Accordingly, we report the different roles of Ca2+-handling in the development of congenital as well as acquired ventricular arrhythmia syndromes. We highlight the therapeutic potential of gene therapy as a novel and innovative approach for future arrhythmia therapy. Furthermore, we discuss various promising cellular and mitochondrial targets for therapeutic gene transfer currently under investigation.Cardiac arrhythmias constitute a major health problem with a huge impact on mortality rates and health care costs. Despite ongoing research efforts, the understanding of the molecular mechanisms and processes responsible for arrhythmogenesis remains incomplete. Given the crucial role of Ca Cardiac arrhythmias constitute a major public health concern worldwide . Several2+) homeostasis plays a crucial role in AP generation and correct cardiac contraction. Several studies have shown that cellular calcium handling is altered in the pathophysiology of heart diseases, such as HF and cardiomyopathies [2+ handling may also be altered by inherited mutations that directly affect Ca2+ channels or Ca2+ handling proteins, such as ryanodine receptor type 2 (RyR2) [2+ handling gets more into focus of current studies [2+ currents or channels and novel therapeutic options.Therefore, investigations on ionic currents and channels, and their influence on action potential (AP) waveforms are of major interest for understanding arrhythmogenesis. Calcium (Caopathies . Additio2 (RyR2) , calsequ2 (RyR2)  and calm2 (RyR2) . Further studies ,14,15. I2+ homeostasis, resulting in a so-called excitation-contraction coupling. Each ventricular AP starts with the influx of sodium (Na+) through voltage-gated Na+ channels that depolarizes the cell. When a certain threshold is reached voltage-sensitive L-type Ca2+ channels (LTCC) are activated that allow the influx of Ca2+ into the cytosol, triggering the much larger Ca2+ release from the sarcoplasmic reticulum (SR), the main intracellular Ca2+ storage organelle [2+ release is mediated by RyR2, which is a massive structure comprising the largest known ion channel-bearing macromolecular complex [2+-induced Ca2+-release (CICR) and is the fundamental link between electrical and mechanical activation in the heart [2+ then binds to troponin C and initiates myocardial contraction, forming the cardiac systole [Cardiac contraction and relaxation are mediated by a precise and coordinated linkage of electrical activation (excitation) and intracellular Carganelle ,17. SR C complex ,19. Thishe heart . The cyt systole .2+-ATPase type-2a (SERCA2a) that pumps Ca2+ back into the SR stores, or Ca2+ is extruded and released into the extracellular space through Na+/Ca2+ exchanger (NCX) that exchanges approximately three Na+ ions entering the cell in exchange for one Ca2+ ion leaving [2+ by generating a net inward depolarizing current, called the transient inward current (Iti). Approximately 63% of cytosolic Ca2+ are taken up by SERCA2a and around 37% Ca2+ are extruded by NCX in humans [During diastole, myofilaments relax, either due to calcium re-uptake into the SR by the SR Ca leaving . Thus, tn humans ,23.2+ into the cytosol and the rapid re-uptake or extrusion results in a Ca2+ wave, which is known as the Ca2+ transient. The amount of Ca2+ released from the SR directly correlates with the Ca2+ transient amplitude and is responsible for the strength of systolic contraction [2+ handling could result in changes of electrical stability and cardiac contractility, which may lead to malignant ventricular arrhythmias.The depolarization and release of Catraction . Consequ2+ handling. β-AR activates a crucial cascade of events, resulting in the phosphorylation of Ca2+-mediating proteins. In principal, two effects follow β-AR signaling: (1) an increased activity of LTCC and (2) an enhanced Ca2+ concentration in the SR due to stimulation of SERCA2a [2+/calmodulin-dependent protein kinase type II (CaMKII) [2+ re-uptake. On the other hand, the activity of PKA and CaMKII phosphorylates RyR2 and causes an activation of the Ca2+ releasing channel. In physiological conditions, this phosphorylation property, due to β-AR stimulation, is an important mechanism to enable SR Ca2+ release. This leads to an improvement of myocardial contractility in response to environmental stressors [Additionally, the activation of the adrenergic nervous system through β-adrenergic receptor (β-AR) stimulation has a great impact on Ca SERCA2a . Both ch SERCA2a  or Ca2+/(CaMKII) . Hence, tressors ,28. The 2+ uptake from the cytosol directly correlates with the amount of energy being produced [2+ uptake is thought to be mostly mediated by the mitochondrial Ca2+ uniporter (MCU) [2+ uptake and ATP synthesis in mitochondria is inhibited [For correct AP progression and myocardial contraction, several adenosine triphosphate (ATP)-consuming proteins, or so-called ion-pumps, such as SERCA2a (as mentioned above), are necessary. Therefore, a continuous supply of energy, in form of ATP, is required and provided by oxidative phosphorylation in myocardial mitochondria . Cardiacproduced . As the er (MCU) ,33,34. Tnhibited ,36.2+-selective protein complex that consists of the pore-forming mitochondrial Ca2+ uniporter protein [2+-dependent inactivation of LTCCs during increased dyadic cleft Ca2+ in UCP2 knock out cardiomyocytes [The MCU is a highly Ca protein ,37, the  protein ,40,41,42 protein ,43,44,45 protein ,48,49,50myocytes . 2+ efflux pathway in the heart [+:1 Ca2+ [2+ overload with consequent effects on ROS generation [+ and Ca2+ concentrations, such as HF [The mitochondrial NCX (mNCX or NCLX) constitutes the major mitochondrial Cahe heart . Similar+:1 Ca2+ . This chneration . As it ich as HF ,55,56. 2+ handling, there is still considerable debate whether mitochondrial Ca2+-uptake might affect cytosolic Ca2+-homeostasis [With respect to cytosolic Caeostasis ,45. Malignant ventricular tachyarrhythmias are caused by either (1) enhanced automaticity, (2) triggered activity or (3) reentry. The first two conditions refer to cellular phenomena, whereas the latter is a matter of the cardiac electrophysiological network ,58,59.2+-mediated premature APs that result from early or delayed afterdepolarization . These premature cellular depolarizations may provoke ventricular contraction or short runs of ventricular tachycardia (VT). The third and most common manifestation is “reentry” where one or several wave fronts circulate around zones of refractory tissue or rotate as spiral waves [Firstly, “enhanced automaticity” is the acceleration of the spontaneous firing rate of cardiomyocytes (CMs). If ventricular CMs, normally quiescent, automatically generate APs due to disease or genetic modifications, irregular activation patterns arise. These abnormal wave fronts then collide against or compete with normal waves originating from the sinoatrial node. Secondly, “triggered activity” refers to Caal waves ,60,61,622+-mediated ventricular arrhythmia and VT may either be caused by heritable mutations in cardiac ion channels and channel-interacting proteins or develop through acquired diseases of the myocardium  or due to acquired conditions, such as chronic HF, injury from myocardial ischemia or ischemia reperfusion (I/R) [2+ waves is related to the balance between the SR Ca2+ concentration and the minimum level of Ca2+, which is able to induce Ca2+ release from the SR, the so-called calcium threshold [In SCRs, Caon (I/R) . The prohreshold . RyR2 anNotably, in inherited genetic mutations, such as catecholaminergic polymorphic VT (CPVT), the fundamental step of arrhythmogenesis is SCR due to mutations in either RyR2 (CPVT-1) or CASQ2 (CPVT-2) ,66. Seve2+ re-uptake due to SERCA2a phosphorylation. However, in pathological conditions, the RyR2 channel can be chronically hyperphosphorylated. Consequently, channel opening probability increases, leading to diastolic Ca2+ release, the so-called Ca2+ leak, from the SR [2+ channel expression is highly dependent on the stage of HF [However, β-AR activity seems to be crucial for the generation of SCRs. As mentioned above, β-AR is responsible for the activation of LTCCs and constitutes a trigger for SR Cam the SR ,71. Howem the SR . On the m the SR . Of notem the SR . LTCC acge of HF ,76, as tge of HF ,78. Of nge of HF ,80,81.Furthermore, mutations and genetic defects in the genes CACNA1C, CACNB2, and CACNA2D1 (encoding components of LTCC) may result in various arrhythmogenic syndromes like the Brugada syndrome ,83, Brug2+ channel expression and function. When abnormal Ca2+ release occurs, intracellular Ca2+ levels increase, potentially leading to cytosolic Ca2+ overload. To maintain Ca2+ homeostasis counterbalancing mechanisms, such as the NCX channel, need to be enhanced [+ channel opening, resulting in a premature beat. Furthermore, if a certain threshold is reached, the electrical wave could spread to downstream cells and trigger premature contraction [In the diseased heart, due to pathophysiological alterations like HF or genetic mutations, cellular remodeling is often accompanied by changes in Caenhanced . This cuenhanced . When a enhanced . The gentraction . Of notetraction . Howevertraction .2+ handling, leading to a reduced cytosolic Ca2+ transient during excitation on the one hand but increased baseline cytosolic Ca2+ level on the other hand is a hallmark of HF. Since mitochondria are located in close proximity of the SR, they are speculated to act as a cytosolic Ca2+ sink in cardiac pathologies. The MCU has a low affinity but a high capacity to transport Ca2+. Therefore, under physiological conditions a rapid uptake of cytosolic Ca2+ into the mitochondria at the plateau phase, and not under basal conditions of the AP, is accomplished. Consequently, with increased cytosolic Ca2+ level at baseline, MCU related Ca2+ uptake could play a crucial role in balancing Ca2+ homeostasis in the failing heart [2+ overload and contribute to mitochondrial dysfunction [As mentioned above, dysregulation of Cang heart ,93. Howefunction ,95.2+ handling regulates mitochondrial energy generation, further Ca2+ dependent alterations might be suggested in mitochondria from the diseased heart. Besides ATP, also reactive oxygen species (ROS) emerge through the reaction of electrons with oxygen, forming superoxide anions (O2−) [2+. Either regulated by the inner membrane anion channel (IMAC) or the Ca2+-dependent PTP, RIRR contributes to mitochondrial dysfunction with consequent generation of an arrhythmogenic substrate [Furthermore, since mitochondrial Cans (O2−) . In healns (O2−) ,100,101 ns (O2−) . Enhanceubstrate ,103. Forubstrate .2+ handling, seem to be a hallmark of the failing heart [2+ levels remains a matter of debate [In summary, mitochondrial dysfunction, including mitochondria-initiated cell death and altered mitochondrial Cang heart . Neverthf debate ,106. 2+ imbalance is one of the main triggers for the generation of malignant ventricular arrhythmias [2+handling in the diseased heart.As summarized in the previous sections and previous reviews ,109,110,hythmias . Therefo2+ channel blockers as new drugs for the treatment of coronary diseases. Indeed, drugs affecting intracellular Ca2+handling have the ability to decrease automaticity of Ca2+ related AP generation and have emerging uses as anti-arrhythmic agents. Classical drugs that target proteins responsible for cellular Ca2+-handling include β-blockers (inhibitor of β-AR activation) [+ channel blocking properties, in mouse models and patients with CPVT it has been shown that it also inhibits RyR2 [Approximately 50 years ago, Fleckenstein  was the ivation) , and theivation)  and diltivation) ,116, as its RyR2 ,118.Unfortunately, current anti-arrhythmic drug therapy often increases or ideally has a neutral effect on cardiac-related mortality ,122,123.2+ related VT, such as gene therapy. Of note, by using various viral vectors  mediated gene transfer of Gem, resulting in a significant decreased LTCC current density in ventricular myocytes. Consequently, this approach successfully promoted shortening of APD with consequent abbreviation of electrocardiographic QTc interval [2+ transient amplitude in neonatal rats. Furthermore, the hypertrophic response in vivo and in vitro was attenuated, without affecting systolic performance [To the best of our knowledge, Muarata et al.  was the interval . In 2007interval  seized oformance .1C-subunit of LTCC. Concerning this, the so-called split-intein protein transsplicing was used. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into AD and applied to adult rat cardiomyocytes in vitro. Of note, triggered recombinant LTCC channel expression promoted Ca2+ transients and supported β-adrenergic regulation of excitation-contraction coupling [As expression dependent manipulation of large cardiac genes (>6 kb) is difficult, due to the limited packaging capacity of viral vectors (4–7 kb), improvements of the delivery approaches were anticipated. In their elegant proof of concept study, Subramanyam et al.  developecoupling .2+ reuptake by SERCA2a is commonly decreased and causes major defects in excitation-contraction coupling [2+ back to the sarcoplasmic reticulum during diastole, promoting SERCA2a expression seems to be a promising goal in HF. Indeed, multiple preclinical studies using SERCA2a adeno-associated virus (AAV) mediated gene transfer [2+ handling is complex. Consequently, besides affecting the heart’s mechanical function, SERCA2a expression might also influence cardiac electrophysiology. Theoretically, the restoration of SERCA2a activity and prevention of cytotoxic Ca2+ overload could affect the generation of beat-to-beat repolarization alternans and consequent DADs. In diseased heart, Cacoupling . Since Stransfer ,148,149 transfer ,51 suppotransfer . Neverth2+ overload of the SR could promote a Ca2+ leak through RYR2 and contribute to an increased susceptibility to arrhythmias.Nevertheless, it remains unclear whether an increase in SERCA2a expression by gene therapy could alter the electrophysiological substrate that triggers the generation of cardiac arrhythmias. Furthermore, SERCA2a overexpression dependent Ca2+ leak through RYR2 and has suggested protection against, not promotion of, arrhythmias  [2+ release events were efficiently suppressed by AIP [Successful results were also achieved by targeting regulatory proteins like CaMKII. Of note, CaMKII dependent phosphorylation is crucial for RyR2-mediated SR CaiPSC-CMs . ImportaiPSC-CMs ,175. TheR176Q/+) . CaMKII R176Q/+) . These rd by AIP . 2+ influx [2+ dependent ventricular arrhythmias is observed [Further studies have addressed the recessive form of CPVT, namely CPVT-2. This form is characterized by mutations in CASQ2, which is an essential regulating part of the RyR2 macromolecular complex. RyR2 channels lacking the CASQ2 protein tend to open spontaneously, without the need for LTCC dependent trigger Ca+ influx . Consequobserved . In CPVTobserved . Therefo2+ transient and reduce the incidence of DADs [R33Q/R33Q (R33Q) mutation) [2+-handling. Importantly, due to successful transfection life-threatening arrhythmias have been abrogated [D307H model supported these enthusiastic results. Interestingly, in the study by Kurtzwald-Josefson et al. [In iPSCs from a patient carrying the homozygous CASQ2-G112+5X mutation, AAV9 dependent gene delivery was able to normalize cellular Ca of DADs . These outation) . Successbrogated . Furthern et al.  antiarrhR33Q/R33Q (R33Q) mutation was also able to restore defective Ca2+ handling and prevent consequent isoproterenol-triggered VT [Further studies have successfully focused on other regulatory proteins of the RyR2 complex, namely CaM. CaM gene transfer into the CPVT knock-in mouse model carrying a CASQ2gered VT .Although transgenic mouse models overexpressing NCX have been studied for a long time ,180,181,+/K+-ATPase (NKA) activity, NCX overexpression resulted in systolic and diastolic dysfunction. These potential alterations observed in transgenic mouse model as well as in transfected CMs are probably related to decreased SR Ca2+ stores with concomitant cellular Ca2+ overload [As in most cardiac pathologies NCX activity seems to be reduced ,190,191,overload ,187. 2+ homeostasis [On the other hand, in some pathological manifestations decreased NCX activity is accompanied by reduction in SERCA2a function. Therefore, the usage of a dual therapy including simultaneous elevation of SERCA2a and NCX function seems favorable. Terracciano and colleagues  have inveostasis . 2+ that is contingent on reduced NCX function.In vitro studies downregulating NCX in rat cardiomyocytes were performed by Tadros et al. . They shA new proof-of-principle study has been published by Gammage et al. . This el2+. This vital process has major regulatory effects on mitochondrial function  but also on the whole cellular metabolism including cellular Ca2+ handling [2+ handling seems to be a promising strategy to suppress cardiac arrhythmias. This concept is supported by several studies, in which MCU activity was suppressed via pharmacological or genetical approaches, resulting in a diminished myocardial damage after I/R [2+ handling. As already described, mitochondria have the ability to transport huge amounts of cellular Cahandling ,30,31,32fter I/R ,193. Tab2+ overload with consequent permeability pore opening, leading to reduced hypoxia-reoxygenation injury indicating a potential strategy for arrhythmia prevention [Wu et al.  were theevention . 2+ uptake is decreased in diabetic cardiomyocytes [2+ handling were detected. These findings support the idea that abnormal Ca2+ handling in CMs could be restored via MCU transgene expression [Since mitochondrial Camyocytes ,198, genpression .2+ mediated ventricular arrhythmias [2+ handling as attractive targets to influence Ca2+ related ventricular arrhythmias.Further studies explored the function of UCPs. As already mentioned, the isoforms UCP2 and UCP3 are suggested to influence MCU activity ,48,49,50hythmias . Therefo+ and Ca2+ concentrations, the mitochondrial NCX (NCLX) may also constitute a promising target for gene therapy [Slc8b1). Due to mitochondrial Ca2+ overload, they observed the development of HF with consequent SCD. On the contrary, overexpression of NCLX resulted in increased mitochondrial Ca2+ clearance with reduced permeability transition formation and decreased tissue necrosis [Since HF is accompanied by alterations in Na therapy ,55,56. I therapy . Consequ therapy  addressenecrosis . Therefo2+ homeostasis could constitute a potential point of application for gene therapy. These potential therapeutic targets are summarized in the following paragraph.Besides the genetic targets mentioned above, further regulatory proteins that mediate cardiac CaThe small ubiquitin-related modifier 1 (SUMO-1) was uncovered as a crucial regulatory part of the SERCA2a complex in HF. Under physiological conditions, SUMO-1 binds to SERCA2a in a process called SUMOylation affecting the ATP-binding domain of SERCA2a. SUMOylation is followed by an increased ATP-binding affinity of SERCA2a with concomitant stabilization of the protein. However, in HF, SUMO-1 expression is decreased. Therefore, multiple studies have aimed for the expression of SUMO using genetic strategies in various HF animal models ,201,202.2+ binding protein S100 calcium-binding protein A1 (S100A1) is a further important regulator of cardiac performance. It directly interacts with SERCA2a and RyR2. Thus, it contributes to improved Ca2+ handling and contractile performance. Consequently, it plays a crucial role in the pathophysiology of HF and might provide a novel therapeutic target for treating acute and chronic cardiac dysfunction [2+ transients and SR load due to an increase in SR Ca2+ uptake and reduced SR Ca2+ leak [2+ related ventricular arrhythmias.The Cafunction . As suspa2+ leak ,206,207 2+ influx into the cytoplasm seems not only to be limited to activation of the LTCC. Interestingly, when Ca2+ is depleted from intracellular stores, such as ER, store-operated Ca2+ entry channels (SOC) open. The most important SOC channel is the Ca2+ release-activated Ca2+ (CRAC) channel, which consist of multimers of ORAI (Ca2+ release-activated Ca2+ channel protein) family proteins, there of the best characterized is ORAI1. ORAI1/CRAC channels are regulated by the stromal interaction molecule 1 (STIM1). Of note, STIM1 is an ER transmembrane protein, which is activated through ER Ca2+ store depletion [2+ cycling this protein is involved in various cellular pathologies including cancerogenesis [2+ mediated ventricular arrhythmias [The regulation of Caepletion ,212,213.ogenesis ,222,223.ogenesis ,226,227.hythmias ,229,230.2+ regulating proteins PLN [2+ handling and cardiac performance.Further potential gene therapy targets could consist of the Caeins PLN ,233,234,eins PLN ,236,237 eins PLN . In expe2+ handling is one the major reasons for congenital but also acquired malignant ventricular arrhythmia syndromes. In recent years, major progresses were achieved to understand the underlying mechanisms of Ca2+ mediated arrhythmogenesis. Consequently, various cellular and mitochondrial targets were identified. The strategy of gene transfer is able specifically to aim disease relevant proteins. Indeed, targeting cardiac Ca2+ handling by somatic gene transfer has been demonstrated to be effective in experimental models. Therefore, various studies have explored this strategy to prevent Ca2+ related arrhythmogenesis. By targeting regulatory proteins of Ca2+ handling, these investigations were already able to uncover promising results by suppressing Ca2+ related congenital but also acquired arrhythmia mechanisms. Therefore, gene transfer to suppress Ca2+ related ventricular arrhythmias seems to be an attractive strategy in in the future. Nevertheless, first investigations in humans will be necessary to uncover the efficiency of this therapeutic approach. A defect in Ca


Neurotransmitter release depends on a complex interplay of Ca2+ fluxes and Ca2+ buffering in the presynaptic terminal that is not fully understood. Here, we show that the angiotensin-receptor-associated protein (ATRAP) localizes to synaptic terminals throughout the central nervous system. In the retinal photoreceptor synapse and the cerebellar mossy fiber-granule cell synapse, we find that ATRAP is involved in the generation of depolarization-evoked synaptic Ca2+ transients. Compared to wild type, Ca2+ imaging in acutely isolated preparations of the retina and the cerebellum from ATRAP knockout mice reveals a significant reduction of the sarcoendoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity. Thus, in addition to its conventional role in angiotensin signaling, ATRAP also modulates presynaptic Ca2+ signaling within the central nervous system.Fast, precise and sustained neurotransmission requires graded Ca One of the main effectors of RAS is angiotensin-2 (AngII or Ang1–8). Perturbations in the retinal RAS change the waveforms of the electroretinogram, which suggests a role of RAS in the signal processing of the retina2. Thus, cells in the retina that express AngII receptors form the signal processing network that is regulated by RAS. AngII-sensitive cells might include a specialized subset of bipolar cells and, better investigated, a subset of ganglion cells1. Binding of AngII to its receptor angiotensin-2-receptor type-1 (AT1R) increases intracellular free Ca2+, thereby suggesting that AngII might affect the electrical activity of retinal neurons1. The angiotensin-receptor-associated protein (ATRAP) is known to interact with and modulate the cytosolic side of AT1R in the systemic RAS network4. Not surprisingly, the vast majority of studies about ATRAP’s functional role are those exclusively related to the modulatory effect of ATRAP on the angiotensin-receptor signaling pathway5. However, in the retinal pigment epithelium (RPE) ATRAP has also been shown to play a role in the Ca2+ store-dependent Ca2+ signaling, as well as in modulating AT1R-evoked Ca2+ through activation of TRPV2 channels6. More recently, Mederle et al.7 showed that ATRAP physically binds to and stimulates the cardiac sarcoendoplasmic reticulum Ca2+-ATPase 2a (SERCA2a). In addition, ATRAP interacts with other molecules such as calcium-modulating cyclophilin ligand in the endoplasmic reticulum to regulate intracellular Ca2+ fluxes8. While investigating the role of ATRAP in the retina, we found that ATRAP was abundantly present in the RPE and in the outer plexiform layer (OPL) of the retina6, a region that contains photoreceptor terminals and postsynaptic horizontal and bipolar cell dendrites. RPE cells from ATRAP knockout mice (Atrap-/-) show decreased amplitudes of AngII evoked Ca2+ signals6. Surprisingly, the role of ATRAP in the photoreceptor synapse has not been investigated yet. Since ATRAP is a protein that modulates intracellular Ca2+ signaling and because of its localization in the OPL, we here tested the hypothesis that ATRAP is involved in Ca2+ signaling at the photoreceptor synapse and potentially in other ATRAP-expressing neurons in the central nervous system.The retina expresses its own intraretinal renin-angiotensin-system (RAS) that includes all metabolites and proteases such as renin or angiotensin-converting enzyme2+ signaling in synapses contributes to triggering depolarization-dependent neurotransmitter release10. Depolarization results by action potentials that arrive at the synaptic terminals or in the photoreceptor by shut-down of the light transduction cascade in the outer segment during transition from light to darkness. The depolarization activates voltage-dependent Ca2+ channels that generate an influx of extracellular Ca2+ into the synapse, which represents the trigger for neurotransmitter release. Store-operated Ca2+ entry represents an important regulation of intracellular free Ca2+ at synaptic terminals. Store-operated Ca2+ entry results from Ca2+-dependent depletion of cytosolic Ca2+ stores which in turn activates an additional type of Ca2+ channel in the plasma membrane as a mechanism to enhance the Ca2+ signal19. At the presynapse of cerebellar interneuron-Purkinje cell junctions, ryanodine-dependent release of Ca2+ from cytosolic stores leads to large miniature inhibitory postsynaptic currents in Purkinje cells19. In the rod synapse, Ca2+ enables recruitment of non-ribbon glutamate release during sustained depolarization24. The increase in presynaptic Ca2+ concentration is terminated by the activity of the plasma membrane Ca2+-ATPase and the uptake of Ca2+ into cytosolic Ca2+ stores by the activity of sarcoplasmic Ca2+-ATPase (SERCA2)29.Cain vitro physiological studies. We found that ATRAP localizes to the synapses of both rod and cone photoreceptors in the retina and also showed abundant expression in other regions of the central nervous system such as the cerebellum. Surprisingly, we found ATRAP expression in the OPL, a region that does not express AT1R, therefore indicating a potentially complete new role for ATRAP, in synapses. Ca2+ imaging experiments demonstrated that in the retina and the cerebellum ATRAP functions as a synaptic protein that affects the magnitude and time course of the presynaptic Ca2+ signal. Our data demonstrate a novel physiological function for ATRAP in the presynaptic terminals of photoreceptors and mossy fibers of the cerebellum, where it modulates depolarization-evoked Ca2+ signals.Given the expression of ATRAP and SERCA2 in the photoreceptor cells, studying the role of ATRAP in the retina provides insights into RAS-mediated retinal function, particularly in the photoreceptor synapse. Thus, we aimed to understand the function of ATRAP and its possible interaction with SERCA in the photoreceptor synapse. To examine the role of ATRAP, we performed molecular, anatomical, and Atrap+/+) mouse retina 6. Because ATRAP has been reported to modulate angiotensin receptor signaling, we examined the expression of angiotensin receptor (AT1R) in the retina. As previously reported30, weak AT1R immunostaining by an antibody that gives no signal in AT1R knockout mouse kidney is seen in the retinal pigment epithelium but is absent from the outer plexiform layer (OPL)  Fig. , suggestAtrap−/− mice we verified that the antibody used to detect ATRAP does not produce false positive signals  cells and cardiac myocytes from ice Fig. . SERCA2 ice Fig. . Thus, p2+ signaling at photoreceptor terminals, we perform Ca2+-imaging experiments on vertical slices of acutely isolated retinae from both Atrap+/+ and Atrap−/− mice. Since our experimental setup does not allow to combine imaging with light responses, we simulate the switch from light to dark with a depolarizing stimulus. We repeatedly expose the retinae to extracellular KCl  depolarize the membrane of photoreceptors to approximately 0 mV, causing the opening of voltage-gated Ca2+ channels. We tested whether the dihydropyridine Ca2+ channel blocker isradipine had a differential effect on Ca2+ influx into photoreceptor terminals of Atrap+/+ and Atrap–/– animals. Slices were incubated with the Ca2+ indicator Fluo4-AM and subjected to extracellular application of 150 mmol/l KCl. KCl-depolarization of Atrap–/– retinal sections induces 2+ responses that are significantly smaller than in Atrap+/+ retinal sections  in Atrap–/– mice.To test whether ATRAP has an effect on Caons Fig. . Applicace  between light and dark conditions is affected in Atrap−/− mice, we compare the shape of SRs between Atrap+/+ and Atrap−/− mice following dark or light adaptation according to Fuchs et al.39. We examine several hundred electron micrographs for each genotype and illumination condition and classified SRs according to their shape into three different categories:rod-shaped, club-shaped, and spherical-shaped , while the remaining SRs appeared club-shaped (4.2% of 320 SRs). Similar results were obtained for Atrap−/− mice, with the majority of SRs being rod-shaped (97.0% of 346 SRs) and only very few club-shaped (2.4% of 346 SRs) and spherical-shaped SRs (0.6% of 346 SRs)  and an increase in the number of club-shaped  and spherical-shaped SRs   Fig. . Light aRs) Fig. .Figure 94, it might be possible that ATRAP has a more generalized function at chemical synapses of the brain. We find strong ATRAP immunolabeling in the cerebral cortex and the cerebellum of Atrap+/+ mice.  in the glomeruli formed by mossy fiber terminals and granule cell dendrites  the lack of ATRAP in RPL cells or in cardiac myocytes reduces the store-mediated Ca2+ response upon extracellular stimulation7 and 2) Ca2+ release from intracellular stores contributes to the depolarization-induced Ca2+ signaling at photoreceptor presynaptic terminals that corresponds with a transition from light to darkness25. To mimic this transition, we used pulses of extracellular high K+ concentrations that lead to photoreceptor cell depolarization. In the presence of the L-type Ca2+ channel blocker isradipine, we observed a similar reduction of the Ca2+ transients (by 85–90%) in Atrap−/− and Atrap+/+ mice. This result indicates that the level of Ca2+ signals were predominantly measured in photoreceptor terminals and that the level of depolarization together with number of activated L-type channels were equal in both Atrap−/− and Atrap+/+ mice. Photoreceptor terminals in retinae from Atrap−/− mice show a smaller and thus faster recovery of depolarization-evoked Ca2+ transients. It was shown38 that the shape of the synaptic ribbon changes between light and dark adaptation through a Ca2+ dependent mechanism. Using electron microscopy techniques, we here showed that the shape of the photoreceptor synaptic ribbons from Atrap+/+ and Atrap−/− mice under light and dark adaptation are similar. Thus, the absence of ATRAP has no influence on the synaptic ribbon, indicating that the function is not influenced as well. In addition, depolarization-evoked Ca2+ transients in retinae from Atrap−/− mice are insensitive to thapsigargin and have similar decay times to that of Atrap+/+ retinae in the presence of the SERCA blocker thapsigargin  in cerebellar Purkinje cells19. Here, the generation of large-amplitude miniature IPSCs in cerebellar Purkinje cells depends on activation of ryanodine receptors at the presynapse of interneurons18. In our experiments, inhibition of SERCA by thapsigargin reduces depolarization-evoked presynaptic Ca2+ signals in mossy fibers, further implying a contribution of Ca2+ release from stores. However, in contrast to wildtype mice, the Ca2+ transients in mossy fibers of Atrap−/− mice are insensitive to thapsigargin. This result equals to that in photoreceptor synapses from Atrap−/−mice and it is tempting to speculate that ATRAP fulfills a related role in Ca2+ signalling in cerebellar synapses. Moreover, because the cerebellum, unlike the photoreceptors, shows a broad AT1R expression46, the function of ATRAP in mossy fibers synapses could also suggest the existence of novel mechanisms of the renin-angiotensin system network in the brain.Since the expression of ATRAP is not restricted to the retina and can be found in the cortex and in the cerebellum, we analyzed KCl-evoked Ca2+ transient amplitude is unchanged in cerebellar mossy fiber cells from Atrap−/− mice. Furthermore, cardiac myocytes from Atrap−/− mice also show unchanged Ca2+ amplitudes in response to depolarization compared to those from wild-type mice7. Thus, the lack of ATRAP has very different effects on depolarization-evoked Ca2+ transients in photoreceptors, mossy fibers and cardiac myocytes. This suggests that, depending on the cell type, ATRAP-dependent modulation of SERCA2 activity affects in different ways the dynamics of Ca2+ signaling.In contrast to photoreceptor terminals, the depolarization-evoked Ca2+ signaling in synapses of the retina and the cerebellum. Since ATRAP expression localizes to different brain areas it is possible that this synaptic ATRAP function is of more broad relevance.To date, ATRAP function was assumed to be limited to modulating the AT1R-mediated angiotensin II signaling cascade. Our study shows that, in addition to its conventional role in angiotensin signaling, ATRAP may be also crucial for the Ca6. Adult male and female ATRAP knockout (Atrap−/−) mice (C57BL/6 × 129SvEv) from a local colony were used in the study4. Atrap−/− and Atrap+/+ littermates were used from heterozygous breeding pairs. All experimental procedures were performed following the guidelines approved by the Institutional Animal Care and Use Committee at University of Regensburg and the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in vision research. All animal experiments were formally approved by the German authorities .Animals and ethical approval followed the conditions as published previously47.GFAP-ECFP/Thy1-EYFP transgenic mice were bred in the animal facility of the University of Saarland in Homburg and used for confocal imaging of the cerebellum after perfusion fixation as previously describedTg(Rac3-EGFP)JZ58Gsat/Mmcd (Rac3-EGFP) mice were obtained from the Mutant Mouse Regional Resource Center, a NCRR-NIH funded strain repository, and were donated to the MMRRC by the NINDS funded GENSAT BAC transgenic project.For laser capture microdissection, unfixed eyes were prepared, frozen and sectioned at a thickness of 25 µm using a cryostat. Defined regions from the outer and inner plexiform layers were dissected and collected on the same day using the PALM Micro Beam system (Carl Zeiss MicroImaging) equipped with a nitrogen laser (337 nm). After microdissection, the samples were ejected from the object plane with a single laser shot and catapulted directly into a microtube cap  for subsequent reverse transcriptase-PCR.et al.48. Freshly micro-dissected tissue was homogenized in RLT buffer  containing 1% β-mercaptoethanol. RNA was isolated using the RNeasy Micro Kit (Qiagen). Poly(dT)- and random hexamer-primed cDNA synthesis (reverse transcriptase reaction) was performed for 30 min at 42 °C using 5x RT-buffer, a mixture of dNTPs, RNAsin  and complete RNA from microdissected tissue. PCR was performed in a volume of 25 µl using 3 µl of prepared cDNA and 0.5 µl of each primer. Cycling conditions were 40 cycles at 95 °C for 45 s, 42–65 °C for 45 s, and 72 °C for 1 min followed by a 5-min 72 °C extension step. PCR product lengths were determined on 1.5–2% agarose gels. Primers specific for mouse ATRAP taken from49 were as follows:RT-PCR was performed using the method as described by Mühlhans Primer ATRAP forward: 5′-TGCTTGGGGCAACTTCACTATC-3′Primer ATRAP reverse: 5′-ACGGTGCATGTGGTAGACGAG-3′Primers to opsin and β-actin used as controls were as follows:Primer opsin forward: 5′-CTCTTCTGCATCTTCTCT-3′Primer opsin reverse: 5′-AGGGTTTACAGATGACAA-3′Primer β-actin forward: 5′-TTCCTCCCTGGAGAAGAG-3′Primer β-actin reverse: 5′-CACTGTGTTGGCATAGAG-3′4, rabbit anti ANG II receptor , rabbit anti-calbindin  and mouse anti-calbindin , mouse anti-CtBP2 , Goα , vGluT1 , anti-SERCA2 . Since it known that anti-AT1R antibodies often lack reliability we checked every batch of antibodies by staining of kidney sections from AT1R/AT2R double knock out mice e.g. see Milenkovic et al.30. Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594  were dissolved in 1% NGS and 0.3% Triton X-100 in PB and applied for 2 h at room temperature. After final washes in PB, samples were mounted in Aqua Poly Mount  and analyzed with a Zeiss Imager Z1 equipped with an ApoTome and a LSM 701 laser scanning microscope . Projections of picture stacks were calculated with AxioVision 4.8 and Zen software (Zeiss). Note that both ATRAP and ANG II receptor antibodies used here are specific as proven by the negative staining in (Atrap−/−) knockout6 and (Atrap−/−/AT1R-R−/−) double knockout mice30, respectively.After dissection, retinas and brain slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 25 min and washed in PB for 30 min. The tissue was frozen and sectioned at a thickness of 12 µm using a cryostat. Sections were incubated in a solution containing 5% normal goat serum (NGS) and 0.3% Triton X-100 in PB for 1 h, and they were subsequently transferred for an overnight incubation to a solution containing primary antibodies. The following primary antibodies were used in separate experiments: rabbit anti-ATRAP  and cut into 100 µm thick sections with a vibratome. After blocking, sections were transferred to a solution containing primary antibody and incubated for 4 days (4 °C). After several rinses in PBS, retinal sections were incubated for 2 h at room temperature in biotinylated goat anti-rabbit IgG . Finally the sections were stained using the VectaStain ABC kit  for 1.5 hr at room temperature.We performed immunoelectron microscopy as previously published2O2. The staining reaction was stopped by Tris-HCl. Subsequently, the sections were rinsed in 0.1 M cacodylate buffer (pH 7.4), post-fixed in 2.5% (v/v) glutaraldehyde in cacodylate buffer (2 hr at 4 °C), and washed in cacodylate buffer overnight at 4 °C. The DAB reaction product was silver-intensified and treated with 0.05% (w/v) gold chloride (Sigma).After several washes in PBS and in 0.05 M Tris-HCl (pH 7.6), the sections were transferred to a solution containing 3,39-diaminobenzidine (DAB) , followed by 0.05% (v/v) DAB with 0.03% (v/v) HThe sections were then post-fixed with 2% (w/v) OsO4 in cacodylate buffer for 1 hr, dehydrated in a graded series of ethanol (30–100%), followed by propylene oxide, and flat-embedded in Epon 812 . Ultrathin sections were cut and stained with uranyl acetate. Control vibratome sections lacking primary antibodies were processed as described above. These produced no staining. Ultrathin sections were examined and photographed with a Zeiss EM10 electron microscope.39: For conventional electron microscopy, retinae were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde for 2 hours at room temperature. Tissue contrasting was carried out by incubation in 4% osmium tetroxide in cacodylate buffer  for 1.5 hours. Retinae were dehydrated using a rising ethanol series and propylene oxide. The tissue was embedded in Epon resin . Ultrathin sections (60 nm) were cut and counterstained with uranyl acetate and lead citrate in an automated Leica EM AC20 contrasting system . Image acquisition was performed using a Zeiss EM10 electron microscope  and a Gatan SC1000 Orius TM CCD camera  in combination with the Digital Micrograph 3.1 software . Images were adjusted for contrast and brightness using Adobe Photoshop CS6. For the quantification of synaptic ribbon shapes, random images of the outer plexiform layer were taken for each genotype and experimental condition. According to their shape, SRs were classified into rod-, club- and spherical-shaped.Sample preparation and image categorization was performed as described earlier2+ signals from retinal slices were recorded according to the method by Regus-Leidig et al.54. For Ca2+ imaging experiments, sagittal slices of Atrap+/+ and Atrap−/− mouse retinas were cut at 200 µm with a vibratome . Subsequently, slices were incubated for 30–60 min at 37 °C in an atmosphere of 5% CO2 / 55% O2 with 1 µM Fluo-4 AM  and 0.5 µl pluronic acid (Life Technologies) in a solution containing (in mM): 117 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 0.4 NaH2PO4, 25 NaHCO3, 15 Glucose (pH 7.4). Following incubation, slices were washed twice and immersed in an extracellular solution containing (in mM): 132 NaCl, 5.4 KCl, 5 CaCl2, 1 MgCl2, 5 Hepes, 10 glucose (pH 7.4). A Zeiss Examiner D1 microscope  equipped with a 63x water immersion objective was used to visualize regions of interest (ROIs), and the images were captured by an AxioCam Hsm camera (Zeiss). Small rectangular ROIs (~25 × 5 µm) were placed in the outer plexiform layer. Each region of interest contained several rod and cone terminals, but individual responses of rods and cones could not be distinguished within a single ROI. Therefore, each response measured within a single ROI represents an average of a small sample of both rods and cones. Photoreceptor terminals in the slice preparation were depolarized with a solution containing 150 mM KCl. By application of Nernst’s equation, we calculated the resulting membrane potential to be near 0 mV. The KCl solution was applied to the preparation with a focal perfusion system  controlled by the Patchmaster software . The tip of the perfusion system was located at a distance of ~500 µm from the tissue and it was operated with minimum pressure to rule out motion artefacts. Under these experimental conditions, a concentration of 150 mM KCl proved to reliably evoke depolarization-induced Ca2+ influx. Retinal slices were incubated with 1 µM thapsigargin for 10 min after loading with Fluo-4 AM. Imaging data were acquired with the Axiovision software  at frame rates ranging from 5 to 20 Hz. Since ratiometric imaging using Fura-2AM led to increased acquisition rates beyond the physiological time frame of synaptic responses, imaging experiments were carried out with Fluo-4 AM. Data analysis was performed with custom-made scripts using the software packages Matlab  and Origin . Time-dependent decrease of mean ΔF/F0 was fitted with a 1st order exponential function using Microcal Origin :1 is the amplitude at t = 0, τ is the time constant of decay, and y0 represents an offset.Ca2+ signal has been used throughout as control.Rundown of fluorescence in imaging experiments was estimated by repeated application of 150 mmol/l KCl to photoreceptor terminals before pharmacological treatment with isradipine or thapsigargin. The fluorescence signal usually decreased by 10–15% before a constant amplitude was obtained. The peak amplitude of the first KCl-induced Ca55, Ca2+ signals were recorded from mouse cerebellar slices. The Atrap+/+ and Atrap−/− mice were anesthetized by isofluran before decapitation, and their cerebella were removed from the skull and immersed in an ice-cold, oxygenated  slice preparation solution containing (in mM) 87 NaCl, 3 KCl, 25 NaHCO3, 1.25 NaH2PO4, 3 MgCl2, 0.5 CaCl2, 75 sucrose and 25 glucose. Sagittal slices of 300 µm were prepared with a vibratome  and transferred to a Nylon basket slice holder for incubation in artificial cerebral spinal fluid (ACSF) containing (in mM) 126 NaCl, 3 KCl, 25 NaHCO3, 15 glucose, 1.2 NaH2PO4, 1 CaCl2, and 2 MgCl2 at 34°C. The slices were allowed to recover in ACSF with continuous oxygenation for at least 0.5 h.Using the methods as previously published3, 15glucose, 1.2 NaH2PO4, 2.5 CaCl2, and 1 MgCl2. To depolarize, KCl (150 mM) in extracellular solution was applied to the cerebellar slices for 5 s by a focal custom-made application system. To block sarco-endoplasmic Ca2+-ATPase (SERCA) activity, slices were incubated with 5 µM thapsigargin for 5 min after loading with Fluo-4 AM; then 10 mM caffeine was applied for 1 min to deplete intracellular calcium. A Zeiss microscope  equipped with a 40x water immersion objective was used to visualize the region of interest and the images were captured by a QuantEM 512SC camera . Imaging acquisition was controlled by Imaging Workbench software 5.2.20.6  at 20 Hz frame rate. Data analysis was performed with custom-made scripts by Matlab  and Graphpad Prism 5.0 . All data were shown as mean ± SEM. Post-hoc Tukey one-way ANOVA was used for multiple group comparison.Before imaging, slices were incubated with 1 µM Fluo-4 AM as described for the retina. Subsequently, the slices were washed twice and immersed in extracellular solution containing (in mM) 126 NaCl, 3 KCl, 25 NaHCOAll data were given as mean and standard error of the mean. Experiments were repeated at least five times. All animal data were obtained from 5–6 animals. If not otherwise stated, test for statistical significance was performed by ANOVA. To test variability the Levene test was used. All statistical analyses were performed by GraphPad, Systat, Sigmaplot or Excel. We used ImageJ to quantify the Pearson’s correlation coefficient of immunostaining co-localization.


STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.Stromal interaction molecule 1 (STIM1) mediates extracellular Ca During skeletal EC coupling, action potential in mature skeletal muscle cells by neural stimulation reaches transverse (t)-tubule. The action potential activates dihydropyridine receptors ) in the t-tubule membrane. Ryanodine receptor 1 molecules  on the membrane of the sarcoplasmic reticulum  are subsequently opened due to the physical interactions with the active DHPRs , which represents a key step in inducing intracellular Ca2+ movement that is required for skeletal muscle contraction. These interactions allow Ca2+ movement from the SR to the cytosol through RyR1s . The Ca2+ ions from the SR are major sources of Ca2+ for skeletal muscle contraction and serve as critical switches that turn on cross-bridge cycles of skeletal muscle. Relaxation of skeletal muscle involves Ca2+-reuptake from the cytosol to the SR4. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) is responsible for Ca2+-reuptake and SERCA1a is the major adult isoform4. The efficient arrangement of DHPR, RyR1, SERCA1a, and their regulatory proteins in a triad junction is crucial for their proper functions and intermolecular interactions3. Junctophilin (JP) and mitsugumin 29 (MG29) participate in the development of the triad junction during the terminal differentiation of myoblasts (proliferative forms of satellite cells) to myotubes  in the tissue6. Various myogenic factors exert their influence to different stages of terminal differentiation, such as a higher expression level of myogenin or myosin heavy chain (MHC) and a lower expression level of MyoD at the late stage of terminal differentiation8. Overall, spatiotemporal distributions of intracellular Ca2+ ions, timely expression of myogenic factors, proper positioning of the proteins mentioned above in the triad junction of skeletal muscle are important factors for contraction and relaxation. A failure at any point induces defective skeletal muscle function such as life-threatening malignant hyperthermia9.Excitation-contraction (EC) coupling is a prerequisite for skeletal muscle contraction2+ entry (SOCE) is a Ca2+ entryway for the sustained presence of Ca2+ in the cytosol to achieve strong and durable cytosolic Ca2+ signals in most cell types12. The main SOCE-mediating proteins are stromal interaction molecule 1  in the ER membrane and Orai1 (an extracellular Ca2+ entry channel) in the plasma membrane12. During SOCE, STIM1 senses the waste of Ca2+ in the ER . This event allows the movement of STIM1s to the ER membranes that are close to the plasma membranes where STIM1s interact with Orai1s to form puncta17. During the formation of puncta, STIM1 self-oligomerizes via its EF–sterile α-motif (EF-SAM) domain, first coiled-coil (CC) domain, and/or Ca2+ release-activated Ca2+-activating domain (CAD)16. Punctum formation activates the entry of extracellular Ca2+ through Orai1 into the cytosol. The cytosolic Ca2+ level that is elevated by SOCE returns to a normal level via the import of cytosolic Ca2+ to the SR by SERCAs or the export of cytosolic Ca2+ to the extracellular area by plasma membrane Ca2+ ATPases 18. In skeletal muscle, interestingly, puncta appear during the terminal differentiation in an SR depletion-independent manner20. This is the reason for a more rapid SOCE in skeletal muscle than in other cells types. In addition to the main role of Orai1 in SOCE21, canonical-type transient receptor potential cation channels (TRPCs) participate in SOCE-mediated Ca2+ entry in skeletal muscle24. Skeletal muscle expresses TRPC1, TRPC3, TRPC4, and TRPC625. Interestingly, STIM1 plays other important roles in skeletal muscle. Via a direct interaction to DHPR, STIM1 attenuates DHPR-RyR1 coupling that is required for skeletal muscle contraction20. Similarly, suppressive activity of STIM1 on another type of voltage-gated Ca2+ channel (Cav1.2) has been reported26. STIM1 is also participates in maintaining the full activity of SERCA1a for the relaxation of skeletal muscle27.Store-operated Ca28. Muscle fibres from a mdx mouse  show increases in SOCE as well as STIM1 expression30. Patients with mutations in STIM1 show the following pathological skeletal muscle conditions: congenital and global muscular hypotonia showing a decrease in muscle tone and progressive muscular dystrophy by a loss-of-function mutation (E136X)32, muscular atrophy, tubular aggregate myopathy, and/or progressive muscle weakness by STIM1 missense mutations 33. A point mutation at R429 of STIM1 (R429C) has been reported in human patients with insufficient immunity and muscular hypotonia34. The abolishment of SOCE by the presence of R429C in T cells is thought to cause insufficient immunity in patients35. However, the pathological mechanism(s) of muscular hypotonia in patients with R429C have not yet been well addressed. Considering that various mutations in STIM1 cause the human skeletal muscle diseases mentioned above, examining the pathological effect(s) of R429C on the major functions of skeletal muscle, such as intracellular Ca2+ movement, which is needed for skeletal muscle contraction, is important and helpful in understanding the multiple physiological roles of STIM1 in skeletal muscle.The conditional deletion of STIM1 in mouse skeletal muscle results in no mediation of SOCE, rapid fatigue, and perinatal death from skeletal myopathyTo study the pathological role(s) of R429C in skeletal muscle Fig. , R429C w2+ in the SR of R429C-expressing myotubes was depleted with thapsigargin (TG) in the absence of extracellular Ca2+ to avoid extracellular Ca2+ entry during depletion, and extracellular Ca2+ was then applied to the myotubes to measure SOCE. Wild-type STIM1 enhanced SOCE. However, SOCE was not enhanced by R429C, suggesting that R429C does not mediate SOCE in skeletal muscle  than those in control or wild-type STIM1 Fig. . R429C w38. In addition, there was also no significant change in the expression level of TRPC1, TRPC3, TRPC4, or TRPC6, which are known to mediate extracellular Ca2+ entry in skeletal muscle  that induces membrane depolarization such as the one during EC coupling was applied to R429C-expressing myotubes, and intracellular Ca2+ movement by R429C was simply due to a change in RyR1 activity rather than a change in DHPR-RyR1 coupling. To examine this scenario, RyR1 activity was assessed by applying a direct agonist of RyR1 (caffeine39) to R429C-expressing myotubes  was measured by depleting the SR with TG in the absence of extracellular Ca2+  that are known to activate Drp-1 by dephosphorylating or phosphorylating Drp-1, respectively, were examined. Expressions of both calcineurin and CaMKII were also increased by R429C  that mediates mitochondrial fission was increased by R429C , it is likely that the proportion of R429C to endogenous STIM1 in the oligomers is low. Second, the oligomerization could interfere with the binding between endogenous STIM1 and DHPR, which could remove the attenuation on DHPR-RyR1 coupling by endogenous STIM1 and result in the increase of intracellular Ca2+ movement in response to KCl in R429C-expressing myotubes 46. Higher cytosolic Ca2+ level increases caffeine-sensitivity of RyR147. In the present study, cytosolic Ca2+ level was increased by R429C  was carried out using a site-directed mutagenesis kit , human STIM1 cDNA as a template (GenBank accession number: NM_003156.3), and a pair of complementary synthetic oligonucleotide primers containing the desired mutation 63. For the differentiation of skeletal myoblasts to myotubes, the myoblasts were re-plated on 96-well plates (for single-cell Ca2+ imaging of myotubes), on 6-well plates (for mitochondria observations), or on 10-cm plates (for other experiments) coated with Matrigel . After three days of culture in differentiation conditions, premature myotubes were transfected with an empty vector or cDNA encoding wild-type STIM1 or R429C  and 20 μg of cDNA per 10-cm dish, or the same ratio of components in the well of other plates) for 3 hr. Mature myotubes were either imaged, observed, or disrupted at 36 hr post-transfection for further experiments, at which time approximately 60% of the myotubes had been transfected, as estimated by the CFP signal. All reagents for the cell cultures were obtained from Invitrogen .Mouse primary skeletal myoblasts were derived from mouse skeletal muscle using a single-cell cloning method and were expanded and differentiated to myotubes, as previously described64. For the co-immunoprecipitation assay63, solubilized myotube lysate  was used. The immunoprecipitate was subjected to immunoblot assays with anti-DHPR, anti-STIM1, or anti-GFP antibody . For the immunoblot assays, the solubilized myotube lysate  was subjected to SDS-PAGE 64. Anti-RyR1, anti-SERCA1a, anti-CSQ1, anti-CaM1, anti-MG29, anti-MG53, anti-JP1, and anti-JP2 antibodies were obtained from Affinity BioReagents . Anti-TRPC1, anti-TRPC3, anti-TRPC4, and anti-TRPC6 antibodies were obtained from Alomone Laboratories . Anti-Orai1, anti-STIM1, anti-STIM2, and anti-α-actin antibodies were obtained from Abcam. Anti-Drp-1, anti-Mfn1, anti-calcineurin and anti-CaMKII antibodies were obtained from Santa Cruz Biotechnology .Mouse primary skeletal myotubes were solubilized in lysis buffer, as previously described65. qRT-PCR was performed using the following primers: MyoD (forward: 5′-GACCTGCGCTTTTTTGAGGACC-3′ and reverse: 5′-CAGGCCCACAGCAAGCAGCGAC-3′); myogenin (forward: 5′-TTGCTCAGCTCCCTCAACCAGGA-3′ and reverse: 5′- TGCAGATTGTGGGCGTCTGTAGG-3′); MHC (type II) (forward: 5′-GGCCAAAATCAAAGAGGTGA-3′ and reverse: 5′-CGTGCTTCTCCTTCTCAACC-3′); and GAPDH as a control (forward: 5′-AGGTCGGTGTGAACGGATTTG- 3′ and reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′). A SYBR Green PCR kit with Taq DNA polymerase  was used with a PCR thermocycler . The PCR was conducted under the following conditions: denaturation at 94 °C for 10 sec, annealing at 58 °C for 10 sec, and extension at 62 °C for 15 sec. Samples of 20 ng cDNA were analyzed in triplicate. The obtained values were analyzed using a software , normalized to GAPDH, and presented as the fold-increase relative to the value of the control.Total RNA was obtained from myotubes. cDNAs were synthesized from the total RNA, as previously described2+ imaging of myotubes experiments were performed using an inverted-stage microscope  equipped with a 40x oil-immersion objective (NA 1.30), a high-speed monochromator with a 75 W xenon lamp , and a 12-bit charge-coupled device camera . Mouse primary skeletal myotubes were loaded with 5 μM fura-2-AM (Invitrogen) for the measurement of cytosolic [Ca2+] or fluo-4-AM (Invitrogen) for other measurements in an imaging buffer  at 37 °C for 45 min, as previously described64. Either KCl (60 mM) or caffeine (20 mM) was dissolved in the imaging buffer and applied to the myotubes via an auto-perfusion system . Before starting the single-cell Ca2+ imaging, images of myotubes were captured for size comparison . The data were displayed and analysed using image acquisition and analysis software . To measure releasable Ca2+ from the SR or total cellular Ca2+ level, TG (2.5 μM) or ionomycin (5 μM) dissolved in dimethyl sulfoxide  was applied to the myotubes, respectively, in the absence of extracellular Ca2+ to avoid the effect of extracellular Ca2+ entry. For the measurement of SOCE, Ca2+ in the SR was depleted with TG (2.5 μM) in the absence of extracellular Ca2+, and once the cytosolic Ca2+ level returned to baseline, 2 mM Ca2+ was added to the myotubes to measure SOCE. To analyse the Ca2+ movement obtained from single-cell Ca2+ imaging of myotubes, the peak amplitudes, which exhibited similar increases or decreases in peak areas, were measured. For long-term Ca2+ movements such as SOCE or the response to TG or ionomycin, the areas under the curves were analysed. To analyze the initial rate of SOCE, the slope at the rising phase of SOCE was examined by a linear equation that was obtained from a linear fitting of the rising phase of SOCE for initial 15 sec.63. Reagents for single-cell Ca2+ imaging of myotubes were obtained from Sigma-Aldrich .Single-cell Ca64. The width at the thickest part of each myotube was measured using the ImageJ program software (http://imagej.nih.gov/ij/).Measurements of the width in myotubes  were performed as previously described63. For TEM observations, the myotubes were fixed, embedded in epoxy resin (Epon 812), sectioned (70–80 nm) using an ultramicrotome , and examined under TEM  at 60 kV, as previously described6. To analyse the length of mitochondria with normal, the length of mitochondria in a unit area (37.5 × 25.0 µm2) was measured using ImageJ software.For immunocytochemistry assays, mouse primary skeletal myotubes were fixed in cold methanol (−20 °C) for 30 min and were permeabilized with 0.05% Tween 20 in phosphate-buffered saline (PBS) for 1 min, as previously describedThe ATP level of the mouse primary skeletal myotubes was measured using myotube lysate and an ATP bioluminescence assay kit  according to the manufacturer’s protocol. Luminescence analysis was performed using a luminescence detector .2+], mean length of mitochondria and [ATP]. Group differences were analysed using an unpaired t-test . The differences were considered to be significant at p < 0.05. For qRT-PCR, the results are presented as the mean ± s.d. for triple experiments, and difference was considered to be considerable at more than 2-fold increase. The graphs were prepared using Origin v7 .The results are presented as the mean ± s.e.m. calculated for the number of myotubes shown in parentheses in the tables or in the figure legends. The values were normalized to the mean value of the corresponding controls, except for those of cytosolic [CaSupplementary Information


Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-β-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.Functional coupling between large-conductance Ca These motifs in the N termini selectively bind to T-tubular membranes and C-terminal transmembrane segments anchor the opposite end to the ER/SR membrane . Among t2+ microdomains, and may play significant roles in the control of Ca2+ signaling, excitability, and contractility by facilitating the functional coupling of ion channel complexes in vascular SMCs (VSMCs)  channels in caveolae. As a result, spontaneous transient outward currents (STOCs) are generated to induce membrane hyperpolarization  tissues ( (VSMCs) , 12. Forrization , 14, learization . Caveolae and SR , 16.Caveolin isoforms  are differentially expressed in tissue-dependent manners and are essential for the stability and function of caveolae . Cav3, w2+ microdomain function within JMCs. Key molecules, such as VDCC, Cav1, BKCa channels, and RyR, may be included in the functional molecular complex in JMCs. Here, fluorescent imaging based on total internal reflection fluorescence (TIRF) microscopy and whole-cell patch clamp recordings reveal that JP2 functions as a critical structural component enabling efficient localized translation of Ca2+ sparks to STOCs, thus effectively regulating resting tone in VSMCs.Based on previous findings , 12, 20,AFig. S1). Western blotting (A) and immunocytochemical staining (B) showed the abundant protein expression of JP2 and its prominent distribution along PM in mouse mesenteric artery SMCs (mMASMCs).JP2 is abundantly expressed in striated muscles such as cardiac muscles , as has been reported in airway SM (C). Moreover, the application of in situ proximity ligation assays  for JP2 and Cav1 in mMASMCs resulted in the appearance of abundant green fluorescent puncta in epifluorescent images, indicating co-localization of these two molecules within 40 nm (D). The number of puncta in MASMCs from WT mice was significantly larger than in Cav1−/− . These results suggest that JP2 and Cav1 closely interact each other in native myocytes.Based on our quantitative PCR analyses, the major Cav isoform expressed in mMASMCs was Cav1 . Some of the fluorescent particles of JP2 co-localized with those of Cav1 in mMASMCs . To investigate this interaction in more detail, the effects of 10 mm methyl-β-cyclodextrin (MβCD), which removes cholesterol from PM and disrupts caveolae, were examined . The ratio of co-localizing particles was significantly reduced by the MβCD treatment .Single molecular imaging using a TIRF microscope was performed to visualize the precise distribution of JP2 and Cav1. These two proteins in freshly isolated mMASMCs were each stained with a specific antibody in SMCs. TIRF images visualized fluorescent particles existing on PM F. Some o(X-Y)-VN) were labeled with VN . The N terminus of Cav1 (VC-Cav1) was labeled with VC. These constructs were co-expressed in HEK293 cells and visualized by confocal microscopy. The consistent detection of fluorescent signals from the reconstituted Venus protein by VN-VC coupling strongly suggested a molecular interaction between JP2 or truncated JP2 and Cav1. The deletion of 271–290 residues within the joining region  removed molecular coupling between JP2 and Cav1, as shown in Fig. 2B. Further molecular analyses using deletion mutants  revealed that 286TTTET290 was important for JP2-Cav1 binding . Collectively, these results strongly suggest that JP2 (286TTTET290) interacts with Cav1 in caveolae in mMASMCs.The specific region of JP2 responsible for the interaction with Cav1 was examined by bimolecular fluorescent complementation (BiFC) analyses . VN and  with VN A, upper.A and B) (B–D) and the number of fluorescent particles of RyR and JP2 (Table S1), suggesting that JP2 binds to RyR in a caveolar-independent manner. The possibility of the co-localization of Cav1 with RyR was also examined in mMASMCs (E and F). The ratio of co-localizing particles was significantly lower in MβCD-pretreated myocytes than in the control . These results suggest that the JP2-mediated co-localization of Cav1 and RyR depends on the integrity of the caveolar structure.The next series of experiments focused on the relationship between JP2 and RyR , which hA and B) . The appA and B) , B–D and mMASMCs , E and F2+ dynamics and the location of JP2 or Cav1 were simultaneously recorded in a mMASMC using TIRF microscopy. To prevent the excessive expression of mCherry-tagged JP2 or Cav1 in mMASMCs, the amount of cDNA for transfection was carefully adjusted as reported previously . JP2 or Cav1 labeled with mCherry was transiently expressed in primary-cultured mMASMCs, and Ca2+ sparks were recorded by fluo-4 imaging . Based on the kinetic analysis, the Ca2+ signals are identified as Ca2+ sparks : 2.4 ± 0.9 μm in mCherry-JP2-expressing cells, 2.3 ± 0.8 μm in mCherry-Cav1–expressing cells). These parameters were very similar to previous findings .2+ spark-initiating point in each movie to the fluorescent peak of the closest mCherry-JP2 particles were measured and summarized in Fig. 4B. We noted that the mean radius of mCherry-JP2 particles was 455 nm . A total of 60.2% of Ca2+ sparks (65/108 sparks from 12 cells) overlapped with mCherry-JP2 particles. Moreover, 61.1% of Ca2+ spark-generating sites (22/36 spark-generating sites from 12 cells) were located within 460 nm of the peak  points of mCherry-JP2 signals. Similarly, 47.3% of Ca2+ sparks  overlapped with the fluorescent particles of mCherry-Cav1 in mMASMCs expressing mCherry-Cav1 . The mean radius of mCherry-Cav1 was 368 nm . Based on analyses of spark-generating sites, 35.8% of the sites (24/67 from 15 cells) were located within 370 nm of the peak point  of mCherry-Cav1 signals. The density of Ca2+ spark-generating sites (sites/μm2) in the JP2 signals (JP2in) and outside of them (JP2out) was 0.276 ± 0.257 and 0.007 ± 0.007, respectively (p < 0.01). The density in the Cav1 signals (Cav1in) and the outside (Cav1out) was 0.517 ± 0.454 and 0.019 ± 0.010, respectively (p < 0.01) (Table S2). These results indicate that a large proportion of Ca2+ spark-generating sites, which may include RyR clusters on SR, closely localized with caveolae (<368 nm) and JP2 signals (<455 nm) in JMC.Two-dimensional distances from spark-generating sites detected as the CaCa channels are preferentially localized in caveolae by the interaction with Cav1  and in HEK293 cells expressing GFP-JP2 and BKCa channels . Our BiFC analyses based on VN-tagged JP2 and the VC-tagged α subunit of BKCa channels (BKα) revealed reconstructed Venus fluorescence along the PM in HEK293 cells, in which VN-JP2 and BKα-VC were both expressed . The results using truncated mutants of VN-JP2 revealed a novel molecular interaction of JP2 joining region with BKα-VC, as well as Cav1-VC in D and EFig. S2, ).Thus, to examine the exact relationship between JP2 and the BKCa channel with JP2. Immunostaining analyses with TIRF microscopy showed that BKCa channels are colocalized with JP2 in freshly isolated mMASMCs (A). We then investigated whether Cav1/caveolae contributed to the molecular complex formation of BKCa channels with JP2 using Cav1 knockout mice (Cav1−/−). The co-localization of JP2 and BKα was also detected in Cav1−/− myocytes (B). The ratio of their co-localization versus all BKα particles was significantly decreased by the Cav1 deficiency , although the number of fluorescent particles of JP2 and BKα were not altered (Table S1). The ratio of co-localizing particles versus all JP2 particles was also decreased by the Cav1 deficiency . Similar results were obtained when caveolae were disrupted by the MβCD pretreatment .We then investigated whether Cav1/caveola contributed to the molecular complex formation of the BK mMASMCs A. We themyocytes B. The ra−/−  was significantly lower than that in WT  (E). The percentage of the integrated area occupied by BKα particles versus the total TIRF image area was also calculated and compared between WT and Cav1−/− (F). The percentage of BKα in WT  was significantly smaller than that in Cav1−/− . In contrast, the number of BKα particles per unit area (100 μm2) of TIRF images and the integrated intensity of BKα particles per unit area were not significantly different between WT and Cav1−/− . These results indicate that BKCa channels are more highly concentrated within local areas (detected as single particles in this experiment) in WT than in Cav1−/−. Therefore, Cav1/caveolae are important for the accumulation of BKCa channels and form a structural basis to facilitate Ca2+ microdomain function by promoting BKCa-JP2 coupling. Moreover, FRET analyses using TIRF microscopy were performed based on acceptor photobleaching methods (I and J) (FRET) in WT myocytes: YFP-JP2 + BKα-CFP, 12.9 ± 9.1%, 37 particles in 5 cells; YFP + BKα-CFP as a negative control, −0.34 ± 6.19%, 51 particles in 11 cells, p < 0.01). This increase in BKα-CFP fluorescence intensity after bleaching was significantly attenuated in Cav1−/− myocytes . These results indicate that Cav1 facilitates the selective localization of BKCa channels in caveolae and enhances the formation of a molecular complex with JP2 in mMASMCs.The average fluorescent intensity per single BKα particle in Cav1 < 0.01) E. The pe Cav1−/− F. The pe methods , I and JI and J) . In WT mA–D).In the next series of experiments, the knockdown effects of JP2 by siRNA were examined to assess the significance of JP2 in molecular coupling between Cav1 and RyR. The mesenteric arterial tissues of mice were organ-cultured for 4 days and concomitantly treated with control siRNA (siControl) or siRNA specific for JP2 (siJP2) using reversible permeabilization procedures . WesternE and F), in a similar manner to that shown in n = 9; siJP2, 34.4 ± 6.9%, n = 8, p < 0.01; for Cav1, siControl, 48.9 ± 9.9%; siJP2, 36.6 ± 5.7%; p < 0.01, G and H), although the number of fluorescent particles of Cav1 and JP2 was not altered (Table S1). These results provide additional evidence for JP2 connecting Cav1 and RyR and, thus, linking caveolae to the SR element.In corresponding TIRF images of single myocytes isolated after the knockdown, fluorescent particles of RyR often co-localized with those of Cav1 in myocytes incorporated with siControl or siJP2 , E and F2+ signaling pathway functionally distinct from those established in striated muscles. Thus, STOCs were measured as the results of functional coupling between Ca2+ sparks and BKCa channel activity at a holding potential of −20 mV in mMASMCs under the whole cell voltage-clamp mode (A). Single mMASMCs were isolated from tissues pre-treated with siControl or siJP2 for 4 days by reversible permeabilization. The principal results obtained were summarized as amplitude histograms of STOCs in Fig. 7B. The averaged STOC amplitude was significantly smaller in siJP2-treated mMASMCs than in siControl-treated myocytes . The averaged frequency was not significantly different between siControl- and siJP2-treated myocytes . The average value of STOCs integrated for 30 s was significantly smaller in siJP2-treated mMASMCs than in siControl-treated myocytes . Although the averaged STOC amplitude and the integrated STOCs were reduced by the treatment with siJP2, the whole-cell BKCa channel current density  and amplitude of Ca2+ sparks were not affected . In contrast, the frequency and area at which Ca2+ sparks spread (FWHM) were significantly increased, and the decay (t½ and FDHM) was prolonged by siJP2 . These results are consistent with previous findings that demonstrated enhanced RyR activity and reduced Na+/Ca2+-exchanger activity in JP2 knockdown cardiac myocytes . Contractions elicited by 1 μm paxilline were significantly weaker in siJP2-treated arteries than in those treated with siControl . In contrast, there was no effect of siJP2 on 40 mm KCl-induced constrictions . These experiments were performed using artery segments, which had a similar maximal passive diameter under fully relaxed conditions . Taken together, it can be suggested that the knockdown of JP2 reduced the contribution of STOCs to the regulation of muscle tone and, thus, the susceptibility of resting tone to paxilline.To examine the contribution of JP2 to muscle contraction, the pressure myography measurements were performed in tissue preparations. The diameter of mouse mesenteric arteries pretreated with siControl or siJP2 was measured before and after the BKel block F. ContraCa channels . These molecular interactions, as well as that between JP2 and RyR . BiFC assays revealed that the 271–290 residues in the JP2 joining region were also an essential site for binding with BKCa channels as well as Cav1 . The joining region also contains some interaction domains for other ion channels, such as VDCC (Cav1.1 and Cav1.2)  , 35, TRP Cav1.2) , and RyR Cav1.2) . In smoo Cav1.2) . We thin Cav1.2) , (ii) Ry Cav1.2) , 32, and/− mouse . TherefoCa channel coupling  a decrease in Cav1-JP2 co-localization , but 2) coupling . Cav1 fachannels , 28 with altered . Thus, tmyocytes , 32. Ourmyocytes  clearly Ca channels  or mCherry-Cav1 particles (39.7%). By taking the expression efficiency of mCherry-labeled JP2 or Cav1 in mMASMCs into account, Ca2+ spark-generating sites distant from mCherry particles may be located close to non-mCherry–tagged, native JP2 or Cav1 . Because JP2 stabilizes RyR and reduces Ca2+ leakage in cardiac myocytes   . Therefo in SMCs . FurtherFig. S1). The physiological impact of the Cav1-JP2 interaction in the caveolae of MASMCs may be more significant with respect to Ca2+ handing than those of Cav3-JP1 . Thus, the Cav1-JP2 interaction in SMCs provides a distinct structural basis essential for the formation of the Ca2+-signaling pathway in JMC and for most efficient signal coupling between Ca2+ sparks and STOCs to regulate membrane potential and, consequently, muscle tone. In addition, the novel Cav1-JP2 interaction and related molecular complex formation are presumably responsible for the Ca2+-induced Ca2+-release mechanism triggered by action potentials in mMASMCs . The major physiological function of the Cav1-JP2 interaction in SMCs appears to differ from that of Cav3-JP1/JP2 in striated muscles. This distinct molecular complex in the JMCs of SMCs enable the effective conversion of Ca2+ sparks to cellular electrical signals, i.e. STOCs and membrane hyperpolarization. More generally, due to these structural and functional mechanisms essentially provided by the Cav1-JP2 interaction, Ca2+-sensitive signal molecules assembled in caveolae and in the site of Ca2+ microdomains can obtain sufficient Ca2+ without affecting other signaling pathways  137 NaCl, 5.4 KCl, 0.17 Na2HPO4, 0.44 KH2PO4, 4.2 NaHCO3, and 5.6 glucose, containing 0.4% collagenase  and 0.1% papain (Sigma) at 37 °C for 40 min. After the incubation, tissues were washed in Ca2+/Mg2+-free Hanks' solution and treated by gentle agitation with a glass pipette. Single mMASMCs were suspended in M-199 (Sigma) supplemented with 2% heat-inactivated FBS (Sigma), 20 units/ml of penicillin (Wako), and 20 μg/ml of streptomycin (Wako), and then cultured on glass-bottomed dishes for 24–48 h. All experiments were approved by the Ethics Committee of Nagoya City University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society.Mesenteric arteries were dissected from male mice or rats  (The full-length cDNAs encoding JP2 (NM_020433.4) and Cav1 (NM_001753) were labeled with fluorescent proteins  at the N terminus. BKα NM_002247) was labeled with fluorescent proteins (using the pECFP-N1 vector from Clontech Laboratories) at the C terminus. In BiFC analyses, constructs were labeled by fragments of the N (1–173: VN173) or C (155–238: VC155) termini of Venus (47 was lam): 120 KCl; 2 MgCl2, 10 EGTA, 5 Na2ATP, and 20 TES, pH 6.8. Thereafter, tissues were placed in a solution containing 10 μm siRNA  at 4 °C for 90 min and then in 10 μm siRNA-containing solution with 10 mm MgCl2 at 22 °C for 30 min. Permeabilization was reversed by placing the tissue at 22 °C for 30 min in MOPS-buffered physiological solution containing (mm): 140 NaCl, 5 KCl, 10 MgCl2, 5 glucose, and 2 MOPS, pH 7.1. Ca2+ was gradually increased over a 45-min period from nominally Ca2+ free to 0.01, 0.1, and 1.8 mm. Following the reversible permeabilization procedures, the mesenteric artery was organ-cultured for 4–5 days in M-199 with 2% FBS. After 2 days, this culture medium was refreshed.siRNA was introduced into mesenteric arteries using a reversible permeabilization procedure . In brie2+ imaging and single-molecular imaging were obtained using a TIRF imaging system , which consists of a fluorescent microscope , objective lens , EM-CCD camera , and AQUACOSMOS software  . Fluo-4/AM was excited with a 488-nm argon laser , and emissions were collected using a dual color filter cube . To observe Ca2+ sparks in a myocyte, 40 mm KCl HEPES-buffered solution (40 KCl), which contained (mm) 102.9 NaCl, 40 KCl, 2.2 CaCl2, 1.2 MgCl2, 14 glucose, and 10 HEPES (pH 7.4 with NaOH), was applied. Fluorescent signals are described as F/F0, where F is the averaged fluorescence intensity in the TIRF area during measurements, and F0 is the baseline F value obtained as the average intensity of the regions of interest (2 μm in diameter). Ca2+ images were collected at 27.3-ms exposure intervals. After Ca2+ spark measurements, mCherry-labeled proteins were excited with a 543-nm He/Ne laser (Coherent). When Ca2+ sparks occurred within 454.9 nm  or 368 nm (distance of the peak points of the nearest mCherry spots) (see x-y) and less than 200 nm (z). All experiments were performed at room temperature (25 °C).Two-dimensional Caotonics) . Regardiots) see , these tots) see . ImmunolFRET was evaluated based on the acceptor photobleaching method, in which the emission of the donor fluorophore was compared before and after the photobleaching of the acceptor , images were acquired using ×20 objective lens . The averaged fluorescence intensity of complimented Venus in the images was divided by that of the Hoechst signals. The Venus/Hoechst ratio of the mutants  was normalized to that of [4-i].Confocal images were obtained using a laser scanning confocal fluorescent microscope  equipped with a fluorescent unit (ECLIPSE Ti), objective lens , and NIS Elements software . In BiFC assay D, imagesSingle MASMCs were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). These cells were treated with 0.2% Triton X-100, 2% bovine serum albumin , and a goat polyclonal anti-JP2 antibody , rabbit polyclonal anti-Cav1 antibody , rabbit polyclonal anti-BKα antibody , or mouse monoclonal anti-RyR antibody  at 4 °C for 12 h. After washing repeatedly, the preparations were treated with Alexa 488- or Alexa 405-conjugated secondary antibodies  at room temperature for 1 h. Fluorescently labeled cells were observed using a confocal imaging system or the TIRF imaging system described above. Alexa 405 and Alexa 488 were excited with the blue diode and argon laser, respectively. The emissions of Alexa 405 and Alexa 488 were collected using CFP-HQ and YFP-HQ filter cubes, respectively.A), (ii) the antibody detected JP2 expression along with PM in immunostaining experiments (B), and (iii) siJP2 clearly decreased the band density (A and B) as well as fluorescent signal (C and D). This antibody has been used in many other studies  was proved by the following data: (i) the antibody detected a single band that corresponded to the predicted molecular mass of JP2 (∼100 kDa) in a Western blot analysis A, (ii) teriments B, and .The specificity of the anti-Cav1 antibody was validated in experiments using mesenteric artery SM cells from Cav1−/− mice.To clarify whether JP2 and Cav1 co-localize within 40 nm of mMASMCs, PLAs were performed using a PLA kit  . Single Fig. S8.Co-IP was performed using the Pierce Co-Immunoprecipitation Kit (number 26149) according to the experimental manual supplied by Thermo Scientific  . The ratm): 140 KCl, 4 MgCl2, 10 HEPES, 0.05 EGTA, and 2 Na2ATP (pH 7.2 with KOH). In BKCa channel current measurements, the pipette solution contained (mm): 140 KCl, 2.8 MgCl2, 10 HEPES, 2 Na2ATP, 5 EGTA, and 3.19 CaCl2 (pCa 6.5). pH was adjusted to 7.2 with KOH. The extracellular solution  had an ionic composition of (mm): 137 NaCl, 5.9 KCl, 2.2 CaCl2, 1.2 MgCl2, 14 glucose, and 10 HEPES. pH was adjusted to 7.4 with NaOH.Electrophysiological studies were performed using a whole-cell voltage-clamp technique with a CEZ-2400 amplifier , analog-digital converter , and pCLAMP software  . In recom): 112 NaCl, 4.7 KCl, 2.2 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 14 glucose, aerated with 95% O2-5% CO2 to obtain pH 7.4. The arterial diameter (D) was measured using a CCD camera. Vasoconstriction was calculated as follows: (1 − D/Dmax), where Dmax is the diameter measured in the presence of Ca2+- and Mg2+-free Krebs solution containing (mm): 112 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 14 glucose, and 1.1 EGTA, aerated with 95% O2, 5% CO2 to obtain pH 7.4.Endothelium-denuded mouse mesenteric arteries (4th branch) were cannulated at each end in a perfusion chamber and loaded with 60 mm Hg. They were placed in standard Krebs solution containing , followed by Tukey's test. Two-way ANOVA was applied for the analysis of FRET data in WT and Cav1−/− (J). In all cases, p values <5% (p < 0.05) were considered to be significant. The mean radius of mCherry particles was calculated by fitting histograms with the Lorenz curve.Pooled data are shown as the mean ± S.D. with the numbers of samples or cells. The significance of differences between two groups was evaluated using the Student's  Cav1−/− J. In allm as a stock solution. We confirmed that up to 0.1% of DMSO did not affect any of the response patterns.Reagents were obtained from Wako Ltd., except for MβCD (Sigma), EGTA, HEPES , and paxilline (Alomone). All hydrophobic compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1–10 mT. S., Y. S., H. Y., H. T., and Y. I. conceptualization; T. S., Y. S., H. Y., and Y. I. resources; T. S. data curation; T. S., Y. S., and Y. I. formal analysis; T. S., Y. S., H. Y., and Y. I. funding acquisition; T. S., Y. S., H. Y., H. T., and Y. I. validation; T. S. and Y. S. investigation; T. S., Y. S., H. Y., and Y. I. visualization; T. S., Y. S., H. Y., H. T., and Y. I. methodology; T. S., Y. S., and Y. I. writing-original draft; Y. S., H. Y., and Y. I. supervision; Y. S., H. Y., H. T., and Y. I. writing-review and editing; H. Y. and Y. I. project administration.