca2+-saturation. Comparison of the two crystal structures reveals the Ca2+-dependent changes experienced by CaM when bound to the NaV1.4 CTerm. The binding data were used to model the populations of four CaM states bound to NaV1.4 and 1.5 CTerm as a function of [Ca2+] and [CaM]. In addition, electrophysiological data show that the CTerm of NaV controls CDI and that deletion of the post-IQ NLBM in NaV1.5 results in robust CDI. These structural, thermodynamic and electrophysiological data all support a mechanism of CDI in NaVs controlled by the positioning of the N-lobe of CaM. Ca2+-N-lobe binding to the post-IQ NLBM (WT NaV1.5) prevents CDI from occurring at elevated Ca2+ levels. The absence of a post-IQ NLBM sequence  leads to CDI when the CaM N-lobe binds Ca2+. Thus, we showed that the structural and thermodynamic determinants of CDI reside in the CTerm and determine the physiological differences between the two NaV isoforms’ response to Ca2+. These findings provide a path to identifying the possibility of Ca2+-inactivation in other NaV isoforms.Here, we present two crystal structures of the NaV1.4 responds to an increase in the Ca2+ concentration by reducing the maximum Na+ current by approximately 30% 13. This effect was observed in HEK293 cells transfected with NaV1.4 as well as in skeletal myotubes derived from mouse GLT cells. CDI was not observed in similar experiments involving expressed NaV1.5. We performed electrophysiology experiments to confirm the NaV-CTerm as the region responsible for the CDI as well as to determine the post-IQ region’s role in physiological NaV function. The sequences of the two isoforms in the IQ and post-IQ regions are shown in Fig. It was previously shown that NaV1.5 (aa 1774–2016) was replaced by its NaV1.4 counterpart (aa 1599–1836). Ca2+ was delivered using voltage pulses that selectively activate voltage-gated Ca2+-channel CaV2.1 which was co-expressed with the NaV channels. The chimeric protein  shows CDI, in presence of CaM  or CaM34 . Only WT CaM and CaM34 were capable of inducing CDI  and Short (residues 1599–1754)—produced the crystals used in the structure determinations.The structure of the NaV1.4 CTerm Long in complex with apo CaM (Rwork/Rfree = 20.3/23.4) contains one complex in the asymmetric unit (ASU) of the cell  followed by a long helix . Helix αVI is in the same orientation relative to the EFL as seen in NaV1.5 CTerm in complex with apo CaM (PDB ID: 4OVN)4. In contrast, there is a rotation of ~90° of helix αVI relative to the EFL in the structure of NaV1.5 CTerm, apo CaM and FHF (PDB ID: 4DCK)5. The apo CaM C-lobe is in a semi-open configuration, and bound to the IQ-motif of helix αVI with a buried surface area (BSA) of 872 Å2  and Tyr1738 (119 Å2 BSA). The CaM C-lobe also forms four Glu–Arg salt-bridges with helix αVI: Glu114–Arg1736, Glu120–Arg1739, Glu123–Arg1745, and Glu127–Arg1745. As expected, in the absence of Ca2+ the CaM N-lobe is in a closed conformation. Its surface contacts the CTerm in such a way that CaM helices A and B of the N-lobe contact helices αIII and αVI of the CTerm burying 495 Å2 . The CaM N-lobe residue Glu11 is involved in salt-bridges with NaV1.4 CTerm: one with Lys1658 and another with Lys1725.The 1.8 Å resolution structure of Na Å2 Fig. 14. The a Å2 Fig. . The apoV1.4 CTerm Short (residues 1599–1754) in complex with (Ca2+)4–CaM was determined to a resolution of 3.30 Å (Rwork/Rfree = 24.0/28.5). Crystals belong to the space group P43212 and contain one complex in the ASU -saturated CaM C-lobe bound to the IQ-motif adopts a semi-open15 configuration and buries 745 Å2  as well as Gln1735 (110 Å2 BSA). Unlike in the complex with the apo–CaM, in this complex the C-lobe does not form any salt bridges with the NaV1.4 CTerm although two residues in the CaM lobe linker are involved in salt bridges: Asp78 and Asp80, both with Arg1740. The N-lobe and EFL interface involves the external regions of the N-lobe, not the hydrophobic cleft, and buries 334 Å2 of total area. This interface involves CaM residues Leu18 (84 Å2 BSA) and Leu39 (95 Å2 BSA) and NaV residues Ile1659 (83 Å2 BSA) and Leu1722 (72 Å2 BSA).The crystal structure of NaASU Fig. , Table 1 Å2 Fig. , anchore2+-saturated CaM N-lobe conformation differs significantly from the one seen in the apo–CaM complex 4–CaM was used to collect data at a wavelength of 2.515 Å, near the anomalous edge of Ca2+ (3.070 Å) 4–CaM crystal shows an intermolecular interaction between the CTerms of symmetry-related molecules: the end of helix αVI interacts with a groove in the EFL of a neighboring CTerm .Inspection of crystal packing in the NaV1.4 and NaV1.5 CTerms for CaM were determined using isothermal titration calorimetry (ITC). Titrations were conducted under both Ca2+-free (with 50 µM EGTA) and Ca2+-saturating conditions (with 1 mM CaCl2). As CaM has two pairs of EF-hands, Ca2+-occupancy of CaM sites is more complex than the binary apo or fully Ca2+-saturated captured in these two crystal structures. Each CaM EF hand is capable of binding one Ca2+ ion so there are 16 possible states of Ca2+-saturation. However, because of the cooperativity between Ca2+ binding to the two EF hands in each lobe , states in which each lobe is either empty or has 2 Ca2+ ions will be the most highly populated. These considerations lead to an approximate model with CaM in 4 states of Ca2+-saturation: apo, (Ca2+)2-N–CaM, (Ca2+)2-C–CaM and (Ca2+)4–CaM. Accordingly, binding measurements were performed using CaM12 or CaM34 under high calcium conditions to capture the CTerm binding affinity for (Ca2+)2-C–CaM and (Ca2+)2-N–CaM, respectively. In total, these binding experiments provided data with CaM’s four most physiologically relevant states. Both lengths of the CTerm used in the structural studies were used in the experiments: Long (residues 1599–1764) and Short (residues 1599–1754). Long is considered to reflect physiological binding more accurately and was used in the thermodynamic modeling; Short crystallized with (Ca2+)4–CaM and Short CTerm binding measurements are also reported here . The intermediate Ca2+-saturated states show similar affinity . (Ca2+)4–CaM shows the weakest binding (Kd = 275 nM) indicating a general trend: the more Ca2+-saturated the species of CaM the more they are (modestly) penalized for NaV1.4 CTerm binding.The ed Table . Apo CaMKds of CaM binding to the cardiac NaV1.5 CTerm Long range from 48 to 90 nM for three of the species: apoCaM, (Ca2+)2-N–CaM, and (Ca2+)4–CaM (Table 2+)2-C–CaM, binds the NaV1.5 CTerm with a Kd of 3.6 µM, or about 40-fold weaker binding than any other CaM species. The dramatically weakened binding of (Ca2+)2-C–CaM to NaV1.5 CTerm contrasts with the trend observed in NaV1.4, for which (Ca2+)2-C–CaM binds with high affinity to the CTerm. For the NaV1.5 CTerm the two tightest binding CaM species are the two physiological endpoint species: apo CaM (Kd = 48 nM) and fully saturated (Ca2+)4–CaM (Kd = 70 nM) which is a trend captured in our thermodynamic model.The poCaM, Ca+2-N–CaM,2+ concentrations and determine the different physiological responses to elevated Ca2+ concentration. We employed a thermodynamic model that provides estimates of the populations of NaV CTerm and CaM species at varying [Ca2+] and [CaM]. To start, the fractions of the CaM species as a function of [Ca2+] can be calculated using the previously reported binding data, which describes Ca2+ affinities for the N- and C-lobe of full-length mammalian CaM19  of all four species as a function of [Ca2+]  can be calculated as a function of the CaM species, using the binding constants reported here 4–CaM and FHF (PDB ID: 4JQ0)6 identified the post-IQ NLBM as residues 1916–1927 (LLQRSLKHASFL) with the N-lobe binding burying two NaV residues in its hydrophobic cleft: Leu1917 and Leu1921. In contrast, the structures and binding model presented here suggest NaV1.4 CTerm lacks such an NLBM. Accordingly, a C-terminally truncated NaV1.5 with the post-IQ NLBM removed  was used in electrophysiology experiments. This truncation, in the middle of the Ca2+-N-lobe binding domain, eliminates the 2nd anchor Leu and any other subsequent downstream NaV-CaM contacts. Photo-uncaging Ca2+ in HEK293 cells expressing the NaV1.5 ΔP-IQ channel leads to robust CDI not observed in full-length NaV1.5  causes the rapid onset of CaM-mediated CDI. In contrast, NaV1.5 which also binds CaM in its CTerm, does not exhibit CDI at elevated Ca2+13. This isoform-specific response has been shown to be CTerm controlled and to require bound CaM. The strongest evidence for the localization of this effect is shown in Fig. V1.5 four transmembrane domains followed by the NaV1.4 C-terminal tail exhibits robust CDI.Na2+] requires the detailed identification of the structural determinants of the CDI as well as the thermodynamics of binding between the two CTerm regions and CaM at different Ca2+ concentrations. We carried out binding and structural studies to characterize the interactions of CaM with the NaV1.4 CTerm. Apo CaM shows high affinity for NaV1.4 CTerm Long . The similarities between the NaV1.4 and NaV1.5 CTerm complexes with apo CaM, in both conformation and binding affinities 4–CaM binds with high affinity to the NaV1.4 CTerm 4–CaM in complex with NaV1.4 CTerm Short shows that the C-lobe of (Ca2+)4–CaM binds the IQ motif of helix αVI, in a pose similar to that seen in the apo CaM CTerm complex  and with a reduced BSA relative to the apo CaM N-lobe EFL interface (334 vs. 495 Å2). The hydrophobic cleft of the Ca2+-saturated N-lobe is open, solvent exposed and unoccupied in this structure , no N-lobe binding post-IQ was observed here. This difference indicates that the post-IQ region is crucial for control of Ca2+–CaM N-lobe binding.The N-lobe of Caure Fig. . Even thV1.4 and 1.5 CTerm is found in the species with Ca2+ only in the C-lobe (Table 2+)2-C–CaM12 species binds NaV1.4 CTerm Long with a Kd = 154 nM and NaV1.5 CTerm Long with a Kd = 3.6 μM. With CTerm Short the situation is similar: (Ca2+)2-C–CaM12 has a Kd = 156 nM for NaV1.4 and Kd = 5.26 μM for NaV1.5. This large difference may, in part, be responsible for the differences in CDI between the isoforms. Nevertheless, a complete explanation of the differences requires an analysis of all the species contributing to the control of the channel’s activity. To this effect, the binding measurements were used to model the populations of NaV CTerm-CaM species as a function of [CaM] and [Ca2+]. Using the experimental affinities, this model provides the relative populations of the five CTerm species in Fig. 2+] and [CaM] that simulate rapid Ca2+ signaling in a cell, or the variation of CaM concentration across cells or experiments  while (Ca2+)4–CaM bound to NaV CTerm is the dominant species at high Ca2+ levels (~100 µM). The isoform populations are most divergent at 10 µM Ca2+ at which concentration NaV1.4 has a high population of half Ca2+-saturated CaM, (Ca2+)2-C–CaM, while NaV1.5 is switching from bound apo CaM to bound (Ca2+)4–CaM with nearly absent intermediate populations.The populations obtained with this model show similar trends for bound-CaM behavior with both Nalls Fig. .21,22 Fo2+ concentration that corresponds to those found in relevant cell Ca2+ signaling events and which leads to CDI. The half Ca2+-saturated species of CaM, (Ca2+)2-C–CaM, predicted bound to NaV1.4 CTerm may then be essential to the molecular mechanism of CDI. However, inspection of the crystal structures presented here shows that Ca2+ binding to the C-lobe does not have a major effect on its conformation nor CaM interaction with the NaV1.4 CTerm. Instead, it is the lack of Ca2+ binding by the N- lobe when the CaM C-lobe is bound to NaV1.4 CTerm, at 10 µM Ca2+ that is mechanistically relevant. The affinity of Ca2+ for the CaM N-lobe is intrinsically low (Kd ≈ 20 µM) and Ca2+ binding is enhanced when bound to a target helix. This lack of Ca2+ binding is consistent with a mechanism in which the NaV1.4 CTerm lacks a functional NLBM and the N-lobe is free to bind another region of the channel. In contrast, with the NaV1.5 CTerm, which does contain the NLBM, the Ca2+-N-lobe binds to this motif and is unavailable. This is supported by the thermodynamic binding model which shows that the N-lobe of CaM binds to NaV1.5 CTerm more readily when it binds Ca2+ 23. In a recent Na+ channel structure, NavPaS (PDB ID: 5 × 0 M)7 as well as in the CaV1.1 structure (PDB ID: 5GJV)24, a portion of the DIII–DIV linker makes contact with the EFL in a pocket formed by helices α I, IV, and V, suggesting the DIII–DIV linker would be near to bound CaM in NaV1.4 11 and NaV1.5 residues L1514–1522K bound to Ca2+-saturated N-lobe (PDB ID: 5DBR)25, show helical portions of the NaV1.5 DIII–DIV linker (NaV1.4 residues 1467–1529) bound to (Ca2+)4–CaM.This isoform-specific tuning of CaM behavior occurs at a Caa2+ Fig. . If the 2+-N-lobe of CaM attached to the NaV1.4 CTerm by the Ca2+-C-lobe binding to the IQ motif can bind the DIII–DIV linker. However, attempts at co-crystallizing a peptide that spans residues 1316–1327 of the DIII–DIV linker (first helix) and the NaV1.4 CTerm (Ca2+)4–CaM complex produced crystals but showed no density that could be attributed to the peptide. Furthermore, ITC experiments titrating the NaV1.4 CTerm and (Ca2+)4–CaM complex with two DIII–DIV linker peptides (12 or 20 residues) exhibit no binding even at high peptide concentrations  stabilization of the helix as required for binding, since the free peptide may not be helical in solution and (2) bringing the peptide and the binder together that in the physiological situation might already be proximal as part of the same complex (chelate effect). Independently of which region is recognized, a proximal binding partner in the NaV1.4 for the CaM–Ca2+ N-lobe brings together the known crystal structures, the thermodynamic model and the existing physiological data.We tested whether the CaV CDI is controlled via a distal binding partner of the Ca2+-N-lobe is provided by electrophysiology experiments using NaV1.5. Unlike NaV1.4, WT NaV1.5 is insensitive to Ca2+ concentrations of up to 10 µM and shows no CDI  in NaV1.4. Leu1921 is buried in the N-lobe’s hydrophobic pocket and makes a close contact with Met73 in Ca2+–CaM (PDB ID: 4JQ0). The change of Leu to Met combined with other differences may lead to steric clashes, resulting in a weaker affinity of the Ca2+-N-lobe for the post-IQ motif of NaV1.4. This proposal identifies a motif that can be used to facilitate the study of Ca2+-regulation in other NaV isoforms. Isoform-specific CDI may represent an additional level of regulation of NaV channels reflecting the different characteristics of their currents: periodicity (or not), frequency and duration.Our results, along with other previously published data, strongly suggest a molecular mechanism of CDI in Na27. The same effects may be operational in neuronal isoforms that mimic the behavior of NaV1.5 leading to neurological disorders such as seizures or neuropathic pain.Also, as the post-IQ is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibilityH. sapiens NaV1.5–eGFP fusion construct was cloned by inserting the coding region of Nav1.5 into EGFP-N3 (Clontech)28. A chimera was created with NaV1.5 up to amino acid 1773 followed by NaV1.4 C-terminal tail (residues 1599–1836) and enhanced green fluorescent protein (NaV1.5-CTail1.4). Approximately 0.75 × 106 HEK293 cells  were cultured in 6-well tissue culture dishes in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (10 mg/mL) and gentamicin(50 mg/ml). The cells were co-transfected with plasmids encoding the appropriate NaV1.5-CTail1.4, CaM, CaV2.1 EFb 43+/44−/47+ α2δ and β2α subunits at 1 µg/µl30. Cells were transfected using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions and were studied 48 to 72 h post-transfection. The total amount of DNA for all transfections was kept constant.The 2, 15; MgCl2, 1; KCl, 4; NaH2PO4, 0.33; HEPES, 10; with pH 7.4 adjusted with NaOH and at 290 mOsm adjusted with NaCl. The pipet solution, “0.5 EGTA” contained (in mM): CsMeSO3, 124; CsCl, 5; MgCl2, 1; MgATP, 4; HEPES (pH 7.4), 10; and EGTA, 0.5; at 290 mOsm adjusted with glucose.The bath solution contained (in mM): NaCl, 130; CaClINa and ICa were recorded under voltage-clamp with an Axopatch 200 A patch-clamp amplifier  at room temperature (22 °C). Voltage command protocols were generated by custom-written software and PCLAMP 10 (Molecular Devices Corp). Briefly the protocol is constituted of three parts: a test pulse P1 at −20 mV to asses INa, steps of 10 mV from −60 mV to +50 mV increase to allow Ca2+ to enter the cell and a final test pulse P2 at −20 mV to evaluate the effect of Ca2+ on INa. The length of P1 and P2 were 15 ms, the steps for Ca2+ entry were 200 ms in length. The holding potential was −120 mV. Capacitance compensation was optimized and series resistance was compensated by 40–80%. Membrane currents were filtered at 5 kHz and digitized with 12-bit resolution through a DigiData-1200 interface (Molecular Devices Corp.).Whole cell H. sapiens NaV1.4 CTerm Long  in a pGEX-6-P1 plasmid with an N-terminal GST tag was purchased from Genscript, together with the gene for full-length mammalian calmodulin  cloned into a pET24b plasmid4 were used to co-transform BL21-CodonPlus RIL E. coli (Agilent) cells. The proteins were co-expressed and purified as a complex, using the purification of NaV1.5 CTerm and apo CaM as a guide4. In brief, the cells were grown overnight at 37 °C in 100 ml of LB medium supplemented with 50 µg/mL kanamycin, 20 µg/mL chloramphenicol and 100 µg/mL ampicillin. Ten mL of the overnight culture was used to inoculate 1 L of LB media containing the same antibiotics. The cells were grown at 37 °C to an absorbance of 0.8 and protein expression was induced with 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG). The cells were grown overnight at 18 °C (approximately 18 h), centrifuged and the cell pellet was frozen at 80 °C.The gene for g. The supernatant was loaded a 30 ml Glutathione Sepharose 4 Fast Flow resin using gravity flow. The column was washed with 200 mL buffer and protein was eluted in aliquots of 8 ml with an elution buffer containing 154 mg of reduced L-glutathione in 50 mM Tris-HCl at pH 8. Eluted fractions containing protein were pooled and 5 µg of PreScission protease was added per mg of NaV CTerm and CaM complex. Dialysis was performed against 500 mL of 20 mM Tris, 50 mM NaCl, 1 mM DTT, pH 7.4. The buffer was changed 3 times and the final dialysis was allowed to proceed overnight. The dialyzed and Precission-cleaved protein was loaded on a Source Q anion exchange column. Elution was performed using a buffer of 20 mM Tris and 1 mM DTT and a gradient of 50–500 mM NaCl. Free cleaved GST tag eluted at ~8 mS/cm and NaV CTerm and CaM in complex eluted at ~10 mS/cm. Fractions were judged to be 98% pure by SDS-PAGE gel then pooled and concentrated to ~40 mg/mL protein.After thawing, pellets were re-suspended at 0.2 g/mL with phosphate-buffered saline (PBS). DNAse was added and the cells were lysed using a microfluidizer  and the lysate clarified at 27,500 × Homo sapiens NaV1.4 CTerm Short  was cloned into a pGEX-6-P1 plasmid with an N-terminal GST tag. This construct contained an additional 7 residues at the C-terminus, LTRAAAS. BL21-CodonPlus RIL E. coli (Agilent) were then co-transformed with NaV1.4 CTerm Short and CALM2 pET-24b plasmids4. The proteins were co-expressed and purified in complex in the same manner as the NaV1.4 CTerm Long and CaM complex.The gene for V1.4 CTerm and CaM were co-expressed in BL21-CodonPlus RIL E. coli (Agilent) cells. Cells were grown and lysed as above. The supernatant was loaded onto a Glutathione Sepharose 4 Fast Flow resin using gravity flow column and washed with 1.5 L of low pH PBS buffer with added trifluoroperazine (TFP) and Ca2+ . This TFP wash removed almost 100% of the CaM bound the NaV CTerm. This was followed by a 4 CV wash of the same buffer lacking TFP and Ca2+, a 4 CV wash of PBS at pH 8.0  and elution with PBS at pH 8.0 containing 10 mM L-glutathione . The NaV CTerm was 70% pure as judged by visual estimation of SDS-PAGE electrophoresis.NaH. sapiens/R. norvegicus CALM2) cloned into a pET24b plasmid4 was used for CaM expression. Two engineered CaM mutants, CaM12 (D20A and D56A) and CaM34 (D93A and D129A), were obtained as a generous gift from the late David Yue lab. CaM12 and CaM34 are lobe-specific Ca2+-binding knockouts have been shown to calcium-insensitive up to 2 mM CaCl231. BL21-CodonPlus RIL E. coli (Agilent) were transformed with the individual plasmids. In brief, the cells were grown overnight at 37 °C in 100 ml of LB medium supplemented with 50 µg/mL kanamycin and 20 µg/mL chloramphenicol. Ten mL of the overnight culture was used to inoculate 1 L of LB media containing the same antibiotics. The cells were grown at 37 °C to an absorbance of 0.8 and protein expression was induced with 0.5 mM IPTG. The cells were grown overnight at 18 °C (approximately 18 h), centrifuged and the cell pellet was frozen at 80 °C.The gene for full-length mammalian calmodulin (g. The supernatant was run through at HiPrep™ Phenyl FF (high sub) 16/10 column (GE Lifesciences) equilibrated in a matched buffer using an ÄKTA FPLC (GE Lifesciences). The eluate was collected and brought up to 10 mM CaCl2. This high Ca2+-eluate was run through a second HiPrep™ Phenyl FF (high sub) 16/10 column (GE Lifesciences) equilibrated in a match high-Ca2+ buffer. The column was washed with 200 mL of buffer containing 50 mM Tris, 10 mM CaCl2, 2 mM DTT, pH 7.4. Calmodulin was eluted with an elution buffer containing 50 mM Tris, 1 mM EDTA, 2 mM DTT, pH 7.4. Fractions were judged to be 98% pure by SDS-PAGE gel.After thawing, pellets were re-suspended at 0.2 g/mL with buffer containing 50 mM Tris, 1 mM EDTA, 2 mM DTT, pH 7.4. DNAse was added and the cells were lysed using a microfluidizer  and the lysate clarified at 27,500×V1.4 residues 1316–1327 (100% identity with NaV1.5 residues 1491–1502) was synthesized with an addition of 2 N-terminal alanines (AAQKKYYNAMKKLG). This peptide has 25% identity with the helical portion of the DIII–DIV linker seen contacting the EFL in NavPaS (PDB ID: 5 × 0 M)7. Synthesis was performed by AnaSpec and the peptide was judged to be over 90% pure by HPLC.The 12-mer peptide corresponding to NaV1.4 residues 1312–1331 (100% identity with NaV1.5 residues 1487–1506) was synthesized (MTEEQKKYYNAMKKLGSKKP). This peptide has 30% identity with the DIII–DIV linker seen in NavPaS. Synthesis was performed by Genscript and judged to be over 95% pure.The 20-mer peptide corresponding to NaV1.4 CTerm Long in complex with apo CaM (50 µM EGTA) was crystallized by hanging drop vapor diffusion at 18 °C with a 1:1 ratio of protein to well solution. The well solution contained 0.1 M MES at pH 6.0, 20% PEG 6000 and 1.0 M LiCl. Crystals were flash-frozen in mother liquor prior to data collection. Diffraction data for the crystals were collected on the 17-ID-1 AMX beamline of the NSLS-II  with a wavelength of 0.918 Å resulting in a 98.0 % complete data set to a resolution of 1.80 Å. Indexing and data reduction were carried out with XDS32.The Na33 using as a search model the NaV1.5 CTerm–CaMC complex 4 followed by the independent placement of the N-lobe 34. One copy of the CTerm-CaM complex was placed in the asymmetric unit. Model building was completed with iterative cycles of manual rebuilding with Coot35 and refinement with Phenix Refine36. The structure was refined to an Rwork of 19.9 (Rfree = 23.2) with excellent geometry (Table 14.The structure was determined by molecular replacement with PhaserV1.4 CTerm Short (residues 1599–1754) in complex with CaM was crystallized by hanging drop vapor diffusion at 18 °C with a 1:1 ratio of protein to well solution. Prior to setting the tray, CaCl2 and MgCl2 were added to a final concentration of 20 mM CaCl2, 20 mM MgCl2, and the DIII–DIV linker peptide was added at a 1:1 ratio with the CaM-NaV1.4 CTerm complex (800 µM). The well solution contained 37. Crystals were flash-frozen in mother liquor prior to data collection. Diffraction data for the crystal were collected at NSLS-II at the 17-ID-2 FMX beamline at a wavelength of 0.979 Å. Diffraction data were then collected from the same crystal at 2.515 Å, near to the Ca2+ anomalous edge. Indexing and data reduction were carried out with XDS32.The Na33 with CaM lobes as search models. Briefly, the coordinates of individual CaM lobes were extracted from PDB depositions containing CaM (at 3.0 Å resolution or better) resulting in 192 N-lobe and 179 C-lobe search models; PDB ID: 2HQW38 chain A for the CaM N-Lobe, PDB ID: 3G4339 chain B for the CaM C-lobe were the most successful. The CTerm of the NaV1.2 CTerm - FHF—(Ca2+)4–CaM complex (PDB ID: 4JPZ)6 was then placed. The crystal contained one copy of the NaV1.4 CTerm-CaM complex in the ASU. No density was observed for the DIII–DIV linker peptide. Model building was completed with iterative cycles of manual model building with Coot35 and refinement with Phenix Refine36. The final structure was refined to an Rwork of 23.8 (Rfree = 29.7) with excellent geometry  and data were analyzed with the Origin-5.0 software and fitted to a single binding site per monomer and the error of this fit is reported. The concentration of CTerm used was always between 2 and 25 µM and the concentration of CaM varied from 30 to 1000 µM, depending on the binding affinity. The results of one replicate are reported in Table 2+. The binding of two Ca2+ ions to either CaM lobe was modeled as a single event and single-ion-bound intermediates were not considered. This model contains four states: apoCaM, (Ca2+)2-N–CaM, (Ca2+)2-C–CaM, and (Ca2+)4–CaM. The free energy of cooperative two-site binding of Ca2+ to either lobe in full-length mammalian CaM was reported by Evans and Shea as −12.82 ± 0.09 kcal mol−1 for the N-lobe and −15.06 ± 0.03 kcal mol−1 for the C-lobe and Linse et al. as −12.7 ± 0.6 and −14.9 ± 0.2 kcal mol−119. Comparing the thermodynamic model results generated with the two sets of binding constants resulted in no discernable difference. The data from Evans and Shea were used in the first binding cycle  and reverse (agctaatctagattaAGAGCGTTGCAGCAGG) primers , 6 µg of eCFP, and 8 µg of rat CaMWT. All of the above cDNA constructs were included within mammalian expression plasmids driven by a cytomegalovirus promoter. To boost expression, cDNA for simian virus 40T antigen (1–2 μg) was co-transfected. Currents were probed ~1–2 days following transfection.For whole-cell patch clamp experiments, HEK293 (ATCC CRL-1573) cells were cultured on 10-cm plates, and channels transiently transfected by calcium phosphate method2+-uncaging experiments, internal solution contained (in mM): CsMeSO3, 120; CsCl, 5; HEPES (pH 7.4 with CsOH), 10; Fluo-4FF pentapotassium salt (Invitrogen), 0.01; Alexa 568 succinimidyl ester (Invitrogen), 0.0025; Citrate, 1; DM-Nitrophen EDTA (DMN) and CaCl2 were adjusted to obtain desired Ca2+ flash. Typically, for flashes in range 0.5–2 μM, DMN, 1 mM; and CaCl2, 0.7 mM. For the 2–8 µM range, DMN, 2 mM; and CaCl2, 1.4 mM. For larger Ca2+ steps, DMN, 4 mM; and CaCl2, 3 mM. Since DMN can bind Mg2+, all experiments were conducted with 0 mM Mg2+ internally. The bath solution contained (in mM): TEA-MeSO3, 45; HEPES (pH 7.4), 10; NaCl, 100; at 300 mOsm, adjusted with TEA-MeSO3.For Ca2+-uncaging experiments were conducted on a Nikon TE2000 inverted microscope with a Plan Fluor Apo 40 × oil objective. Ca2+-uncaging was performed using a classic Cairn UV flash photolysis system. UV pulses of ~1.5 ms in duration were powered by a capacitor bank of 4000 µF charged to 200–280 V. PMTs were protected during UV pulse to prevent photo-damage. For simultaneous Ca2+ imaging, Fluo4FF and Alexa568 dyes  were dialyzed into the cell and then probed with Argon laser excitation (514 nm). Autofluorescence for each cell was obtained prior to pipet dialysis of dyes. A field-stop aperture was used to isolate fluorescence from a single cell. Two-color fluorescence measurements were attained using a 545DCLP dichroic mirror, paired with a 545/40BP filter for detecting Fluo4FF, and a 580LP filter for detecting Alexa568. Ca2+ measurements were determined from ratio of Fluo4FF/Alexa fluorescence intensities (R), according to the relation [Ca2+] = Kd · (R − Rmin)/(Rmax − R). All three parameters Kd, Rmin, Rmax were experimentally determined in HEK293 cells dialyzed with reference Ca2+ solutions. Briefly, Rmin was determined with internal solution containing 40 mM EGTA, and Rmax using 4 mM Ca2+/1 mM EGTA (~3 mM free Ca2+) solution. An R20µM measurement was obtained with internal solution containing [Ca2+] = 20 µM (buffered using NTA). Kd was experimentally determined by solving the equation above. Calibration measurements were repeated at 1 or 4 mM DMN to account for minor differences in Rmax. Uncaging experiments were conducted following 2 min of dialysis of internal solution upon reaching stable current levels. In all cases, the steady-state [Ca2+] step amplitude was measured at 150 ms after the instant of uncaging.All CaFurther information on experimental design is available in the Supplementary InformationReporting SummarySource Data File


Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called “bystander effects”. To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2−∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2−∙ production in these bystander cells. Our results support a key role for ER–mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca Primary products of PS activation are singlet oxygen (1O2) and O2−∙, which in turn initiate a cascade of secondary reactions, leading to the formation of H2O2, hydroxyl radical (OH∙), and oxidation of substrates ER) reached a steady state (see n = 6 cells).Depletion of ER CaPS-loaded cells (expressing G-CEPIA1er or CEPIA2mt or loaded with MitoSOX Red) were superfused with Tg-containing NES for 6 minutes and then focally irradiated and imaged as described above.Two-photon excitation of both biosensors was provided by the beam of an optical parametric oscillator  tuned to 1025 nm, coupled to the Bergamo II microscope  and fed by a femtosecond pulsed Titanium-sapphire pump laser . The microscope was equipped with a water immersion objective designed for multiphoton imaging . Fluorescence emission signals were selected by band-pass filters  and detected by cooled GaAsP photomultiplier modules . Images were acquired simultaneously in these two emission channels at 3 frames/s (Hz).Matlab  and the open-source software ImageJ/Fiji (ImageJ-win64). Fluorescence signals were extracted from sequences of recorded frames as the average pixel values within selected regions of interest (ROIs) after uniform background subtraction. Image background was computed as the average pixel value within a square ROI, placed in a region of the image where there were no detectable fluorophores. Fluorescence traces were computed as relative changes of the instantaneous fluorescence emission intensity (F(t)) with respect to the average prestimulus value (F0), that is as:Image processing and data analysis were carried out using 2−∙ concentration was evaluated as the time derivative of the acquired dF/F0 fluorescence trace using the Matlab function diff. Photobleaching correction was performed by fitting a single exponential function to the prestimulus time course (baseline) of each trace, and extrapolating the fitting function to the overall acquisition time interval. In the graphs, mean fluorescence traces are shown as point-by-point mean ± standard error of the mean (s.e.m.) for the indicated number of independent experiments. In histograms, mean values are quoted ± s.e.m.For the irreversible indicator MitoSOX Red, the change in mitochondrial Oα = 5% for the type I error in the ANOVA test. Then, fixing β = 4α = 20% so as to obtain a test power of 1 − β = 80%, we computed the number n of each of the two samples to be compared using the formula:α/2 = 1.96 and zβ = 1.28. We quantified the variability of the data  and established the minimum difference Δ = μ1 − μ2 between averages that had a biological significance. Statistical comparisons of means were made by ANOVA (independent samples) or by paired sample t-test (dependent samples), where p-value (P) < 0.05 indicates statistical significance. Asterisks were used to indicate significant differences as follows: *, P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.In order to construct the optimal experimental design and estimate the sample size of the groups for each type of experiment, we set a probability To investigate the effects of PS activation, we exposed B16-F10 cell cultures to focal irradiation under SS conditions  in C26GM and MCA-203 tumor cells propagate as a wave from the irradiated cell to bystander cells, while NO diffuses rapidly out of the irradiated cell [2+ signaling with Fluo-4 in B16-F10 cultures. As shown in 2+ responses were detected in all cells of the field of view  within one minute of PS photoactivation under SS conditions. To determine whether these [Ca2+]c elevations were due to Ca2+ influx and/or release from intercellular Ca2+ stores, we repeated focal PS activation experiments in a Ca2+-free extracellular medium , the propagation speed was larger  within cells of the 3rd order  and lower in bystander cells of higher orders . In 0 mM [Ca2+]e, the difference between the wave speeds in 3rd and 5th order cells approached statistical significance . To a first approximation, the kinetics of wave propagation were fitted to a logarithmic function for the 2 mM condition and to a linear function for the 0 mM condition  increased in each order of bystander cells within the field of view and was progressively delayed with increasing distance from the site of photostimulation . At this time point, [Ca2+]m was still elevated in cells of higher orders , a specific and irreversible inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps  after the onset of photostimulation.The results reported above suggested a dependence of mitochondrial Canditions a. Import2−∙. The response of MitoSOX Red was extremely rapid in the irradiated cell, as expected, since O2−∙ is a product of primary (type I) photochemical reactions triggered by PS activation c in a plethora of cellular systems e compared with 2 mM [Ca2+]e in all cells. In addition, the peak amplitude, the area subtended by the signal, and the slope of the linear rising signal in furthest bystander cells were all significantly larger in 0 mM [Ca2+]e than in 2 mM [Ca2+]e., indicating that the propagation of the cytosolic Ca2+ wave was strengthened in the absence of extracellular Ca2+.The excitation of photosensitized cells causes the rise of e ER stress would be stronger, resulting in delayed reactions towards recovery of Ca2+ homeostasis, larger cytosolic Ca2+ signals, and possibly enhanced cellular damage. Several studies support the idea that Ca2+ concentration in the ER controls cellular sensitivity to apoptosis depending on the amount of Ca2+ released following a Ca2+-mobilizing stimulus e and whether inhibition of external Ca2+ influx could enhance cytotoxic stimuli transmission.It is well known that different mechanisms contribute to the spatiotemporal shaping of cytosolic Ca signals ,36. Amonle cells . When thER lumen . It has ytoplasm . This prstimulus ,41, inclstimulus ,42. Henc2+ release from the ER following PS activation was nonlinearly dependent on the irradiance. In fact, the lowest irradiance we tested (~7∙103 mW/cm2) was comparatively more efficient at depleting ER Ca2+ store than the medium irradiance, which was 3 orders of magnitude higher (~4∙106 mW/cm2), albeit in the first case ER emptying proceeded far more slowly. The simplest explanation is that efficiency of photochemical reactions triggered by PS excitation depends on O2 availability, which limits the rate of 1O2 and O2−∙ production, and, ultimately, sets an upper boundary for photodynamic cell-killing efficiency c, overcoming the opening threshold of the mitochondrial Ca2+ uniporter (MCU), the main Ca2+-selective channel responsible for fast Ca2+ accumulation in the IMM m progressively propagated to nonexposed bystander cells up to a distance of ~80 μm within 30 s of PS activation. The [Ca2+]m increase lasted more than 60 s in bystander cells. In contrast, it rose to a peak and subsequently displayed a descending phase decreasing below prestimulus levels in the irradiated cell. Both Ca2+ overload and ROS elevation in mitochondria promote the opening of mitochondria permeability transition pores (mPTPs) [2+ efflux, and finally matrix swelling and outer mitochondrial membrane rupture, with consequent release of cytochrome c and other pro-apoptotic factors [c release in bystander cells [c release from mitochondria and activation of caspase-9/-3 [2+ signaling and bystander cell killing.Besides ER and extracellular space, mitochondria are known to shape the spatiotemporal profile of cytoplasmic Ca2+ sinks ,49. In oganelles . During  the IMM ,50. We h (mPTPs) ,52. The  factors ,54. In per cells . The miter cells . Our finase-9/-3 . Further2+ and ROS signaling pathways are tightly interconnected in a mutual interplay: Ca2+ increase in the mitochondrial matrix drives ROS formation as a product of upregulated oxidative metabolism [2+ signaling through interactions with PM Ca2+ channels, activation of intercellular Ca2+ release channels and inhibition of Ca2+ ATPases [2−∙ production spread from the irradiated cell towards nearby bystander cells within seconds of PS excitation. Importantly, we determined that Ca2+ efflux from the ER was required for mitochondrial Ca2+ overload and O2−∙ production in both the irradiated and bystander cells. Therefore, our data indicate that ER–mitochondria Ca2+ transfer is crucial for the spreading of cytotoxic effects triggered by PS activation by promoting additional mitochondrial ROS production in bystander cells. It is even more likely to expect exacerbation of oxidative stress following PS activation because of the hypoxic condition due to photodynamic O2 consumption. In fact, recently published data reported that MCU is targeted by ROS in hypoxia conditions, stimulating mitochondrial Ca2+ accumulation [2O2 level in bystander cells after PS activation.Catabolism ,48 and,  ATPases ,57. More ATPases ,58. Seve ATPases ,23. Heremulation . Accordi2+-driven second generation of ROS, which can be crucial for the induction of apoptotic pathways in bystander cells. In this process, ER stress is key to the modulation of Ca2+ signaling triggered by PS activation and, therefore, to the propagation of cytotoxic stimuli to nonexposed bystander cells.Collectively, our data support the hypothesis of a central role for ER–mitochondria communication in the induction of a mitochondrial Ca


Ca2+) from the endoplasmic reticulum via the inositol 1, 4, 5-trisphosphate (IP3) pathway. This results in gliotransmission which elevates the presynaptic transmission probability rate (PR) causing weight potentiation and a gradual increase in postsynaptic neuronal firing, that eventually stabilizes. However, by capturing the complex interactions between IP3, generated from both GABA and the 2-arachidonyl glycerol (2-AG) pathway, and PR, this paper shows that this interaction not only gives rise to an initial weight potentiation phase but also this phase is followed by postsynaptic bursting behavior. Moreover, the model will show that there is a presynaptic frequency range over which burst firing can occur. The proposed model offers a novel cellular level mechanism that may underpin both seizure-like activity and neuronal synchrony across different brain regions.It is now widely accepted that glia cells and gamma-aminobutyric acidergic (GABA) interneurons dynamically regulate synaptic transmission and neuronal activity in time and space. This paper presents a biophysical model that captures the interaction between an astrocyte cell, a GABA interneuron and pre/postsynaptic neurons. Specifically, GABA released from a GABA interneuron triggers in astrocytes the release of calcium ( Spiking neural networks (SNNs) are considered to be the most biologically plausible representation of brain function , and connect to several neighboring neurons (four-eight). The interplay between an astrocyte and the neighboring neurons is believed to occur at the tripartite synapse  interneurons participate in astrocyte-mediated control of excitatory synaptic transmission  a novel biophysical model that describes the signaling pathways at the tripartite synapse and (ii) a novel mechanism that can potentially explain postsynaptic neuron burst firing. The rest of paper is organized as follows. Section 2 presents the biophysical model while section 3 provides simulation results that demonstrate the bursting. Section 4 concludes the paper and discusses future work.Ca2+ dynamics model shows that calcium (Ca2+) oscillations only occur over a range of inositol 1, 4, 5-trisphosphate (IP3) concentrations and furthermore this paper will show that Ca2+ oscillations are periodic and this behavior is key to the bursting behavior.In this section, a detailed discussion of the signaling pathways at the tripartite synapse is presented with a specific focus on GABA signaling between the presynaptic terminal and the nearby astrocyte. It will be shown that this interplay acts as a frequency dependent switch, which modulates the probability of release (PR) at the presynaptic terminal. Our fpre) is present at the excitatory presynaptic axon, neurotransmitter (glutamate) is released into the cleft and subsequently binds to receptors at the postsynaptic dendrite causing the depolarization of the postsynaptic neuron. While the authors accept that fast-spiking interneurons can fire at much higher frequencies than glutamatergic neurons, in this work we assume for simplicity that the firing rate of the GABA interneuron (fGABA) follows fpre, as the most likely physiological condition would be the activation of GABA interneuron by activation of glutamatergic axons  on the Endoplasmic Reticulum (ER). When the total cytosolic IP3 is sufficiently high, Ca2+ is released from the ER  occurs from the ER  into the synapse [see Glu (e-SP) pathway in As  Dawson, . However Dawson,  where CaPR≥PR* (PR* is defined as the plasticity activation level) the plasticity window fully opens and with PR<PR* the plasticity window closes. The decision on whether potentiation or depression occurs is governed by the STDP rule -type glutamate receptors  to below TCICR. In this instance, both the oscillatory Ca2+ transient and Glu (e-SP) pathways cease  pathway is re-established, PR increases and the level of neurotransmitter in the cleft is raised. In this post-learning phase PR cannot be elevated to a level where the plasticity window opens (PR<PR*), as the postsynaptic neuron is active and therefore the 2-AG pathway leads to a reduction in PR due to the DSE pathway. Consequently, the postsynaptic neuron firing rate reaches a maximum when the TCICR level is reached but it subsequently falls afterwards: a postsynaptic burst has occurred. This is followed by repeated bursts at each Ca2+ oscillatory period. We therefore propose that neuronal burst firing directly correlates with astrocytic Ca2+ oscillation.During the synapse learning phase, the frequency of the postsynaptic neuron, fpre at the excitatory presynaptic axon. As fpre increases so does the rate of increase of IP3 and the burst period is reduced. Therefore, network bursting is fpre dependant and will only occur over a range of fpre.Moreover, it should be noted that the duration of the burst correlated with the frequency of presynaptic terminal In this paper, the Leaky Integrate and Fire (LIF) model . The release of 2-AG correlates with the postsynaptic neuron activity , such as the phosphoinositide-specific phospholipase C (PLC) isoenzyme of PLCδ |x = K = 1/2.where IP3 mainly occurs through phosphorylation into inositol 1, 3, 4, 5-tetrakisphosphate (IP4), catalyzed by IP3 3-kinase (3K), and dephosphorylation by inositol polyphosphate 5-phosphatase (5P). The rate of IP3 degradation by The degradation of IP3 degradation rate by IP-5P. The activity of Ca2+ in a complex fashion  pumps. The Ca2+ model in the approach of De Pittà et al.  rate, C0 is the total free Ca2+ cytosolic concentration, C1 is the ER/cytoplasm volume ratio, and m∞ and n∞ are the IP3 Induced Calcium Release (IICR) and CICR channels respectively, which are given bywhere andJleak and Jpump are described byandrL is the Ca2+ leakage rate, vER is the maximum SERCA pump uptake rate and kER is the SERCA pump activation constant.where Ca2+ crosses the CICR threshold, a quantity of glutamate is released  is the initial PR for each synapse. As discussed in section 2.1, the PR can switch on/off learning at the synaptic terminal by modulating the height of the plasticity learning window. The authors are not aware of any biophysical model that relates PR to the plasticity window weighting parameter A0 and therefore in this work it is assumed that A0 is modulated according towhere PR* is the learning activation level and r is a constant value which controls the maximum height of the learning window. The STDP rule used in this approach to update the synaptic weights according to the timing difference between the post and presynaptic spikes is described bywhere w(Δt) is the weight update, Δt is the time difference between the post and presynaptic spikes, A0 is the height of the plasticity window which limits the maximum levels of weight potentiation and depression, and τ+ and τ− control the width of the plasticity window. A symmetrical plasticity window is assumed in this approach, and τ+ = τ− = 40ms.where δfpre is large enough, IP3 is generated sufficiently to cause Ca2+ oscillations. Then the Ca2+-induced glutamate binds to the mGluRs receptors at the presynaptic terminal resulting in an increase of the synaptic transmission probability PR. Note that the authors wish to point out that astrocytes are believed to gate LTP and LTD by regulating glutamate levels in the synaptic cleft  pathway and PR falls causing a reduction in fpost. Therefore, the bursting activity of the postsynaptic neuron is regulated by the GABA interneuron and the astrocyte cell. The results in the next section show the signaling pathways leading to a bursting postsynaptic neuron.From the proposed models, it can be seen that if the This section provides simulation results which highlight the dynamic behavior at the synapse terminals and how the interactions between an astrocyte and GABA interneuron can give rise to bursting behavior. The MATLAB simulation platform is used in this work together with the Euler method with the time step of 1 ms. fpre = fGABA which causes the release of GABA and glutamate , the STDP learning window opens (A0>0) at ~80 s, as shown in vth.In this simulation both the presynaptic excitatory neuron and the GABA interneuron are stimulated by the same spike train at frequency >PR* see , a signi>PR* see  and weig>PR* see  resultinc weight . In our A0 = 0) and the weight stabilizes to ~610 at 110 s, as shown in PR<PR* for all subsequent Ca2+ oscillations as the DSE pathway is also active. M under the input spike stimulus. M which is consistent with the input stimuli profile. IP3 sources that contribute to the total IP3 in the cytosol.After a period of learning the postsynaptic neuron activity has stabilized and PR drops sufficiently, toward the end of the first set of PR “spikes” see , closingIP3 increases with TCICR level is reached triggering the release of Ca2+, as shown in TCICR is consistent with an IP3 level of approximately 0.5 μM and whenever IP3 exceeds this threshold a transient elevation in Ca2+ occurs, as can be seen in IP3 level increases with IP3 due to elevated Ca2+/IP3 levels is insufficient to reduce IP3 to below 0.5 μM and consequently the transient elevations of Ca2+ stops just after 100 s followed by a relatively slow degradation of IP3 and Ca2+: these periodic bursts in Ca2+ gives rise to a Ca2+ oscillatory wave where the initial Ca2+ burst is longer due to synaptic potentiation. At the onset of each subsequent Ca2+ burst the IP3 level drops sharply and we attribute this to strong dependence of Ca2+ (Equation 10). As the Ca2+ level drops Jchan (Equation 17) reverses direction perturbing the rate of change in Ca2+ (Equation 12) and this causes a rapid increase in IP3 (Equation 11).Initially the total Ca2+ oscillation is initiated at ~20 s  pathway to restrict PR to a relatively stable low value for all subsequent Ca2+ oscillations that occur post-learning, as shown in The fpre of 20, 40, and 80 Hz where clearly only fpre = 40Hz results in repeated Ca2+ oscillations. This is because at 20Hz the TCICR level cannot be reached whereas at 80Hz the astrocyte cytosol is quickly swamped with both IP3 and Ca2+ and subsequent degradation in IP3 is insufficient to allow further CICR. Consequently our model shows presynaptic frequency selectivity which is consistent with work reported elsewhere  is used in this experiment, however the same results are observed for other fpre values such as 50 Hz, and the burst frequency variation is not constrained for specific fpre values. The results in ried out  which shIP3 secondary messenger and thus the probability of release (PR) at the presynaptic terminal. In our model we propose that PR modulates the height of the plasticity window and therefore controls when synaptic potentiation/depression occurs. Specifically, the simulations show that during the weight potentiation phase, increasing IP3 leads to a cycle of CICR events where each is followed by rapid degradation in IP3. Over time the firing frequency of the postsynaptic neuron continually increases and eventually the synaptic weights stabilize. Postsynaptic firing results in the release of 2-AG into the extracellular space and this messenger binds to CB1R receptors on the astrocyte membrane. The associated IP3 and eventually Ca2+ oscillations, and therefore the Glu (e-SP) pathway ceases: 2-AG also binds to CB1Rs on the presynaptic terminal causing a decrease of the synaptic transmission PR via the DSE pathway. PR therefore decreases at the presynaptic terminal which reduces the level of neurotransmitter in the cleft, and consequently the firing frequency of the postsynaptic neuron diminishes, as does IP3/Ca2+ degrades significantly over time but is replenished by PR<PR*. The postsynaptic neuron firing rate increases again until the Ca2+ transients stop and thereafter the activity of the postsynaptic neuron falls off again. A network burst has occurred and this is followed by repeated bursts where each coincides with Ca2+ transients: the network burst frequency correlates with the Ca2+ oscillatory wave. In addition, the GABA released by the GABA interneuron controls the frequency range within which the network bursts can occur. Future work will further explore other neurotransmitters released by astrocytes such as D-serine and ATP, and also slow inward currents at the postsynaptic terminal as a result of glutamate release by astrocytes.In this paper, a biophysical model is proposed where it is shown that GABA interneuron regulates the astrocytic IP3 receptor and GABA-B receptor, are dysregulated in human epileptic brain tissue or animal models  and investigating whether this alters the frequency or occurrence of pathophysiological neuronal firing and seizures.The authors recognize that this study is based on biological findings of the simplest signaling mechanisms involving astrocytic GABA responses and astrocytic glutamate signaling in presynaptic terminals that regulate network function. Other factors, such as astrocytic ATP/adenosine release from astrocytes (Covelo and Araque, The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.JL, LM, AA, JW, JHa, SK, DCH, NC, AT, JT, and DMH investigated and proposed the biophysical model. JL, LM, AA, JW, JHa, DCH, NC, and DMH wrote and revised the manuscript. JL, LM, AA, JW, JHa, DCH, NC, AJ, AT, JT, AM, JHi, and DMH analyzed the results and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most intracellular Ca2+ signals involve the release of Ca2+ from the endoplasmic reticulum (ER) through Inositol 1,4,5-Trisphosphate Receptors (IP3Rs). The non-uniform spatial organization of IP3Rs and the fact that their individual openings are coupled via cytosolic Ca2+ are key factors for the variety of spatio-temporal distributions of the cytosolic [Ca2+] and the versatility of the signals. In this paper we combine experiments performed in untreated and in progesterone-treated Xenopus laevis oocytes and mathematical models to investigate how the interplay between geometry  and dynamics  affects the resulting signals. Signal propagation looks more continuous and spatially uniform in treated (mature) than in untreated (immature) oocytes. This could be due to the different underlying IP3R spatial distribution that has been observed in both cell types. The models, however, show that the rate of cytosolic Ca2+ removal, which is also different in both cell types, plays a key role affecting the coupling between Ca2+ release sites in such a way that the effect of the underlying IP3R spatial distribution can be modified.Calcium (Ca IP3Rs need to bind IP3 and Ca2+ on the cytosolic side to become open   and dynamics .Calcium  and in progesterone-treated (mature) Xenopus laevis oocytes. It is known that the IP3R spatial distribution is different in these two situations  model was introduced some years ago , τ is the mean duration during which a site releases Ca2+, D is the Ca2+ (effective) diffusion coefficient, d is the mean separation between release sites, [Ca]b is the basal Ca2+ concentration, and [Ca]T is a threshold Ca2+ concentration above which a site starts to release Ca2+. Γ determines whether the wave can propagate (if it is sufficiently large) or not. β rules whether the propagation is saltatory (β ≤ 1) or continuous which means that Ca2+ release from individual  sites is apparent or is smeared out, respectively. In this paper signals that we observe experimentally in eggs and in oocytes have distinctive features that lead us to classify them as continuous and saltatory, respectively. Namely, the global waves that we observe in immature oocytes are preceded by localized signals (puffs) while those in eggs start, within the time resolution of the experiments, as soon as IP3 is released. In our experiments we also observe differences in the way that Ca2+ is cleared in the two cell types once the IP3 release is stopped which can be related to the existence of larger Ca2+ concentration gradients  in oocytes than in eggs. This distinction between saltatory and continuous signals is consistent with the differences between the waves elicited in immature oocytes  than the former (~ 20μ/s)  could be accommodated within the fdf model in Dawson et al. , that Γ was only twice as large in eggs than in oocytes but that β was very different in both cases (0.08 in oocytes and 50 in eggs). It is interesting to notice that even if in these examples it is in principle “easier” to induce Ca2+ release from neighboring sites in the egg due to the larger Γ and D values, the wave propagates more slowly than in the oocyte. This shows that the mode of propagation  is not the only difference between the waves elicited in immature eggs and the fertilization wave. The latter is slower . In this paper the signal properties were contrasted against the structural changes that they observed occurred in the cells. Based on experimental observations with marked IP3Rs and ER, it was reported in Sun et al. . In order to obtain waves that propagated without failure at approximately the same speed as the Ca2+ fertilization wave, on the other hand, they used clusters with about 1,000 IP3Rs each and a larger (17.5μm) inter-cluster distance. They found propagation failure, however, if the distance was increased to 20μm, which seems to indicate that the propagation was saltatory for slightly smaller inter-cluster distances. To mimic the actual situation observed in Sun et al.  and a 12-h light-dark cycle. Each frog was kept in an individual tank with filtered water and was fed twice a week. Frogs were anesthetized for surgery by immersion in 0.3% tricaine (MS222) and oocytes were removed and prepared as previously described in Goldman et al.  was used to probe cytosolic [Ca2+]. Caged IP3 -(1-(2-Nitrophenyl)ethyl) Ester) was used to induce IP3Rs opening. Final intracellular concentrations of the different compounds were calculated assuming a 1μl cytosolic volume. Final intracellular concentration of IP3 and Fluo-4 were 9 and 36 μM, respectively, in all the experiments. Fluo-4 and IP3 were from Molecular Probes Inc. Recordings were made at room temperature.Experiments were performed in both immature and mature g/ml concentration at 18°C during 12–16 h. Eggs with white dots in the animal hemisphere were chosen since this indicates germinal vesicle break down (GVBD). Experiments were performed between 3 and 4 h after the white's dot appearance. At this time it is supposed that the egg is at metaphase II of meiosis and that maturation is complete  with a 4μs time per pixel and 0.56s by frame. The Ca2+ signals were elicited photolyzing the caged IP3 with the UV part of the spectrum of a mercury lamp that comes with the microscope using the modification introduced in Sigaut et al. . The emitted fluorescence was detected in the 500–600 nm range with PMT detectors. Images were acquired in the frame mode over regions of 250 × 250 pixels  of the k-th subregion and NTk = Nk · Nk, with Nk = 50 ∀k ∈ , is the total number of pixels of the k-th subregion.where the sum is over the pixels ] in the following way. We computed the rise time as FM the maximum value of F(t), Fb the value of F(t) immediately before the UV flash and Δt the time elapsed between the initiation of the flash and the occurrence of the fluorescence maximum. The decay time was obtained by fitting F(t) over the time interval that went from the first frame-time after the UV flash had been turned off (to) to the time at which F(t) reached the value 1.5 × Fb. We tried three types of fittings:Both tdf and tds the fast and slow decay times obtained after fitting with Equation (4), tdm the decay time of the monoexponential fit (Equation 5) and tdl the decay time obtained when fitting with Equation (6).i.e., bi-exponential, mono-exponential, and linear, respectively. For each case we obtained the characteristic decay times, 3Rs were initially closed. Briefly, the simulation domain was a πR2 circular region with R = 10μm. N IP3R clusters were initially placed at random in the domain choosing their positions with uniform distribution over the circle. For each simulation, the number, N, was chosen from a Poisson distribution with mean, dm. The number of IP3Rs in each cluster was chosen from a Poisson distribution with mean, NIP3R, at the beginning of the simulation. All IP3Rs were assumed to be IP3-bound and initially active. In the model an event is a sequence (or cascade) of IP3R openings coupled via CICR. The time propagation of the signal is not described, it is instantaneous  level that depends on the latest No and that subsequently decreases exponentially with timescale 1/δCa ; Noi is the number of channels of that cluster that participated of the signal and the function that multiplies Noi is a Gaussian approximation of the [Ca2+] contribution of a 0.1pA Ca2+ point source. The amplitude of this Gaussian is D = 100μm2/s is the Ca2+ diffusion coefficient, dr determines the “coarse-graining” with the [Ca2+] contribution of the point source is computed (dr = 0.16μm) and σ2 = 2μm2 gives the width of the Gaussian.where X. laevis and in oocytes of the same species maturated with progesterone (eggs) as described in section 2. We also show the results of numerical simulations of the model described in Materials and Methods. The aim of these studies is to analyze the differences and similarities between the IP3R-mediated signals evoked in eggs and in immature oocytes and to establish the factors that are key to determine their differences.Here we present the results of experiments performed in immature oocytes of m, the scale is indicated with a white line. The color bar represents the fluorescence value, F, at each pixel of the image in arbitrary units. In these two examples the UV light used to uncage the IP3 was turned on at t = 11.2 s and turned off at t = 28 s and then turned back on at t = 173.6 s and off at t = 190.4 s in the case of the oocyte . Differently from the case of the oocyte, by t ~ 44.8 s the egg has not reached the basal level yet  and σF(t) as defined in Equations (2) and (3). The number of subregions with similar basal fluorescence levels was n = 20 in the case of the oocyte  distribution would look like during signals with the number and location of the IP3Rs that participated of an event according to the simulations. We show plots of the distributions for three such events in 1, i.e., a situation where the IP3Rs were spatially clustered and the Ca2+ removal rate was high. The example of 3, i.e., a situation where the IP3Rs were more uniformly distributed in space and the Ca2+ removal rate was high. The example of 4, i.e., a situation with uniformly distributed IP3Rs and low Ca2+ removal rate.We used Equation (7) to estimate how the  is more spatially uniform during the signals evoked in the eggs than in the oocytes. We could also observe other differences in the experiments, some of which reinforce this conclusion. In particular, we observed differences in the growth of the fluorescence between the egg and the oocyte as illustrated in Fm, showed a marked peak that occurred approximately at the same time as the localized spots observed in s), while they increased much more slowly and reached smaller values in the egg . We can interpret this lower elevation as being due to the existence of a subset of IP3Rs that, when the second flash was applied, still remained inactive after the signal evoked by the first flash. The absence of this difference in the egg indicates that the pool of activatable IP3Rs was approximately the same for both flashes in this example. However, we did not obtain this same behavior in other experiments performed in eggs. Although the experiments performed with varying durations of the UV flash showed smaller fluorescence amplitudes for eggs than oocytes  indicates that there is still Ca2+ release during that time but that this release is unable to overcome the processes that remove Ca2+ from the observed region. Given that, for the same amount of IP3 released, it takes longer for 3Rs in the latter. After having been open, IP3Rs typically enter an inhibited state. Thus, the more efficient IP3R recruitment in oocytes implies that IP3Rs become inhibited faster in these cells than in eggs which could explain the faster decay of 2+ release has usually a smaller amplitude than the first one in oocytes, even for short durations of the IP3 release and that this occurs for eggs for long enough UV flashes. An efficient recruitment would imply that the number of IP3Rs that become open during the first round of IP3 release is so large that a significant amount of them is still inhibited when the second UV flash is shone which, in turn, would lead to a lower amplitude Ca2+ pulse. Now, the decay of 3. In order to discard this possibility all the experiments were repeated for a new round 10 min after the second UV flash obtaining larger amplitudes in the third flash than in the second one (data not shown).We can interpret some of these observations in terms of the IP2+ gradient dissipates, the role of pumps (and buffers) is more important. Thus, we can expect that the slow component of the decay in oocytes and perhaps all of the decay in the case of eggs is dominated by this process. Given the smaller [Ca2+] that we observe in eggs . However, the good linear fits that we obtain  is completely internalized so that Ca2+ cannot be removed to the extracellular medium. This limitation to remove cytosolic Ca2+ together with the permanent loss of Ca2+ from the ER through IP3Rs could explain why Ca2+ could remain at relatively large concentrations for a longer time in eggs than in oocytes  due to CICR. In particular, we studied how the distribution of No values varied depending on the IP3R spatial distribution and the rate of Ca2+ removal for a fixed mean value of IP3-bound IP3Rs . For the case in which the rate of Ca2+ removal was low, the distribution was approximately symmetric around a mean value, 〈No〉 ~ 0.02NT with NT the total number of IP3Rs with IP3 bound. This happened both when the spatial distribution of IP3Rs was approximately uniform  was higher than in all the examples of No = 38, No = 44, and No = 48) but had a much more localized Ca2+ spatial distribution. The maximum values of 2+] values in the examples of 2+ distribution looked locally more uniform in 3R distribution, bur did not spread over the whole domain as observed in the examples of 2+] spatial distribution was apparent even for events with similar values of No .We subsequently analyzed how the Ca case 1, ; more un case 3,  and more case 4, . The exaibutions . We obse2+ concentration before the start of the cascade was quite similar in the S4, i.e., the conditions for which the No distribution was Gaussian-like about the mean 〈No〉 = 56  [Ca2+], then, distant uninhibited IP3-bound IP3Rs will be coupled via CICR. In this way, the number, No, of IP3Rs that participate of an event will be mostly determined by the number of simultaneously active IP3Rs, no matter how far away from one another they are (within certain limits). In this case, spatial heterogeneities do not play much of a role: the slow Ca2+ removal smears out inhomogeneities and couples relatively distant regions. If there are sufficiently many IP3Rs in the whole system, it is likely that several IP3Rs will be simultaneously uninhibited. Thus, the signal will then spread throughout the domain. In summary, we associate the exponential part of the No distribution to those cascades which occurrence is limited by CICR, i.e., by the IP3Rs binding the Ca2+ that is released by other IP3Rs. On the other hand, we interpret the Gaussian-like distribution as being the consequence of a long-distance coupling where space inhomogeneities are less visible.The CaNo〉 = 56 . This prcase S1,  or for acase S3, . The sig2+ removal rate is decreased, to a bistable situation with one fixed point corresponding to basal Ca2+ and no IP3Rs open and the other corresponding to a higher Ca2+ level and No open IP3Rs with No equal to the value at which the Gaussian has its maximum. We recall that such a situation could be maintained provided that the turn-over time of luminal Ca2+ was fast enough to guarantee Ca2+ release every time there is an open IP3R. So, the actual situation could be one in which the release starts eventually to decline. In any case, we do not want to analyze this possibility here. The exponential like distribution (No) while less often events are evoked that involve the opening of many IP3Rs (large No). The latter sends most of the IP3Rs of the system into an inhibited state which delays the occurrence or reduces the number of participating IP3Rs of the subsequent event (as in Fraiman et al., 3Rs that participate of each event fluctuates around a mean that is only a fraction of the total number of IP3Rs of the system. Thus, IP3Rs do not enter the inhibited state simultaneously as they do after the largest events of the excitable case. This means that active IP3Rs are readily available to become open at any given time which guarantees the spatial spread of the Ca2+ signal. This could explain the observations of 3Rs in both. This is in fact observed in the simulations as illustrated in the 2+ removal rate and that, after a transient, the fraction of IP3Rs that participate of the events fluctuates very little around the mean that can be associated to the high [Ca2+] fixed point.The Gaussian like event size distribution  is indicribution , 7A is i3Rs is relevant for the different types of signals observed in eggs and oocytes, the rate of Ca2+ removal is key since it can smear out spatial inhomogeneities. All our results indicate that Ca2+ removal due to pumps and buffering occurs much more slowly in eggs than in oocytes. This is consistent with previous observations. As we have already mentioned, the PMCA is completely internalized in eggs (El-Jouni et al., 2+ diffusion coefficient that was twice as large in the former case. This larger effective diffusion coefficient is, in turn, compatible with less effective Ca2+ buffers. The results of 2+ buffers disrupts the steps that are necessary for development to advance. In fact, [Ca2+] needs to reach relatively high values for the steps that follow fertilization to take place. Simulations of the fertilization wave in X. laevis oocytes (Wagner et al., 2+ release increased with maturation. A similar transition could be explained, however, for a decreasing rate of Ca2+ removal (to which the efflux of Ca2+ from mitochondria observed in eggs could also contribute; Falcke et al., The results of the experiments and of the numerical simulations presented in this paper show that even though the non-uniform distribution of IPAll datasets generated for this study are included in the manuscript and/or the EP and CV performed the experiments. EP and SP performed the simulations. EP, CV, and SP analyzed the experiments and simulations and wrote the paper. SP conceived the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The EL Ca2+ store releases Ca2+ through a variety of Ca2+-permeable channels, including Transient Receptor Potential (TRP) Mucolipin 1-3 (TRPML1-3) and two-pore channels 1-2 (TPC1-2), whereas EL Ca2+ refilling is sustained by the proton gradient across the EL membrane and/or by the endoplasmic reticulum (ER). EL Ca2+ signals may be either spatially restricted to control vesicle trafficking, autophagy and membrane repair or may be amplified into a global Ca2+ signal through the Ca2+-dependent recruitment of ER-embedded channels. Emerging evidence suggested that nicotinic acid adenine dinucleotide phosphate (NAADP)-gated TPCs sustain multiple cancer hallmarks, such as migration, invasiveness and angiogenesis. Herein, we first survey the EL Ca2+ refilling and release mechanisms and then focus on the oncogenic role of EL Ca2+ signaling. While the evidence in favor of TRPML1 involvement in neoplastic transformation is yet to be clearly provided, TPCs are emerging as an alternative target for anticancer therapies.The acidic vesicles of the endolysosomal (EL) system are emerging as an intracellular Ca Typically, extracellular agonists, including growth factors, cytokines, neurotransmitters and hormones, bind to their specific tyrosine kinase receptors (TKRs) or Gq-protein coupled receptors (GPCRs), thereby recruiting, respectively, phospholipase Cγ (PLCγ) and PLCβ, which in turn cleave phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (InsP3) and diacyl-glycerol (DAG) ER) which triggers a signaling cascade leading to the opening of a Ca2+-permeable pathway on the PM, known as store-operated Ca2+ entry (SOCE) ER, which undergoes a dramatic conformational change and sub-cellular repositioning. The depletion of ER Ca2+ induces STIM1 to oligomerize and translocate from its reticular location in the bulk ER to specialized ER-PM junctions (≈20 nm) where it forms STIM1 puncta and interacts with the pore-forming subunit of store-operated channels (SOCs), Orai1 i i ,26, alth see the .2+ exchange with the ER, thereby regulating pleoiotropic functions across the phylogenetic tree i i in a wide number of cells types i ,13. The vacuoles . GPN is ls types ,39,40,41 and LEs ,29. It hs fusion . In addi of TFEB , as desc2+-dependent manner i concentration by further mobilizing lysosomal Ca2+ through TRPML1 i i . Moreoveus [2+]i . Subsequus [2+]i . In thist manner ,102.Arabidopsis thaliana i that drive a variety of cellular processes, ranging from fertilization to muscle contraction i ,14. Acco process ,162,163.al Ca2+) . Ultrastal Ca2+) . As revial Ca2+) ,165. Notal Ca2+) ,166. MCSal Ca2+) ,167 and ent, GPN . Thereforocesses ,51. Thesrocesses , neurotrrocesses , exocytorocesses , neuriterocesses , cardiacrocesses  and vascrocesses  contractrocesses , vasculorocesses , and nitrocesses ,173.2+ release from the EL store can also be effected by other members of the TRP superfamily, such as TRPA1 and TRPM2, ATP-gated ionotropic P2X4 receptors and voltage-gated Ca2+ channels  and hydrogen peroxide (H2O2), and positively modulated by NAADP and cyclic ADP-ribose [2+ release channels in mouse pancreatic β cells, in which it mediates H2O2–induced apoptosis [TRPM2 is a CaP-ribose . Additiopoptosis , and in poptosis .2+-permeable, non-selective cation current that is activated by noxious cold temperatures and mechanical stimuli and by a wealth of irritant compounds in peripheral somatosensory neurons [2+ sparks that stimulate vesicle exocytosis and calcitonin gene-related peptide release. It has, therefore, been proposed that EL TRPA1 further increases the excitability of dorsal root ganglia [TRPA1 belongs to a distinct sub-family of TRP channels and mediates a Ca neurons . Herein, ganglia .2+-permeable, non-selective channels [2- and COOH-terminal tails [2-terminus [P2X receptors are homo- or heterotrimers which serve as ATP-gated ionotropic, Cachannels . Seven sal tails . Earlierterminus . Neverthterminus . A subseterminus . Notablyterminus .2+ channels mediate extracellular Ca2+ entry in response to membrane depolarization to regulate multiple functions, including muscle contraction and gene expression (CaV1), neurotransmitter release (CaV2), and repetitive firing in sino-atrial cells and thalamic neurons (CaV3) [V2.1, which mediates voltage-gated P/Q-type Ca2+ currents at cerebellar terminal synapses and is encoded by the CACNA1A gene [V2.1 prevented autophagy by inhibiting the fusion between lysosomes and endosomes and/or autophagosomes [2+ currents were not measured, this finding raises the possibility that lysosomal Ca2+ release could effectively occur in response to a positive shift in ΔΨ [Voltage-gated Cas (CaV3) . SurprisA1A gene , is alsoA1A gene . Mutatioagosomes . Althougft in ΔΨ .2+ content drive or are tightly involved in the development of neurodegenerative disorders, such as MLIV, Parkinson’s disease, Alzheimer’s disease, and Niemann Pick disease, type C [The lysosomal membrane serves as a signaling platform that regulates multiple functions, including endocytic traffic, autophagy, and lysosomal function, thereby impacting on the metabolism and fate of the entire cell ,185. The, type C ,146,186., type C ,188. Bri, type C . In neop, type C . As a co, type C ,192. Cat, type C . Finally, type C . It shou, type C ,195.2+-permeable channels, i.e., TRPML1, or defects in the acidic Ca2+ store underlie severe neurodegenerative disorders, such as MLIV and Niemann Pick disease, type C, respectively [2+ toolkit supports many cancer hallmarks. For instance, the reduction in ER Ca2+ concentration is responsible for the increased resistance to pro-apoptotic stimuli [2+ machinery was implicated in malignant transformation [2+ channels in malignant transformation [2+-dependence of the subcellular localization of TFEB in cancer cells. TRPML2 is the only member of this sub-family to have been clearly associated to malignant transformation. A recent investigation revealed that TRPML2 transcript and protein were highly expressed in glioma tissues and that their expression increased during tumor progression [2+ signalling in the oncogenic effect of TRPML2 was not explored. Conversely, emerging evidence clearly hints at TPCs as main drivers of multiple cancer hallmarks in a growing number of solid malignancies , which is indispensable for cell migration [The first report about the pro-tumorigenic role of TPCs was, however, provided by Favia and coworkers . This stma cells . Accordima cells . A more 2+ store . NAADP-eigration .2+ response to NAADP stimulated angiogenesis through the paracrine release of growth factors from the cancer cells. In addition, the same group previously reported that NAADP sustains vascular endothelial growth factor (VEGF)-dependent angiogenesis in vitro and neovessel formation in vivo [2+ signals were attenuated by NED-19 and v-ATPase inhibition with bafilomycin A1. Likewise, the pharmacological blockade of TPCs with NED-19 blocked cell proliferation, migration and tube formation by suppressing VEGF-induced phosphorylation of ERK1/2, Akt, JNK and endothelial nitric oxide synthase (eNOS) [−/−TPCN2, but not −/−TPCN1, mice [2+ signals in HUVECs and prevented their assembly into capillary-like bidimensional structures in Matrigel scaffolds [2+ store enriched with TPC1 is also expressed in human endothelial colony forming cell (ECFCs) [2+/CaM-dependent manner [This study further suggested that the Ca in vivo . Accordie (eNOS)   ,171, a s (ECFCs) ,216. Nott manner ,171. Tumt manner ,218,219.2+ response to NAADP was inhibited by antagonists of L-type CaV channels, such as verapamil and diltiazem and dihydropyridines  [V antagonists within their selectivity filter, which is not surprising when considering the strong molecular similarity between TPCs and CaV [V antagonists, such as nitrendipine, nifedipine and verapamil, are routinely employed to treat life-threatening disorders [V antagonists could also be probed in preclinical trials assessing whether they are able to induce cancer shrinkage by blocking TPCs.The studies described strongly suggest that TPC1 and TPC2 represent suitable molecular targets to interfere with tumor growth, vascularization, and metastasis. Pharmacological blockade of TPCs could thus be regarded as a promising, alternative anticancer approach ,21. Thrordipine) ,224. Sub and CaV . As revi and CaV , CaV antisorders , such as2+ release. The most efficient inhibitors were PF-543, a cell permeant blocker of sphingosine kinase 1, SKF96365, a non-specific antagonist of Ca2+-permeable channels that was already known to affect NAADP signalling [2+ signals [2+ release in rat Jurkat T-lymphocytes in vitro and was effective in an EAE model of multiple sclerosis in vivo. Another agent obtained by this screening, termed 3 or BZ194 [2+ response to NAADP in vitro [A larger arsenal of drugs is currently under intense scrutiny to design alternative treatments of diseases associated to mutated or dysfunctional TPCs, including neurodegenerative diseases, or requiring a functional EL trafficking, such as viral infections. A recent screening campaign conducted on sea urchin egg homogenates disclosed a novel pharmacopeia of 18 drugs effectively (>80%) inhibiting NAADP-induced Cagnalling , and racgnalling . The sub signals  and atte signals . Paralle signals . This th signals . Notably signals . Finally signals . One of or BZ194 , blockedin vitro  and cardin vitro .2+-permeable channels, such as TPCs, is now coming of age and is predicted to rapidly enlarge our arsenal of selective TPC inhibitors, which could be also testable for anticancer treatments.Only NED-19, tetrandrin and naringenin were hitherto probed to halt tumor growth and vascularization. Unfortunately, tetrandrine and naringenin may target multiple signalling pathways and are not approved for human use worldwide . However2+ signalling through TRPML1-3 and TPC1-2 finely regulates cellular fate and behavior by driving the EL trafficking machinery and the autophagic flux, thus enabling the cells to adapt to stressful conditions, such as those imposed by lack of nutrients. In addition, local EL Ca2+ signals control trafficking and correct positioning of signaling proteins involved in cell proliferation and migration, such as integrins and EGF receptors, and the endocytosis of bacteria, toxins and viruses, which may cause cell death. Moreover, TPCs may interact with ER-embedded InsP3Rs and RyRs to generate global Ca2+ signals that further expand the repertoire of cellular functions regulated by the EL Ca2+ store. Herein, we discussed the evidences supporting the role of EL Ca2+ signalling in cancer. While the oncogenic function of TRPML1, the main driver of TFEB activation in healthy cells, remains obscure, TPC1 and TPC2 are standing out as crucial drivers of many cancer hallmarks, including excessive proliferation, aberrant migration and sustained vascularization. Many issues remain to be elucidated. For instance, is TPC expression actually altered in cancer cells? Is NAADP the main ligand of tumor TPCs or is there a role also for PIP2? Is the EL Ca2+ store altered by neoplastic transformation, just as it happens for the ER? Which is the main source of Ca2+ responsible for EL Ca2+ refilling in cancer cells: the cytosol, the ER or the extracellular milieu? Does CICR support the Ca2+ response to NAADP also in cancer cells (EL Caer cells ? With al


This study was performed to identify the effect of verapamil, which is an L-type Ca2+ channel blocker and a prophylaxis for migraines, on the TRESK channel in trigeminal ganglion (TG) neurons, as well as in a heterologous system. Single-channel and whole-cell currents were recorded in TG neurons and HEK-293 cells transfected with mTRESK using patch-clamping techniques. In TG neurons, changes in [Ca2+]i were measured using the fluo-3-AM Ca2+ indicator. Verapamil, nifedipine, and NiCl2 inhibited the whole-cell currents in HEK-293 cells overexpressing mTRESK with IC50 values of 5.2, 54.3, and >100 μM, respectively. The inhibitory effect of verapamil on TRESK channel was also observed in excised patches. In TG neurons, verapamil (10 μM) inhibited TRESK channel activity by approximately 76%. The TRESK channel activity was not dependent on the presence of extracellular Ca2+. In addition, the inhibitory effect of verapamil on the TRESK channel remained despite the absence of extracellular Ca2+. These findings show that verapamil inhibits the TRESK current independently of the blockade of Ca2+ influx in TG neurons. Verapamil will be able to exert its pharmacological effects by modulating TRESK, as well as Ca2+ influx, in TG neurons in vitro. We suggest that verapamil could be used as an inhibitor for identifying TRESK channel in TG neurons.Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord K Of the K2P channels, TRESK is known as the only member activated by an increase in the intracellular Ca2+ concentration ([Ca2+]i) [2+]i activates calcineurin, a calcium-activated serine–threonine phosphatase, and the calcineurin dephosphorylates TRESK to activate it. The Ca2+-induced activation of mouse and human TRESK depends on direct targeting of calcineurin to the nuclear factor of activated T-cell-like consensus motif [Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord KCa2+]i) . Increas+i) . Incus motif .2+ influx by ionomycin, an effective Ca2+ ionophore, activates the TRESK channel i [This study shows an advance in the understanding the mechanism of action of verapamil on TRESK channel. We first assumed that Ca Ca2+]i ,2. As ex+i ,2. As2+ influx. Verapamil showed an inhibitory effect on TRESK currents despite the absence of extracellular Ca2+. In addition, TRESK was inhibited by verapamil under inside-out and outside-out patch modes. These results indicate that verapamil inhibits TRESK current independently of the blockade of Ca2+ influx. Similar to TRESK channels, Kv channels expressed in smooth muscle cells isolated from a rabbit coronary artery are inhibited by verapamil independently of any Ca2+ channel inhibition  in a heterologous system i, increases the TRESK activity in rat DRG neurons, but the effect was much less than that in a heterologous system [2+]i. These differences between DRG and TG could be from changes in gene expression between them. Comprehensive RAN-Seq expression analysis of DRG and TG shows different expression patterns in the ion channels and G protein-coupled receptors (GPCRs) i was measured as described previously [2+]i was represented as FI.Changes in [Caeviously  with slip < 0.05 was considered as the criterion for significance.Differences among groups were analyzed using one-way ANOVA test with post hoc comparisons using Tukey’s test . Data are represented as mean ± SD. A 50 (5.2 μM), independently of the blockade of Ca2+ influx. Verapamil will be able to exert its pharmacological effects by modulating TRESK as well as Ca2+ influx in TG neurons.In conclusion, verapamil reversibly inhibits TRESK currents in a dose-dependent manner with a low IC