Checking your SOCCs and feet: the molecular mechanisms of Ca2+ entry in skeletal muscle

It has long been known that skeletal muscle contraction persists in the absence of extracellular Ca²⁺. Nevertheless, recent evidence indicates that multiple distinct Ca²⁺ entry pathways exist in skeletal muscle: one active at negative potentials that requires store depletion (store-operated calcium entry or SOCE) and a second that is independent of store depletion and is activated by depolarization (excitation-coupled calcium entry or ECCE). This review highlights recent findings regarding the molecular identity, subcellular localization, and inter-relationship between SOCE and ECCE in skeletal muscle. The respective roles of ryanodine receptors (RyRs), dihydropyridine receptors (DHPRs), inositol-1,4,5-trisphosphate receptors (IP₃Rs), canonical transient receptor potential channels (TRPCs), STIM1 Ca²⁺ sensor proteins, and Orai1 Ca²⁺ permeable channels in mediating SOCE and ECCE in skeletal muscle are discussed. Differences between SOCE and ECCE in skeletal muscle with Ca²⁺ entry mechanisms in non-excitable cells are also reviewed. Finally, potential physiological roles for SOCE and ECCE in skeletal muscle development and function, as well as other currently unanswered questions and controversies in the field are also considered.     

Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels

Agonist-activated Ca²⁺ signals in non-excitable cells are profoundly influenced by calcium entry via both store-operated and store-independent conductances. Recent studies have demonstrated that STIM1 plays a key role in the activation of store-operated conductances including the Ca²⁺-release-activated Ca²⁺ (CRAC) channels, and that Orai1 comprises the pore-forming component of these channels. We recently demonstrated that STIM1 also regulates the activity of the store-independent, arachidonic acid-regulated Ca²⁺ (ARC) channels, but does so in a manner entirely distinct from its regulation of the CRAC channels. This shared ability to be regulated by STIM1, together with their similar biophysical properties, suggested that these two distinct conductances may be molecularly related. Here, we report that whilst the levels of Orai1 alone determine the magnitude of the CRAC channel currents, both Orai1 and the closely related Orai3 are critical for the corresponding currents through ARC channels. Thus, in cells stably expressing STIM1, overexpression of Orai1 increases both CRAC and ARC channel currents. Whilst similar overexpression of Orai3 alone has no effect, ARC channel currents are specifically increased by expression of Orai3 in cells stably expressing Orai1. Moreover, expression of a dominant-negative mutant Orai3, either alone or in cells expressing wild-type Orai1, profoundly and specifically reduces currents through the ARC channels without affecting those through the CRAC channels, and siRNA-mediated knockdown of either Orai1 or Orai3 markedly inhibits ARC channel currents. Importantly, our data also show that the precise effects observed critically depend on which of the three proteins necessary for effective ARC channel activity (STIM1, Orai1 and Orai3) are rate limiting under the specific conditions employed.     

The molecular physiology of CRAC channels

Summary:  The Ca²⁺release-activated Ca²⁺ (CRAC) channel is a highly Ca²⁺-selective store-operated channel expressed in T cells, mast cells, and various other tissues. CRAC channels regulate critical cellular processes such as gene expression, motility, and the secretion of inflammatory mediators. The identification of Orai1, a key subunit of the CRAC channel pore, and STIM1, the endoplasmic reticulum (ER) Ca²⁺ sensor, have provided the tools to illuminate the mechanisms of regulation and the pore properties of CRAC channels. Recent evidence indicates that the activation of CRAC channels by store depletion involves a coordinated series of steps, which include the redistributions of STIM1 and Orai1, direct physical interactions between these proteins, and conformational changes in Orai1, culminating in channel activation. Additional studies have revealed that the high Ca²⁺ selectivity of CRAC channels arises from the presence of an intrapore Ca²⁺ binding site, the properties of which are finely honed to occlude the permeation of the much more prevalent Na⁺. Structure-function studies have led to the identification of the potential pore-binding sites for Ca²⁺, providing a firm framework for understanding the mechanisms of selectivity and gating of the CRAC channel. This review summarizes recent progress in understanding the mechanisms of CRAC channel activation, pore properties, and modulation.     

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca²⁺ release activated Ca²⁺ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca²⁺ sensor required for activation of CRAC current ( I _CRAC) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca²⁺ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I _CRAC. In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I _CRAC. The results show that cells expressing low Orai1 : STIM1 ratios produce I _CRAC with strong fast Ca²⁺-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I _CRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca²⁺, Ba²⁺ and Sr²⁺ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca²⁺ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.     

The molecular architecture of the arachidonate-regulated Ca2+-selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits

The activation of Ca²⁺ entry is a critical component of agonist-induced cytosolic Ca²⁺ signals in non-excitable cells. Although a variety of different channels may be involved in such entry, the recent identification of the STIM and Orai proteins has focused attention on the channels in which these proteins play a key role. To date, two distinct highly Ca²⁺-selective STIM1-regulated and Orai-based channels have been identified – the store-operated CRAC channels and the store-independent arachidonic acid activated ARC channels. In contrast to the CRAC channels, where the channel pore is composed of only Orai1 subunits, both Orai1 and Orai3 subunits are essential components of the ARC channel pore. Using an approach involving the co-expression of a dominant-negative Orai1 monomer along with different preassembled concatenated Orai1 constructs, we recently demonstrated that the functional CRAC channel pore is formed by a homotetrameric assembly of Orai1 subunits. Here, we use a similar approach to demonstrate that the functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. Expression of concatenated pentameric constructs with this stoichiometry results in the appearance of large currents that display all the key biophysical and pharmacological features of the endogenous ARC channels. They also replicate the essential regulatory characteristics of native ARC channels including specific activation by low concentrations of arachidonic acid, complete independence of store depletion, and an absolute requirement for the pool of STIM1 that constitutively resides in the plasma membrane.     

ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond

Summary:  Store-operated Ca²⁺ entry (SOCE) is a mechanism used by many cells types including lymphocytes and other immune cells to increase intracellular Ca²⁺ concentrations to initiate signal transduction. Activation of immunoreceptors such as the T-cell receptor, B-cell receptor, or Fc receptors results in the release of Ca²⁺ ions from endoplasmic reticulum (ER) Ca²⁺ stores and subsequent activation of plasma membrane Ca²⁺ channels such as the well-characterized Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Two genes have been identified that are essential for SOCE: ORAI1 as the pore-forming subunit of the CRAC channel in the plasma membrane and stromal interaction molecule-1 (STIM1) sensing the ER Ca²⁺ concentration and activating ORAI1-CRAC channels. Intense efforts in the past several years have focused on understanding the molecular mechanism of SOCE and the role it plays for cell functions in vitro and in vivo . A number of transgenic mouse models have been generated to investigate the role of ORAI1 and STIM1 in immunity. In addition, mutations in ORAI1 and STIM1 identified in immunodeficient patients provide valuable insight into the role of both genes and SOCE. This review focuses on the role of ORAI1 and STIM1 in vivo , discussing the phenotypes of ORAI1- and STIM1-deficient human patients and mice.     

Capacitative calcium entry: from concept to molecules

Summary:  Rapid to moderately rapid changes in intracellular Ca²⁺ concentration, or Ca²⁺ signals, control a variety of critical cellular functions in the immune system. These signals are comprised of Ca²⁺ release from intracellular stores coordinated with Ca²⁺ influx across the plasma membrane. The most common mechanisms by which these two modes of signaling occur is through inositol 1,4,5-trisphosphate (IP₃)-induced release of Ca²⁺ from the endoplasmic reticulum (ER) and store-operated Ca²⁺ entry across the plasma membrane. The latter process was postulated over 20 years ago, and in just the past few years, the key molecular players have been discovered: STIM proteins serve as sensors of Ca²⁺ within the ER which communicate with and activate plasma membrane store-operated channels composed of Orai subunits. The process of store-operated Ca²⁺ entry provides support for oscillating Ca²⁺ signals from the ER and also provides direct activator Ca²⁺ that signals to a variety of downstream effectors.     

Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells

We have investigated the nature of the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca²⁺ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca²⁺]_i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca²⁺. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca²⁺ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca²⁺ extrusion with a high concentration of Gd³⁺, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca²⁺]_i oscillations, and this was reversed by use of high concentrations of Gd³⁺; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca²⁺ entry in HEK293 cells. These findings indicate that the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in HEK293 cells depends upon the Ca²⁺ sensor, Stim1, and calcium release-activated Ca²⁺ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.     

The functional network of ion channels in T lymphocytes

Summary:  For more than 25 years, it has been widely appreciated that Ca²⁺ influx is essential to trigger T-lymphocyte activation. Patch clamp analysis, molecular identification, and functional studies using blockers and genetic manipulation have shown that a unique contingent of ion channels orchestrates the initiation, intensity, and duration of the Ca²⁺ signal. Five distinct types of ion channels – Kv1.3, KCa3.1, Orai1+ stromal interacting molecule 1 (STIM1) [Ca²⁺-release activating Ca²⁺ (CRAC) channel], TRPM7, and Cl_swell– comprise a network that performs functions vital for ongoing cellular homeostasis and for T-cell activation, offering potential targets for immunomodulation. Most recently, the roles of STIM1 and Orai1 have been revealed in triggering and forming the CRAC channel following T-cell receptor engagement. Kv1.3, KCa3.1, STIM1, and Orai1 have been found to cluster at the immunological synapse following contact with an antigen-presenting cell; we discuss how channels at the synapse might function to modulate local signaling. Immuno-imaging approaches are beginning to shed light on ion channel function in vivo . Importantly, the expression pattern of Ca²⁺ and K⁺ channels and hence the functional network can adapt depending upon the state of differentiation and activation, and this allows for different stages of an immune response to be targeted specifically.     

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca²⁺ stores, following the release of Ca²⁺ during intracellular signalling, triggers the Ca²⁺ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca²⁺]_i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca²⁺ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca²⁺ entry in the form of motes.     

CRAC channels and Ca2+ signaling in mast cells

Summary:  Mast cells are integral members of the immune system. Upon activation by a rise in cytoplasmic Ca²⁺, they release a battery of paracrine signals, chemokines, and cytokines, which help sculpt the subsequent immune response. Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels in the plasma membrane is central for driving most of these responses. The molecular basis of the CRAC channel has been identified, with Orai1 forming the channel pore. Recent work has revealed that a range of mast cell responses are activated by spatially restricted Ca²⁺ signals just below the plasma membrane. These Ca²⁺ microdomains can activate cytosolic enzymes, leading to the generation of intracellular messengers as well as proinflammatory molecules like LTC₄. In this review, we describe key features of CRAC channels in mast cells, how they generate local Ca²⁺ signals, and how the cell can decode these restricted signals to generate a raft of responses.     

Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

Unloading of endoplasmic reticulum (ER) Ca²⁺ stores activates influx of extracellular Ca²⁺ through 'store-operated' Ca²⁺ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca²⁺ stores. Here, using high resolution imaging with the membrane-associated Ca²⁺ indicator, FFP-18, it is shown that store-operated Ca²⁺ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane–ER junctions. In the absence of extracellular Ca²⁺, depletion of ER Ca²⁺ stores using cyclopiazonic acid, an ER Ca²⁺-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca²⁺ causes localized Ca²⁺ influx that first increases [Ca²⁺]_SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs , markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca²⁺ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca²⁺ stores.     

Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca²⁺ sensor stromal interacting molecule 1 (STIM1) and the Ca²⁺ channel Orai1 mediate the ubiquitous store-operated Ca²⁺ entry (SOCE) pathway activated by depletion of internal Ca²⁺ stores and mediated through the highly Ca²⁺-selective, Ca²⁺ release-activated Ca²⁺ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca²⁺ currents regulated by either arachidonate or its metabolite, leukotriene C₄. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca²⁺ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.     

Cell proliferation depends on mitochondrial Ca2+ uptake: inhibition by salicylate

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca²⁺ influx pathway involved in control of multiple cellular and physiological processes including cell proliferation. Recent evidence has shown that SOCE depends critically on mitochondrial sinking of entering Ca²⁺ to avoid Ca²⁺-dependent inactivation. Thus, a role of mitochondria in control of cell proliferation could be anticipated. We show here that activation of SOCE induces cytosolic high [Ca²⁺] domains that are large enough to be sensed and avidly taken up by a pool of nearby mitochondria. Prevention of mitochondrial clearance of the entering Ca²⁺ inhibited both SOCE and cell proliferation in several cell types including Jurkat and human colon cancer cells. In addition, we find that therapeutic concentrations of salicylate, the major metabolite of aspirin, depolarize partially mitochondria and inhibit mitochondrial Ca²⁺ uptake, as revealed by mitochondrial Ca²⁺ measurements with targeted aequorins. This salicylate-induced inhibition of mitochondrial Ca²⁺ sinking prevented SOCE and impaired cell growth of Jurkat and human colon cancer cells. Finally, direct blockade of SOCE by the pyrazole derivative BTP-2 was sufficient to arrest cell growth. Taken together, our results reveal that cell proliferation depends critically on mitochondrial Ca²⁺ uptake and suggest that inhibition of tumour cell proliferation by salicylate may be due to interference with mitochondrial Ca²⁺ uptake, which is essential for sustaining SOCE. This novel mechanism may contribute to explaining the reported anti-proliferative and anti-tumoral actions of aspirin and dietary salicylates.     

钙离子感受蛋白STIM1与Orai1在钙敏感受体介导钙内流及NO生成中的相互作用

目的:研究Ca~(2+)感受蛋白[基质相互作用分子1(STIM1)与钙释放激活钙通道蛋白1(Orai1)]在人脐静脉内皮细胞(HUVECs)钙敏感受体(Ca SR)介导的钙内流和一氧化氮(NO)生成中的相互作用。方法:将Ca SR激动剂精胺[钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)均激活]单用或与ROC模拟剂12-O-十四烷酰佛波醇-13-醋酸酯(TPA)+Ca SR负性变构调节剂Calhex 231(激活ROC、阻断SOC)、蛋白激酶C(PKC)抑制剂Ro 31-8220和PKCα/β1选择性抑制剂Go 6976(激活SOC、阻断ROC)联合孵育HUVECs;利用免疫荧光技术检测HUVECs中STIM1和Orai1的蛋白表达和共定位;免疫共沉淀法检测STIM1和Orai1之间的相互作用;取2~3代HUVECs随机分为特异性的质粒转染组(sh STIM1+sh Orai1组)、空质粒组(vehicle-STIM1+vehicle-Orai1组)和未转染组(control组),将3组细胞分别加入上述4组不同药物刺激,采用荧光探针Fura-2/AM检测HUVECs中Ca~(2+)浓度的变化,NO荧光探针DAF-FM DA负载方法同步检测HUVECs中NO生成的变化。结果:STIM1和Orai1蛋白表达共定位于胞浆,与control组相比,加入Calhex 231+TPA、Ro 31-8220和Go 6976刺激后,STIM1和Orai1在胞浆中的定位均减少,且二者的相互作用均减弱;在4种不同处理因素作用下,sh STIM1+sh Orai1组细胞内Ca~(2+)浓度和NO净荧光强度均明显降低(P0.05)。结论:STIM1与Orai1以二元复合物的形式共同调节Ca SR,并通过激活SOC和ROC介导钙内流及NO生成。     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

Sarcoplasmic reticulum Ca2+ release and depletion fail to affect sarcolemmal ion channel activity in mouse skeletal muscle

In skeletal muscle, sarcoplasmic reticulum (SR) Ca²⁺ depletion is suspected to trigger a calcium entry across the plasma membrane and recent studies also suggest that the opening of channels spontaneously active at rest and possibly involved in Duchenne dystrophy may be regulated by SR Ca²⁺ depletion. Here we simultaneously used the cell-attached and whole-cell voltage-clamp techniques as well as intracellular Ca²⁺ measurements on single isolated mouse skeletal muscle fibres to unravel any possible change in membrane conductance that would depend upon SR Ca²⁺ release and/or SR Ca²⁺ depletion. Delayed rectifier K⁺ single channel activity was routinely detected during whole-cell depolarizing pulses. In addition the activity of channels carrying unitary inward currents of ∼1.5 pA at −80 mV was detected in 17 out of 127 and in 21 out of 59 patches in control and mdx dystrophic fibres, respectively. In both populations of fibres, large whole-cell depolarizing pulses did not reproducibly increase this channel activity. This was also true when, repeated application of the whole-cell pulses led to exhaustion of the Ca²⁺ transient. SR Ca²⁺ depletion produced by the SR Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) also failed to induce any increase in the resting whole-cell conductance and in the inward single channel activity. Overall results indicate that voltage-activated SR Ca²⁺ release and/or SR Ca²⁺ depletion are not sufficient to activate the opening of channels carrying inward currents at negative voltages and challenge the physiological relevance of a store-operated membrane conductance in adult skeletal muscle.