RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (Ca_V1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca²⁺ channel, and (3) voltage-sensor for slow depolarization-dependent Ca²⁺ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of Ca_V1.1. However, it is not known whether the latter function that has been attributed to Ca_V1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca²⁺ entry that is dependent on the voltage-sensing capability of Ca_V1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca²⁺ entry. Our observations provide the first evidence of modulation of this enigmatic Ca²⁺ entry pathway peculiar to skeletal muscle.     

RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (Ca_V1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca²⁺ channel, and (3) voltage-sensor for slow depolarization-dependent Ca²⁺ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of Ca_V1.1. However, it is not known whether the latter function that has been attributed to Ca_V1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca²⁺ entry that is dependent on the voltage-sensing capability of Ca_V1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca²⁺ entry. Our observations provide the first evidence of modulation of this enigmatic Ca²⁺ entry pathway peculiar to skeletal muscle.     

A CaV1.1 Ca Channel Splice Variant with High Conductance and Voltage-Sensitivity Alters EC Coupling in Developing Skeletal Muscle

The Ca²⁺ channel α_1S subunit (Ca_V1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca²⁺ release from internal stores and conducts a slowly activating Ca²⁺ current. However, this Ca²⁺ current is not essential for skeletal muscle EC coupling. Here, we identified a Ca_V1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged α_1S subunit and expressed it in dysgenic (α_1S-null) myotubes. GFP-α_1SΔ29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca²⁺ currents through GFP-α_1SΔ29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca²⁺ influx contributed substantially to the depolarization-induced Ca²⁺ transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary α₂δ-1 subunit. Thus, characterizing the Ca_V1.1Δ29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca²⁺ channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.     

Distinct Components of Retrograde CaV1.1-RyR1 Coupling Revealed by a Lethal Mutation in RyR1

The molecular basis for excitation-contraction coupling in skeletal muscle is generally thought to involve conformational coupling between the L-type voltage-gated Ca²⁺ channel (Ca_V1.1) and the type 1 ryanodine receptor (RyR1). This coupling is bidirectional; in addition to the orthograde signal from Ca_V1.1 to RyR1 that triggers Ca²⁺ release from the sarcoplasmic reticulum, retrograde signaling from RyR1 to Ca_V1.1 results in increased amplitude and slowed activation kinetics of macroscopic L-type Ca²⁺ current. Orthograde coupling was previously shown to be ablated by a glycine for glutamate substitution at RyR1 position 4242. In this study, we investigated whether the RyR1-E4242G mutation affects retrograde coupling. L-type current in myotubes homozygous for RyR1-E4242G was substantially reduced in amplitude (∼80%) relative to that observed in myotubes from normal control (wild-type and/or heterozygous) myotubes. Analysis of intramembrane gating charge movements and ionic tail current amplitudes indicated that the reduction in current amplitude during step depolarizations was a consequence of both decreased Ca_V1.1 membrane expression (∼50%) and reduced channel P_o (∼55%). In contrast, activation kinetics of the L-type current in RyR1-E4242G myotubes resembled those of normal myotubes, unlike dyspedic (RyR1 null) myotubes in which the L-type currents have markedly accelerated activation kinetics. Exogenous expression of wild-type RyR1 partially restored L-type current density. From these observations, we conclude that mutating residue E4242 affects RyR1 structures critical for retrograde communication with Ca_V1.1. Moreover, we propose that retrograde coupling has two distinct and separable components that are dependent on different structural elements of RyR1.     

Physiological and Pharmacological Modulation of the Embryonic Skeletal Muscle Calcium Channel Splice Variant CaV1.1e

Ca_V1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult Ca_V1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant Ca_V1.1a, the embryonic Ca_V1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike Ca_V1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in Ca_V1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (Ca_V1.1-null) myotubes reconstituted with either Ca_V1.1a or Ca_V1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric Ca_V1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in Ca_V1.1a had abolished the calcium currents, substitution of the II-III loop in Ca_V1.1e did not significantly reduce current amplitudes. This indicates that Ca_V1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in Ca_V1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, Ca_V1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, Ca_V1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant Ca_V1.1e in the skeletal muscles of myotonic dystrophy patients.     

The cardiac α1C subunit can support excitation-triggered Ca2+ entry in dysgenic and dyspedic myotubes

Depolarization-induced entry of divalent ions into skeletal muscle has been attributed to a process termed Excitation-Coupled Ca²⁺ Entry (ECCE), which is hypothesized to require the interaction of the ryanodine receptor (RyR1), the L-type Ca²⁺ channel (DHPR) and another unidentified cation channel. Thus, ECCE is absent in myotubes lacking either the DHPR (dysgenic) or RyR1 (dyspedic). Furthermore, ECCE, as measured by Mn²⁺ quench of Fura-2, is reconstituted by expression of a mutant DHPR α_1S subunit (SkEIIIK) thought to be impermeable to divalent cations. Previously, we showed that the bulk of depolarization-induced Ca²⁺ entry could be explained by the skeletal L-type current. Accordingly, one would predict that any Ca²⁺ current similar to the endogenous current would restore such entry and that this entry would not require coupling to either the DHPR or RyR1. Here, we show that expression of the cardiac α_1C subunit in either dysgenic or dyspedic myotubes does result in Ca²⁺ entry similar to that ascribed to ECCE. We also demonstrate that, when potentiated by strong depolarization and Bay K 8644, SkEIIIK supports entry of Mn²⁺. These results strongly support the idea that the L-type channel is the major route of Ca²⁺ entry in response to repetitive or prolonged depolarization of skeletal muscle.     

Ca2+ Binding/Permeation via Calcium Channel,CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein,CD36, and Fatty Acid Metabolism

Ca²⁺ permeation and/or binding to the skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) facilitates activation of Ca²⁺/calmodulin kinase type II (CaMKII) and Ca²⁺ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α₁ subunit of Ca_V1.1) gene that abolishes Ca²⁺ binding within the Ca_V1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that Ca_V1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Ca_v1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized Ca_V1.1-mediated pathway that regulates energy utilization in skeletal muscle.     

The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

The term excitation-coupled Ca²⁺ entry (ECCE) designates the entry of extracellular Ca²⁺ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca²⁺ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn²⁺. Here, we have examined the effects of these agents on the L-type Ca²⁺ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La³⁺, and Gd³⁺) and dantrolene all inhibited the L-type Ca²⁺ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca²⁺ channel displays permeability to Mn²⁺ in the absence of external Ca²⁺ and produces a Ca²⁺ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca²⁺ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca²⁺; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α_1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca²⁺-free solution and were unaffected by Ca²⁺ removal; and (3) after block of SR Ca²⁺ release by 200 μM ryanodine, normal myotubes still displayed a large Ca²⁺ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca²⁺ entry attributed to ECCE.     

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca²⁺ channel (Ca_V1.1) to be communicated to the type 1 ryanodine-sensitive Ca²⁺ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca_V1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α_1S subunit of Ca_V1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β_1a subunit of Ca_V1.1 is a conduit for this intermolecular communication. However, a direct role for β_1a has been difficult to test because β_1a serves two other functions that are prerequisite for conformational coupling between Ca_V1.1 and RYR1. Specifically, β_1a promotes efficient membrane expression of Ca_V1.1 and facilitates the tetradic ultrastructural arrangement of Ca_V1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca²⁺ transients without greatly affecting Ca_V1.1 targeting, intramembrane gating charge movement, or releasable SR Ca²⁺ store content. In contrast, a β_1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca²⁺ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca_V1.1 from RYR1-mediated SR Ca²⁺ release via its ability to interact with β_1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β_1a subunit in skeletal-type EC coupling.     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.     

Impaired Gating of an L-Type Ca2+ Channel Carrying a Mutation Linked to?Malignant Hyperthermia

Recently, we characterized the functional properties of a mutant skeletal muscle L-type Ca²⁺ channel (Ca_V1.1 R174W) linked to the pharmacogenetic disorder malignant hyperthermia. Although the R174W mutation neutralizes the innermost basic amino acid in the voltage-sensing S4 helix of the first conserved membrane repeat of Ca_V1.1, the ability of the mutant channel to engage excitation-contraction coupling was largely unaffected by the introduction of the bulky tryptophan residue. In stark contrast, the mutation ablated the ability of Ca_V1.1 to produce L-type current under our standard recording conditions. In this study, we have investigated the mechanism of channel dysfunction more extensively. We found that Ca_V1.1 R174W will open and conduct Ca²⁺ in response to strong or prolonged depolarizations in the presence of the 1,4-dihydropyridine receptor agonist ±Bay K 8644. From these results, we have concluded that the R174W mutation impedes entry into both mode 1(low P_o) and mode 2 (high P_o) gating states and that these gating impairments can be partially overcome by maneuvers that promote entry into mode 2.     

STAC proteins: The missing link in skeletal muscle EC coupling and new regulators of calcium channel function

Excitation-contraction coupling is the signaling process by which action potentials control calcium release and consequently the force of muscle contraction. Until recently, three triad proteins were known to be essential for skeletal muscle EC coupling: the voltage-gated calcium channel Ca_V1.1 acting as voltage sensor, the SR calcium release channel RyR1 representing the only relevant calcium source, and the auxiliary Ca_V β_1a subunit. Whether Ca_V1.1 and RyR1 are directly coupled or whether their interaction is mediated by another triad protein is still unknown. The recent identification of the adaptor protein STAC3 as fourth essential component of skeletal muscle EC coupling prompted vigorous research to reveal its role in this signaling process. Accumulating evidence supports its possible involvement in linking Ca_V1.1 and RyR1 in skeletal muscle EC coupling, but also indicates a second, much broader role of STAC proteins in the regulation of calcium/calmodulin-dependent feedback regulation of L-type calcium channels.     

Calciseptine, a Ca2+ Channel Blocker, Has Agonist Actions on L-type Ca2+ Currents of Frog and Mammalian Skeletal Muscle

Calciseptine is a natural peptide consisting of 60 amino acids with four disulfide bonds. The peptide is a natural L-type Ca²⁺-channel blocker in heart and other systems, but its actions in skeletal muscle have not been previously described. The aim of this study is to characterize the effects of calciseptine on L-type Ca²⁺ channels of skeletal muscle and on contraction. Whole-cell, patch-clamp experiments were performed to record Ca²⁺ currents (I _Ca) from mouse myotubes, whereas Vaseline-gap voltage-clamp experiments were carried out to record I _Ca from frog skeletal muscle fibers. We found that calciseptine acts as a channel agonist in skeletal muscle, increasing peak I _Ca by 37% and 49% in these two preparations. Likewise, the peptide increased intramembrane charge movement, though it had little effect on contraction. The molecular analysis of the peptide indicated the presence of a local, electrostatic potential that resembles that of the 1,4-dihydropyridine agonist Bay K 8644. These observations suggest that calciseptine shares the properties of 1,4-dihydropyridine derivatives in modulating the permeation of divalent cations through L-type channels. Received: 18 December 2000/Revised: 16 July 2001     

Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+-release Channel RyR1 by S-Palmitoylation

The ryanodine receptor/Ca²⁺-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca²⁺ release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca²⁺ channel Ca_V1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in “hot spot” regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca²⁺ release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca²⁺-ATPase 1A and the α_1S subunit of the L-type Ca²⁺ channel Ca_V1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca²⁺ flux in skeletal muscle that mediates excitation-contraction coupling.     

Rem inhibits skeletal muscle EC coupling by reducing the number of functional L-type Ca2+ channels

In skeletal muscle, the L-type voltage-gated Ca²⁺ channel (1,4-dihydropyridine receptor) serves as the voltage sensor for excitation-contraction (EC) coupling. In this study, we examined the effects of Rem, a member of the RGK family of Ras-related monomeric GTP-binding proteins, on the function of the skeletal muscle L-type Ca²⁺ channel. EC coupling was found to be weakened in myotubes expressing Rem tagged with enhanced yellow fluorescent protein (YFP-Rem), as assayed by electrically evoked contractions and myoplasmic Ca²⁺ transients. This impaired EC coupling was not a consequence of altered function of the type 1 ryanodine receptor, or of reduced Ca²⁺ stores, since the application of 4-chloro-m-cresol, a direct type 1 ryanodine receptor activator, elicited myoplasmic Ca²⁺ release in YFP-Rem-expressing myotubes that was not distinguishable from that in control myotubes. However, YFP-Rem reduced the magnitude of L-type Ca²⁺ current by ∼75% and produced a concomitant reduction in membrane-bound charge movements. Thus, our results indicate that Rem negatively regulates skeletal muscle EC coupling by reducing the number of functional L-type Ca²⁺ channels in the plasma membrane.     

Calcium current kinetics in young and aged human cultured myotubes

There is evidence that the complex process of sarcopenia in human aged skeletal muscle is linked to the modification of mechanisms controlling Ca²⁺ homeostasis. To further clarify this issue, we assessed the changes in the kinetics of activation and inactivation of T- and L-type Ca²⁺ currents in in vitro differentiated human myotubes, derived from satellite cells of healthy donors aged 2, 12, 76 and 86 years. The results showed an age-related decrease in the occurrence of T- and L-type currents. Moreover, significant age-dependent alterations were found in L-(but not T) type current density, and activation and inactivation kinetics, although an interesting alteration in the kinetics of T-current inactivation was observed. The T- and L-type Ca²⁺ currents play a crucial role in regulating Ca²⁺ entry during satellite cells differentiation and fusion into myotubes. Also, the L-type Ca²⁺ channels underlie the skeletal muscle excitation–contraction coupling mechanism. Thus, our results support the hypothesis that the aging process could negatively affect the Ca²⁺ homeostasis of these cells, by altering Ca²⁺ entry through T- and L-type Ca²⁺ channels, thereby putting a strain on the ability of human satellite cells to regenerate skeletal muscle in elderly people.     

The couplonopathies: A comparative approach to a class of diseases of skeletal and cardiac muscle

ConclusionsWe define a novel category of diseases of striated muscle, the couplonopathies, as those that have in common a substantial disruption of the functional unit of Ca²⁺ release for EC coupling, the couplon. Consideration of similarities and differences between the couplons of skeletal and cardiac muscle affords insights into the pathogenesis of several couplonopathies, including MH and CPVT. Specifically, we argue that the allosteric connection among couplon proteins Ca_V1.1 and RyR1 is required for the MH phenotype usually linked to mutations in the RyR channel to also associate with mutations in Ca_V1.1. As an extension of this idea, we propose that the same allosteric interaction underpins the beneficial effects of dantrolene. The absence of a corresponding mechanical connection in cardiac muscle explains the absence of CPVT diseases caused by mutations in Ca_V1.2. Based on mechanistic considerations applicable to both couplons, we identify the plasmalemma as a site of alterations in transport properties, typically consisting of an increase in store-operated calcium entry, secondary to couplon mutations that promote Ca²⁺ release. These secondary changes constitute significant factors in the pathogenesis of MH. Mutations in triadin and calsequestrin have tissue-specific consequences: in the heart they cause couplonopathies associated with either loss of the allosteric control putatively exerted by these proteins on the Ca²⁺ release channel or loss of Ca²⁺ buffer capacity in the SR. In skeletal muscle, the phenotypes are milder or nonexistent because of the narrower range of physiological [Ca²⁺]_SR visited during function, as well as the much greater functional reserve of Ca storage that is present in this tissue. Finally, the effects of variants or ablation of JP-45 demonstrate a control of the DHPR that is unique to skeletal muscle and may be prescribed by the separate channel and sensor functions of the skeletal muscle DHPR.     

Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating

The voltage-gated calcium channel Ca_V1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of Ca_V1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire Ca_V1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for Ca_V1.1 and Ca_V1.2 L-type calcium channels. Moreover, in Ca_V1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-Ca_V1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V_1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-Ca_V1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V_1/2, whereas no further shift of V_1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in Ca_V1.1.     

Differential Effects of RGK Proteins on L-Type Channel Function in Adult Mouse Skeletal Muscle

Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small GTP-binding proteins profoundly inhibit L-type Ca²⁺ channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of P_o without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca²⁺ channel (Ca_V1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of Ca_V1.1’s voltage-sensors, whereas Rad utilizes at least one mode that limits voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of Ca_V1.1.