Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with EGTA and A23187. Ca²⁺ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca²⁺ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca²⁺ indicator, demonstrated that verapamil inhibited Ca²⁺ release from them induced by valinomycin in the presence of K⁺ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca²⁺ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.     

Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid worm,Phascolosoma scolops

Summary Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K⁺-contracture was dependent on external Ca²⁺ concentration and the contracture disappeared in Ca²⁺-free solution. The K⁺-contracture was suppressed by application of procaine and Mn²⁺. Caffeine induced contracture even when external Ca²⁺ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO₄ solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca²⁺ as well as by the release of Ca²⁺ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca²⁺ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate.2. Plots relating the initial rate of calcium ion (Ca²⁺) uptake and the decrease in membrane potential (Δψ) to the Ca²⁺ concentration show a half-maximal change at less than 10 μM Ca²⁺ and a saturation above 20 μM Ca²⁺.3. Plots relating the initial rate of Ca²⁺ uptake to Δψ are linear.4. Addition of Ca²⁺ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca²⁺ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca²⁺ efflux.5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca²⁺ ion is transferred across the membrane with one net positive charge in present conditions.     

Oxysterols and calcium signal transduction

Ionised calcium (Ca²⁺) is a key second messenger, regulating almost every cellular process from cell death to muscle contraction. Cytosolic levels of this ion can be increased via gating of channel proteins located in the plasma membrane, endoplasmic reticulum and other membrane-delimited organelles. Ca²⁺ can be removed from cells by extrusion across the plasma membrane, uptake into organelles and buffering by anionic components. Ca²⁺ channels and extrusion mechanisms work in concert to generate diverse spatiotemporal patterns of this second messenger, the distinct profiles of which determine different cellular outcomes. Increases in cytoplasmic Ca²⁺ concentration are one of the most rapid cellular responses upon exposure to certain oxysterol congeners or to oxidised low-density lipoprotein, occurring within seconds of addition and preceding increases in levels of reactive oxygen species, or changes in gene expression. Furthermore, exposure of cells to oxysterols for periods of hours to days modulates Ca²⁺ signal transduction, with these longer-term alterations in cellular Ca²⁺ homeostasis potentially underlying pathological events within atherosclerotic lesions, such as hyporeactivity to vasoconstrictors observed in vascular smooth muscle, or ER stress-induced cell death in macrophages. Despite their candidate roles in physiology and disease, little is known about the molecular mechanisms that couple changes in oxysterol concentrations to alterations in Ca²⁺ signalling. This review examines the ways in which oxysterols could influence Ca²⁺ signal transduction and the potential roles of this in health and disease.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca²⁺ concentration, [Ca²⁺]_i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca²⁺-activated K⁺ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca²⁺]_i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca²⁺-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca²⁺]_i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca²⁺]_i and of cell migration. Thus, BK channels function to maintain elevations in [Ca²⁺]_i needed to sustain increases in DBTRG cell migration.     

Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca²⁺ flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca²⁺ channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca²⁺ channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca²⁺ entry in VSM is described in the present review.     

Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca²⁺ flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca²⁺ channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca²⁺ channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca²⁺ entry in VSM is described in the present review.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Longitudinal smooth muscle of the mammalian intestine

Ca²⁺ mobilization in muscle cells from the circular muscle layer of the mammalian intestine is mediated by IP₃-dependent Ca²⁺ release. Ca²⁺ mobilization in muscle from the adjacent longitudinal muscle layer involves a distinct, phosphoinositide-independent pathway. Receptors for contractile agonists in longitudinal muscle cells are coupled via a pertussis toxinsensitive G protein to activation of PLA₂ and formation of arachidonic acid (AA). The latter activates Cl⁻ channels resulting in depolarization of the plasma membrane and opening of voltage-sensitive Ca²⁺ channels. Ca²⁺ influx via these channels induces Ca²⁺ release by activating sarcoplasmic ryanodine receptor/Ca²⁺ channels. The increase in [Ca²⁺]_i activates membrane-bound ADP ribosyl cyclase, and the resultant formation of cADPR enhances Ca²⁺-induced Ca²⁺ release.     

Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52

The Ca²⁺-ATPase of cardiac muscle cells transports Ca²⁺ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca²⁺ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22-Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore. ¹³C MAS NMR measurements are also presented on phospholamban that is 1-¹³C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca²⁺ regulation that may involve phospholamban as both an inhibitor of the Ca²⁺ ATPase and as an ion channel.     

The role of calcium ions in the maintenance of resting potentials and ionic gradients of mollusk neurons

We studied on apple snail neurons the connection between K⁺ and Na⁺ concentration gradients, transmembrane difference of potentials, and concentrations of Ca²⁺ in the external medium. Sensitivity of the resting potential (RP) of neurons to the influence of temperature and to metabolic poisons rose considerably with a decrease of Ca²⁺ concentration in the solution surrounding a ganglion. An excess of Ca²⁺ in the external medium did not affect the RP or ion concentration in nerve cells. Removal of Na²⁺ from this solution causes hyperpolarization of the membrane which disappears when active transport of sodium ions through the membrane is suppressed. Sodium enrichment and potassium impoverishment of the neurons are observed in potassium-free solutions at 4°C. Reaccumulation of K⁺ and exclusion of Na⁺ from the solutions of 21°C depends on the concentration of Ca²⁺ in the medium. The ionic composition of the neurons is not restored upon removal of Ca²⁺ from the solution. Upon increasing the amount of Ca²⁺, movement of ions against the concentration gradients is intensified. Thus, it may be concluded that Ca²⁺ ions on the one hand participate in the maintenance of normal passive permeability of ions through the membrane, and on the other accelerate active transport of K⁺ and Na⁺ against the concentration gradients. The mechanisms of these processes are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 323–330, November–December, 1969.     

Mitochondrial Ca2+ uptake in skeletal muscle health and disease

Muscle uses Ca²⁺ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca²⁺ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca²⁺ from their surroundings, a process called mitochondrial Ca²⁺ uptake. Under physiological conditions, Ca²⁺ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca²⁺ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca²⁺ uptake could shape spatio-temporal patterns of intracellular Ca²⁺ signaling. Malfunction of mitochondrial Ca²⁺ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca²⁺ levels. Besides the sudden elevation of Ca²⁺ level induced by action potentials, Ca²⁺ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca²⁺ uptake is fast and big enough to shape intracellular Ca²⁺ signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca²⁺ inside mitochondria. This review focuses on characterization of mitochondrial Ca²⁺ uptake in skeletal muscle and its role in muscle physiology and diseases.     

Single-cell screening of cytosolic [Ca2+] reveals cell-selective action by the Alzheimer’s Aβ peptide ion channel

Interaction of the Alzheimer’s Aβ peptides with the plasma membrane of cells in culture results in chronic increases in cytosolic [Ca²⁺]. Such increases can cause a variety of secondary effects leading to impaired cell growth or cell degeneration. In this investigation, we made a comprehensive study of the changes in cytosolic [Ca²⁺] in single PC12 cells and human neurons stressed by continuous exposure to a medium containing Aβ42 for several days. The differential timing and magnitude of the Aβ42-induced increase in [Ca²⁺] reveal subpopulations of cells with differential sensitivity to Aβ42. These results suggest that the effect produced by Aβ on the level of cytosolic [Ca²⁺] depends on the type of cell being monitored. Moreover, the results obtained of using potent inhibitors of Aβ cation channels such as Zn²⁺ and the small peptide NA7 add further proof to the suggestion that the long-term increases in cytosolic [Ca²⁺] in cells stressed by continuous exposure to Aβ is the result of Aβ ion channel activity.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Ca signaling and fluid secretion by secretory cells of the airway epithelium

Cytoplasmic Ca²⁺ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca²⁺ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca²⁺. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca²⁺ as a second messenger. Changes in intracellular Ca²⁺ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca²⁺-activated K⁺ channels and Cl⁻ channels. We also review evidence of interactions of Ca²⁺ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca²⁺ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca²⁺ stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca²⁺ influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca²⁺ pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca²⁺ pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca²⁺ which was not observed in control cells. Ca²⁺ measurements indicated that resting Ca²⁺ and the rate of Ca²⁺ increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca²⁺ transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca²⁺ leak channel in skeletal muscle.