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Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as l-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature325, 717–720). In addition to “receiving” the EC coupling signal from the DHPR, RyR-1 also “transmits” a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature 380, 72–76). A similar kind of retrograde signaling (from RyRs tol-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719–722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635–2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+channel function, whereas a chimera containing adjacent RyR-1 residues (2,659–3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR.

Expression of cardiac L-type Ca 2+ channels in dysgenic myotubes results in large Ca 2+ currents and electrically evoked contractions resulting from Ca 2+ -entry dependent release of Ca 2+ from the sarcoplasmic reticulum. By contrast, expression of either P/Q-type or N-type Ca 2+ channels in dysgenic myotubes does not result in electrically evoked contractions despite producing comparably large Ca 2+ currents. In this work we examined the possibility that this discrepancy is caused by the preferential distribution of expressed L-type Ca 2+ channels in close apposition to sarcoplasmic reticulum Ca 2+ release channels. We tagged the N termini of different α 1 subunits (classes A, B, C, and S) with a modified green fluorescent protein (GFP) and expressed each of the fusion channels in dysgenic myotubes. Each GFP-tagged α 1 subunit exhibited Ca 2+ channel activity that was indistinguishable from its wild-type counterpart. In addition, expression of GFP-α 1S and GFP-α 1C in dysgenic myotubes restored skeletal- and cardiac-type excitation-contraction (EC) coupling, respectively, whereas expression of GFP-α 1A and GFP-α 1B failed to restore EC coupling of any type. Laser-scanning confocal microscopy revealed a distinct expression pattern for L-type compared with non-L-type channels. After injection of cDNA into a single nucleus, GFP-α 1S and GFP-α 1C were present in the plasmalemma as small punctate foci along much of the longitudinal extent of the myotube. In contrast, GFP-α 1A and GFP-α 1B were not concentrated into punctate foci and primarily were found adjacent to the injected nucleus. Thus, L-type channels possess a targeting signal that directs their longitudinal transport and insertion into punctate regions of myotubes that presumably represent functional sites of EC coupling.

Publisher Summary Two calcium channels, the dihydropyridine (DHP) receptor, which is a component of the surface membrane/T tubules, and the ryanodine receptor (RyR) of the SR, have been identified, purified, sequenced, and functionally characterized. This chapter describes approaches that have contributed to the understanding of the roles of DHPRs and RyRs in excitation-contraction (e-c) coupling. A major experimental strategy that has been employed is to analyze e-c coupling in developing muscle cells from mice. The rationale for this approach is that during development the components of e-c coupling are being actively assembled. A longitudinal analysis of e-c coupling during this developmental period can thus be likened to a comparative analysis of different muscle types from different species. This period of active development makes it feasible to use molecular genetic techniques to manipulate the expression of key proteins. These molecular strategies are discussed in this chapter. In addition, methods are presented for the basic physiological measurements of e-c coupling in developing muscle.

Hypokalemic periodic paralysis (HypoPP) is the most common form of the periodic paralysis. It is caused by mutations in two voltage-gated ion channels of skeletal muscle, Cav1.1 (HypoPP-1) and Nav1.4 (HypoPP-2). Almost all HypoPP-causing mutations replace positive amino acids in the S4 voltage sensor of the channels. S4 mutations in Nav1.4 channels have already been shown to conduct sodium or protons through the omega pore which cause depolarization and impair the action potential generation. A leak current conducted by muscle fibers from HypoPP-1 patients was shown; however it was not formally demonstrated to be conducted by an omega pore. A novel mutation K1245Q in S4 of domain IV of Cav1.1 was identified in a patient who expressed weakness attacks with low serum potassium suggestive of HypoPP. These attacks have also been provoked by carbohydrate application. Further investigations revealed this mutation in other affected family members. To investigate possible gating alterations in the Cav1.1-K1245Q mutation, we performed whole-cell patch clamp measurements. A GLT cell line from muscular dysgenic mice (mdg) was cultured and transiently transfected with cDNA of wild type (WT) or mutant (K1245Q) Cav1.1. Calcium current amplitudes and voltage dependencies of activation of WT and K1245Q did not show significant differences in absolute or relative values. This data does not explain the patients pathogenesis. Omega currents produced by HypoPP Nav1.4 mutations might also exist in this Cav1.1-K1245Q mutant which could contribute to the phenotype of the patients. The corresponding measurements are currently performed, the results will be presented.

In skeletal muscle, CaV1.1 serves dual functions as the voltage sensor for excitation-contraction (EC) coupling and as an L-type Ca2+ channel. It has been long established that L-type Ca2+ current activates at potentials ∼20 mV more depolarized than intramembrane charge movement or EC coupling. We recently characterized a CaV1.1 mutation (R174W) which affects the innermost basic residue of the voltage-sensing S4 helix of Repeat I and results in malignant hyperthermia susceptibility in humans. Interestingly, R174W abolishes activation of the L-type current in response to 200 ms depolarizations without affecting the voltage dependence of intramembrane charge movement or of SR Ca2+ release. In this study, we have made corresponding mutations in Repeats II (K537W), III (R906W) and IV (K1245W) and expressed these mutants in dysgenic (CaV1.1 null) myotubes. Confocal imaging of affixed YFP tags indicated that all the mutants were correctly targeted to membrane-SR junctions. In striking contrast to the virtual loss of channel function observed with R174W, the K537W and K1245W mutants produced enormous L-type currents with peak amplitudes nearly 8-fold greater than wild-type CaV1.1 and greatly impaired deactivation. Even though these two gain of channel function mutants activated at substantially more hyperpolarized potentials (∼20 mV shift), the voltage-dependence of charge movement and SR Ca2+ release were largely unaffected. On the other hand, R906W produced currents of similar amplitude to wild-type CaV1.1 but with a consistent ∼15 mV depolarizing shift in activation. We are currently investigating the effects of the R906W mutation on the voltage dependence of charge movement and SR Ca2+ release in order to determine whether RIII plays a more critical role in engaging EC coupling than the other conserved Repeats of CaV1.1. Supported by NIH AR055104 (KGB) and AG038778 (RAB), and MDA176448 (KGB).

The multisubunit (α 1S , α 2 /δ, β 1 , and γ) skeletal muscle dihydropyridine receptor transduces transverse tubule membrane depolarization into release of Ca 2+ from the sarcoplasmic reticulum, and also acts as an L-type Ca 2+ channel. The α 1S subunit contains the voltage sensor and channel pore, the kinetics of which are modified by the other subunits. To determine the role of the β 1 subunit in channel activity and excitation-contraction coupling we have used gene targeting to inactivate the β 1 gene. β 1 -null mice die at birth from asphyxia. Electrical stimulation of β 1 -null muscle fails to induce twitches, however, contractures are induced by caffeine. In isolated β 1 -null myotubes, action potentials are normal, but fail to elicit a Ca 2+ transient. L-type Ca 2+ current is decreased 10- to 20-fold in the β 1 -null cells compared with littermate controls. Immunohistochemistry of cultured myotubes shows that not only is the β 1 subunit absent, but the amount of α 1S in the membrane also is undetectable. In contrast, the β 1 subunit is localized appropriately in dysgenic, mdg/mdg , (α 1S -null) cells. Therefore, the β 1 subunit may not only play an important role in the transport/insertion of the α 1S subunit into the membrane, but may be vital for the targeting of the muscle dihydropyridine receptor complex to the transverse tubule/sarcoplasmic reticulum junction.

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.

EXCITATION-CONTRACTION coupling in skeletal muscle involves a voltage sensor in the plasma membrane which, in response to depolarization, causes an intracellular calcium-release channel to open. The skeletal isoform of the ryanodine receptor (RyR-1) functions as the Ca2+-release channel1–3 and the dihydropyridine receptor (DHPR) functions as the voltage sensor and also as an L-type Ca2+ channel4,5. Here we examine the possibility that there is a retrograde signal from RyR-1 to the DHPR, using myotubes from mice homozygous for a disrupted RyR-1 gene (dyspedic mice)3. As expected, we find that there is no excitation–contraction coupling in dyspedic myotubes, but we also find that they have a roughly 30-fold reduction in L-type Ca2+-current density. Injection of dyspedic myotubes with RyR-1 complementary DNA restores excitation–contraction coupling and causes the density of L-type Ca2+ current to rise towards normal. Despite the differences in Ca2+-current magnitude, measurements of charge movement indicate that the density of DHPRs is similar in dyspedic and RyR-1-expressing myotubes. Our results support the possibility of a retrograde signal by which RyR-1 enhances the function of DHPRs as Ca2+ channels.

In an attempt to identify and characterize functional domains of the rabbit skeletal muscle dihydropyridine receptor alpha 1 subunit II-III loop, we synthesized several peptides corresponding to different regions of the loop: peptides A, B, C, C1, C2, D (cf. Fig. 1). Peptide A (Thr671-Leu690) activated [3H]ryanodine binding to, and induced Ca2+ release from, rabbit skeletal muscle triads, but none of the other peptides had such effects. Peptide A-induced Ca2+ release and activation of ryanodine binding were partially suppressed by an equimolar concentration of peptide C (Glu724-Pro760) but were not affected by the other peptides. These results suggest that the short stretch in the II-III loop, Thr671-Leu690, is responsible for triggering SR Ca2+ release, while the other region, Glu724-Pro760, functions as a blocker of the release trigger. A hypothesis is proposed to account for how these subdomains interact with the sarcoplasmic reticulum Ca2+ release channel protein during excitation-contraction coupling.

CONTRACTION of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) after depolarization of transverse tubules1,2. The ryanodine receptor exists as a 'foot' protein in the junctional gap between the sarcoplasmic reticulum and the transverse tubule in skeletal muscle, and is proposed to function as a calcium-release channel during excitation-contraction (E-C) coupling3–6. Previous complementary DNA-cloning studies have defined three distinct subtypes of the ryanodine receptor in mammalian tissues, namely skeletal muscle, cardiac and brain types7–12. We report here mice with a targeted mutation in the skeletal muscle ryanodine receptor gene. Mice homozygous for the mutation die perinatally with gross abnormalities of the skeletal muscle. The contractile response to electrical stimulation under physiological conditions is totally abolished in the mutant muscle, although ryanodine receptors other than the skeletal-muscle type seem to exist because the response to caffeine is retained. Our results show that the skeletal muscle ryanodine receptor is essential for both muscular maturation and E-C coupling, and also imply that the function of the skeletal muscle ryanodine receptor during E-C coupling cannot be substituted by other subtypes of the receptor.

The beta-subunit is an integral component of purified voltage-sensitive Ca2+ channels. Modulation of Ca2+ channel activity by the beta-subunit, which includes significant increases in transmembrane current and/or changes in kinetics, is observed on coexpression of six alpha 1-subunit genes with four beta-subunit genes in all alpha 1-beta combinations tested. Recent reports suggest that this regulation is not due to targeting of the alpha 1-subunit to the plasma membrane but is probably a result of a conformational change induced by the beta-subunit. Here we report that the beta-subunit binds to the cytoplasmic linker between repeats I and II of the dihydropyridine-sensitive alpha 1-subunits from skeletal (alpha 1S) and cardiac muscles (alpha 1C-a), and also with the more distantly related neuronal alpha 1A and omega-conotoxin GVIA-sensitive alpha 1B-subunits. Sequence analysis of the beta-subunit binding site identifies a conserved motif (QQ-E--L-GY--WI--E) positioned 24 amino acids from the IS6 transmembrane domain in each alpha 1-subunit. Mutations within this motif reduce the stimulation of peak currents by the beta-subunit and alter inactivation kinetics and voltage-dependence of activation. Conservation of the beta-subunit binding motif in these functionally distinct calcium channels suggests a critical role for the I-II cytoplasmic linker of the alpha 1-subunit in channel modulation by the beta-subunit.

Expression studies with skeletal and cardiac muscle cDNAs have suggested that the putative cytoplasmic loop region of the dihydropyridine receptor (DHPR) alpha 1 subunit between transmembrane repeats II and III (DCL) is a major determinant of the type of excitation-contraction coupling (skeletal or cardiac) in rescued dysgenic muscle cells (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569). In this study, the possibility of a direct functional interaction with the sarcoplasmic reticulum ryanodine receptor/Ca2+ release channel has been tested by expressing the DCLs of the mammalian skeletal and cardiac muscle DHPR alpha 1 subunit in Escherichia coli. The purified peptides activated the skeletal muscle ryanodine receptor/Ca2+ release channel in single channel and [3H]ryanodine binding measurements, by increasing channel open probability and the affinity of [3H]ryanodine binding, respectively. The two peptides did not activate the cardiac muscle Ca2+ release channel. Other proteins (polylysine, serum albumin) also increased [3H]ryanodine binding and Ca2+ release channel activity, but their activation mechanisms were distinguishable from DCLs. These results show that the II-III cytoplasmic loop of the skeletal and cardiac DHPR alpha 1 subunit functionally interacts with the skeletal, but not cardiac, muscle Ca2+ release channel. Furthermore, our studies suggest that in addition to the DHPR, the sarcoplasmic reticulum Ca2+ release channel may determine the type of E-C coupling that exists in muscle.

THE skeletal muscle dihydropyridine (DHP) receptor is essential in excitation–contraction (EC) coupling1–4. The receptor is postulated to be the voltage sensor giving rise to the intramembrane current, termed charge movement5. We have now tested this hypothesis using myotubes from mice with the muscular dysgenesis mutation, which alters the skeletal muscle DHP receptor gene and prevents its expression3,4,6. Our results indicate that charge movement is deficient in dysgenic myotubes but is fully restored following injection of an expression plasmid carrying the rabbit skeletal muscle DHP receptor complementary DNA, strongly supporting the hypothesis that the DHP receptor is the voltage sensor for EC coupling in skeletal muscle. Additionally, our data obtained for normal and chimaeric DHP receptor constructs demonstrate that DHP receptors with widely differing abilities to function as calcium channels and to mediate EC coupling produce very similar charge movements

There are dihydropyridine (DHP)-sensitive calcium currents in both skeletal and cardiac muscle cells, although the properties of these currents are very different in the two cell types (for simplicity, we refer to currents in both tissues as L-type). The mechanisms of depolarization-contraction coupling also differ. As the predominant voltage-dependent calcium current of cardiac cells, the L-type current represents a major pathway for entry of extracellular calcium. This entry triggers the subsequent large release of calcium from the sarcoplasmic reticulum (SR). In contrast, depolarization of skeletal muscle releases calcium from the SR without the requirement for entry of extracellular calcium through L-type calcium channels. To investigate the molecular basis for these differences in calcium currents and in excitation-contraction (E-C) coupling, we expressed complementary DNAs for the DHP receptors from skeletal and cardiac muscle in dysgenic skeletal muscle. We compared the properties of the L-type channels produced and showed that expression of a cardiac calcium channel in skeletal muscle cells results in E-C coupling resembling that of cardiac muscle.

The architecture of the junctional sarcoplasmic reticulum (SR) and transverse tubule (T tubule) membranes and the morphology of the two major proteins isolated from these membranes, the ryanodine receptor (or foot protein) and the dihydropyridine receptor, have been examined in detail. Evidence for a direct interaction between the foot protein and a protein component of the junctional T tubule membrane is presented. Comparisons between freeze-fracture images of the junctional SR and rotary-shadowed images of isolated triads and of the isolated foot protein, show that the foot protein has two domains. One is the large hydrophilic foot which spans the junctional gap and is composed of four subunits. The other is a hydrophobic domain which presumably forms the SR Ca2+-release channel and which also has a fourfold symmetry. Freeze-fracture images of the junctional T tubule membranes demonstrate the presence of diamond-shaped clusters of particles that correspond exactly in position to the subunits of the feet protein. These results suggest the presence of a large junctional complex spanning the two junctional membranes and intervening gap. This junctional complex is an ideal candidate for a mechanical coupling hypothesis of excitation-contraction coupling at the triadic junction.

Single muscle fibers continue to twitch for up to 20 min when immersed in ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) solutions containing less than 10−8 M free calcium. Failure of the twitch results from reversible depolarization, which occurs after 15–20 min in EGTA. The results make it clear that external calcium or calcium in the transverse tubules play no essential part in action potential propagation or excitation-contraction coupling.

Skeletal muscle knockout cells lacking the beta subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca(2+) current, charge movements, and excitation-contraction coupling, despite having a normal Ca(2+) storage capacity and Ca(2+) spark activity. In this study we identified a specific region of the missing beta1a subunit critical for the recovery of excitation-contraction. Experiments were performed in beta1-null myotubes expressing deletion mutants of the skeletal muscle-specific beta1a, the cardiac/brain-specific beta2a, or beta2a/beta1a chimeras. Immunostaining was used to determine that all beta constructs were expressed in these cells. We examined the Ca(2+) conductance, charge movements, and Ca(2+) transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca(2+) current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length beta1a. In addition, all constructs except beta2a mutants recovered charge movements with a density similar to full-length beta1a. Thus, all beta constructs became integrated into a skeletal-type DHPR and, except for beta2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca(2+) transient was not affected by separate deletions of the N-terminus of beta1a or the central linker region of beta1a connecting two highly conserved domains. Also, replacement of the N-terminus half of beta1a with that of beta2a had no effect. However, deletion of 35 residues of beta1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca(2+) transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from beta1a. The identified domain at the C-terminus of beta1a may be responsible for colocalization of DHPRs and ryanodine receptors (RyRs), or may be required for the signal that opens the RyRs during excitation-contraction coupling. This new role of DHPR beta in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca(2+) release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490-524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490-508) is sufficient to reproduce activating effects of β1a490-524. Here we examined the effects of β1a490-508 on Ca(2+) release and Ca(2+) currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490-508 (25 nM) increased the peak Ca(2+) release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490-508 in fibers. β1a490-508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490-508 peptide was used as negative control and produced negligible effects on Ca(2+) release flux and RyR1 activity. Our results show that the β1a490-508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.

Voltage-gated Ca2+ channels couple membrane depolarization to Ca2+-dependent intracellular signaling events. This is achieved by mediating Ca2+ ion influx or by direct conformational coupling to intracellular Ca2+ release channels. The family of Cav1 channels, also termed L-type Ca2+ channels (LTCCs), is uniquely sensitive to organic Ca2+ channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming alpha1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Cav1.1 alpha1), incomplete congenital stationary night blindness (CSNB2; Cav1.4 alpha1), and Timothy syndrome (Cav1.2 alpha1; reviewed separately in this issue). Cav1.3 alpha1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Cav2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function.

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.

Contraction of a vertebrate skeletal muscle fibre is triggered by electrical depolarization of sarcolemmal infoldings termed transverse-tubules (t-tubules), which in turn causes the release of calcium from an internal store, the sarcoplasmic reticulum (SR)1,2. The mechanism that links t-tubular depolarization to SR calcium release remains poorly understood. In principle, this link might be provided by the prominent slow calcium current that has been described in skeletal muscle cells of adult frogs3,4 and rats5. However, blocking this current does not abolish the depolarization-induced contractile responses of frog muscle6, and the function of this slow calcium current is unknown. Here we describe measurements of calcium currents in developing skeletal muscle cells of normal rats and mice, and of mice with muscular dysgenesis, a mutation7 that causes excitation–contraction (E–C) coupling to fail8. We find that a slow calcium current is present in skeletal muscle cells of normal animals but absent from skeletal muscle cells of mutant animals. The effect of the mutation is specific to the slow calcium current of skeletal muscle; a fast calcium current is present in developing skeletal muscle cells of both normal and mutant animals, and slow calcium currents are present in cardiac and sensory neurones of mutant animals. We believe this to be the first report of a mutation affecting calcium currents in a multicellular organism. The effects of the mutation raise important questions about the relationship between the slow calcium current and skeletal muscle E–C coupling.

The transduction of action potential to muscle contraction (E-C coupling) is an example of fast communication between plasma membrane events and the release of calcium from an internal store, which in muscle is the sarcoplasmic reticulum (SR). One theory is that the release channels of the SR are controlled by voltage-sensing molecules or complexes, located in the transverse tubular (T)-membrane, which produce, as membrane voltage varies, 'intramembrane charge movements', but nothing is known about the structure of such sensors. Receptors of the Ca-channel-blocking dihydropyridines present in many tissues, are most abundant in T-tubular muscle fractions from which they can be isolated as proteins. Fewer than 5% of muscle dihydropyridines are functional Ca channels; there is no known role for the remainder in skeletal muscle physiology. We report here that low concentrations of a dihydropyridine inhibit charge movements and SR calcium release in parallel. The effect has a dependence on membrane voltage analogous to that of specific binding of dihydropyridines. We propose specifically that the molecule that generates charge movement is the dihydropyridine receptor.

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.

Communication in our body runs on electricity. Between the exterior and interior of every living cell, there is a difference in electrical charge, or voltage. Rapid changes in this so-called membrane potential activate vital biological processes, ranging from muscle contraction to communication between nerve cells. Ion channels are cellular structures that maintain membrane potential and help ‘excitable’ cells like nerve and muscle cells produce electrical impulses. They are specialized proteins that form highly specific conduction pores in the cell surface. When open, these channels let charged particles (such as calcium ions) through, rapidly altering the electrical potential between the inside and outside the cell. To ensure proper control over this process, most ion channels open in response to specific stimuli, which is known as ‘gating’. For example, voltage-gated calcium channels contain charge-sensing domains that change shape and allow the channel to open once the membrane potential reaches a certain threshold. These channels play important roles in many tissues and, when mutated, can cause severe brain or muscle disease. Although the basic principle of voltage gating is well-known, the properties of individual voltage-gated calcium channels still vary. Different family members open at different voltage levels and at different speeds. Such fine-tuning is thought to be key to their diverse roles in various parts of the body, but the underlying mechanisms are still poorly understood. Here, Fernandez-Quintero, El Ghaleb et al. set out to determine how this variation is achieved. The first step was to create a dynamic computer simulation showing the detailed structure of a mammalian voltage-gated calcium channel, called CaV1.1. The simulation was then used to predict the movements of the voltage sensing regions while the channel opened. The computer modelling experiments showed that although the voltage sensors looked superficially similar, they acted differently. The first of the four voltage sensors of the studied calcium channel controlled opening speed. This was driven by shifts in its configuration that caused oppositely charged parts of the protein to sequentially form and break molecular bonds; a process that takes time. In contrast, the fourth sensor, which set the voltage threshold at which the channel opened, did not form these sequential bonds and accordingly reacted fast. Experimental tests in muscle cells that had been engineered to produce channels with mutations in the sensors, confirmed these results. These findings shed new light on the molecular mechanisms that shape the activity of voltage-gated calcium channels. This knowledge will help us understand better how ion channels work, both in healthy tissue and in human disease.

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

The voltage-gated calcium channel CaV1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant CaV1.1e, which lacks ...

Human induced pluripotent stem cells-derived myogenic progenitors develop functional and ultrastructural features typical of skeletal muscle when differentiated in culture. Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca2+ signaling were examined, along with myofibrillogenesis. Pax7+-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca2+ channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca2+ transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca2+ imaging, and myocyte contraction was captured by digital photomicrography. We show that the E-C coupling machinery occurs and operates within a few days post-differentiation, as soon as the myofilaments align. However, Ca2+ transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels. The E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling.

The CaV1.1 calcium channel is the voltage sensor of skeletal muscle excitation-contraction (EC) coupling. The current of the adult CaV1.1a splice variant is slowly activating, small and has poor voltage-sensitivity, whereas that of the embryonic CaV1.1e splice variant, lacking exon 29 in the IVS3-S4 linker, has an 8-fold higher amplitude, activates fast and at 30mV less depolarizing potentials (Tuluc et al.,2009). Here we created intra-molecular chimeras to test the hypothesis that the voltage sensors of homologous repeats I to IV differentially control current kinetics, voltage-dependence, and EC-coupling. Inserting the IVS3-S4 linker (mainly coded by exon 29) plus IVS4 into the corresponding region of repeat I fully restored CaV1.1a amplitude and voltage-sensitivity to CaV1.1e. Transferring the IVS3-S4 linker plus IVS4 into the homologous region of repeat II enhanced the voltage-sensitivity of the calcium current but fully abolished skeletal muscle EC-coupling. Any substitution in IS3-S4 resulted in accelerated activation-kinetics. However, slow activation-kinetics could not be transferred from the Ist to the IVth repeat. Interestingly, inserting IS3 plus the IS3-S4 linker into repeat IV fully restored the poor CaV1.1a voltage-dependence and small amplitude in the absence of exon 29. Secondary structure prediction revealed a beta-sheet in exon 29 that is missing in CaV1.1e. Point mutations which abolish this beta-sheet partially recapitulate the effects of deleting exon 29. Together these findings suggest a model according to which the voltage sensor of repeat IV controls the voltage-dependence and amplitude and its properties can be transferred to repeat I; the voltage sensor of repeat I determines the current kinetics exclusively in the context of this repeat; and the voltage sensor of repeat II controls EC-coupling. Funded by the Austrian Science Fund P20059-B05, P23479-B19 and MFI-2007-417.

In adult skeletal muscle, the majority of the L-type Ca2+ channel CaV1.1 subunit is truncated post-translationally at residue 1664 (PNAS, 102:5274-79), raising the question of the functional role of the distal residues (1665-1873). It has been suggested (J Neurosci.17:1243-55; J Biol Chem. 277:4079-87) that (i) the distal C-terminus is non-covalently associated with the remainder of the channel, (ii) reduces channel open probability, and (iii) loses this inhibitory effect as a result of phosphorylation during strong depolarization. In regard to point (ii), previous analysis of L-type ionic conductance (G) and membrane gating charge movements (Q) demonstrated that channel open probability (i.e., G/Q ratio) was indistinguishable for full-length or truncated (at 1662) CaV1.1 expressed in dysgenic myotubes (Nature 360:169-171). Here we have investigated the effects of removing the distal C-terminus on depolarization-induced potentiation of CaV1.1. Specifically, tail currents were measured for repolarization to -30 mV following a 200 ms depolarization to either +40 or +90 mV. For both full-length and truncated CaV1.1, tail currents were both larger (∼2.5-fold) and more slowly decaying (∼2-fold) following the +90 mV depolarization. Thus, we find no evidence for a role of the CaV1.1 distal C-terminus in depolarization-induced potentiation.We are currently examining the role of the CaV1.2 distal C-terminus by co-expression of full-length or truncated (1669) CaV1.2 in tsA-201 cells together with β2a and α2δ1. In agreement with previous work (J Physiol. 576:87-102, and in contrast to CaV1.1, truncation of CaV1.2 resulted in ∼4-fold increase in the G/Q ratio. We are currently investigating the ability of the truncated CaV1.2 to undergo depolarization-induced potentiation. Supported by NIH (NS24444) and MDA grants to KGB.

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.

The Ca2+ channel CaV1.1 is the voltage sensor of skeletal muscle excitation-contraction coupling. The classical skeletal muscle CaV1.1 isoform has poor voltage sensitivity and conducts a small, slowly activating Ca2+ current. In contrast, a splice variant lacking exon 29 (α1S-ΔE29) (Tuluc et al.,2009) has an 8-fold higher current amplitude, fast activation-kinetics, and a 30mV left-shifted voltage-dependence of activation. Therefore, the extracellular loop in repeat IV (IVS3-IVS4) mainly coded by exon 29 is a critical determinant of the characteristic gating properties of CaV1.1. Here we used intramolecular chimeras between repeats I and IV to characterize the structural basis of the gating properties of CaV1.1.Inserting exon 29 (alone or in combination with IVS3) into the corresponding region of repeat I was ineffective. However, in combination with the voltage sensor (IVS4) it fully restored α1S-like amplitude and voltage-sensitivity to α1S-ΔE29. Interestingly, all three chimeras exhibit faster activation kinetics. Secondary structure predictions showed that the long IVS3-IVS4 loop contains a beta-sheet while the short loop forms a coil. Point mutations in exon 29 which abolish the beta-sheet fully mimic the effects of deleting exon 29 regarding the kinetic properties and increase the current amplitude by 3-fold and left-shift the voltage dependence by −15mV. Together with previous findings (Nakai et al., 1994) our data suggest that the S3-S4 loop of the first repeat determines activation kinetics, while the corresponding loop plus voltage sensor in the fourth repeat with its unique secondary structure dictate the voltage-dependence, amplitude, and kinetics of skeletal muscle Ca2+ currents.Grants: PT (MFI-2007-417), BEF (FWF-P20059-B05).

CaV1.1e is the calcium channel splice variant of embryonic skeletal muscle. It functions as voltage sensor in EC coupling, but in contrast to the adult CaV1.1a variant it also supports sizeable calcium currents activating at the same membrane potential as SR calcium release. Mis-splicing of CaV1.1 results in elevated levels of the CaV1.1e variant in mature muscle that correlate with the severity of muscle weakness in patients suffering from dystrophic myotonia. Therefore we hypothesize that exclusive expression of CaV1.1e in exon 29 knockout mice will cause muscle weakness. In muscles of wildtype mice expression of CaV1.1e declines after birth. However, two weeks after sciatic nerve resection CaV1.1e transcript levels are upregulated in paralyzed muscles, indicative of an activity-dependent regulation of CaV1.1e splicing. Quantitative RT-PCR analysis demonstrates that exon 29 knockouts express exclusively the CaV1.1e splice variant at levels comparable to total CaV1.1 in wildtype muscle. Combined voltage clamp and fluorescence calcium recordings of isolated muscle fibers from exon 29 knockout mice revealed a substantial influx-dependent component of the calcium transient. This not only alters myoplasmic calcium handling, but is further expected to prolong the muscle action potential and consequently reduce the tetanic fusion frequency. Surprisingly, these severe alterations of the muscle fiber properties were not accompanied by a dystrophic myotonia phenotype in knockout mice. A battery of behavioral tests (home-cage activity, voluntary and forced running, grip strength and rotarod tests) revealed no signs of muscle weakness or altered motor activity compared to wildtype siblings. Thus, development of dystrophic myotonia may require the combinatorial action of several mis-spliced genes. Alternatively, strong compensatory mechanisms may cause the absence of the disease phenotype in exon 29 knockout mice. Support: FWF P23479, W1101, OTKA-NN107765, TAMOP-4.2.2./B.-10/1-2010-0024.

In adult muscle fibers, tetanic electrical stimulation induces two separate calcium signals, a fast one related to contraction and dependent on Cav1.1 (DHPR) and RyRs and a slow signal, dependent on DHPR and IP3Rs. We have recently shown that the amplitude of this slow signal is dependent on frequency of stimulation, having a maximum at 20 Hz. Importantly, this signal participates in the activation of genes related to slow muscle fiber type phenotype. This signal is inhibited by nifedipine, supporting a role for the DHPR in its onset. We have stimulated adult muscle fibers with different concentrations of extracellular K+, and found calcium signals at more negative membrane potential than those described for triggering calcium release associated with RyR. Moreover, in the same conditions, we observe an increase in mRNA of the slow isoform of TroponinI, as seen in fibers stimulated with a 20Hz train of pulses. These signals have an intracellular origin, because they are still present when working in solutions with no extracellular calcium and 0.1 mM EGTA. Interestingly, they are inhibited by incubation of fibers with 20 uM XestosponginB, a specific inhibitor of IP3R. These results strongly suggest that the slow signal fires at membrane potential values more negative (Nernst potential estimated −50 mV) than the fast signal (E-C coupling related), where the fast signal threshold (−30 to −20 mV) has not yet been reached. This opens the possibility for a different part of the DHPR molecule being involved in the signal transmission of depolarization to IP3-production machinery, independently of the process of E-C coupling.FONDECYT 1080120, FONDAP 15010006.

In adult muscle fibers, Cav1.1 acts as voltage sensor for both excitation-contraction coupling and the activation of a signaling cascade that regulates gene expression. We have shown that ATP is released trough pannexin-1 channels after electrical stimulation at 20 Hz, having a key role in the induction of transcriptional changes related to fast-to-slow muscle fiber phenotype transition. Myotubes lacking the Cav1.1-α1 subunit displayed almost no ATP release after electrical stimulation. The same was observed in adult fibers treated with the Cav1.1 antagonist nifedipine (25µM), showing that Cav1.1 has a central role controlling ATP release.We examined the activation of this signaling cascade in muscle fibers where a knock-down of the α1s subunit of Cav1.1 was obtained by a U7-exon skipping strategy using adenovirus-associated viral vectors (AAV-U7delα1s).Four months after AAV-U7delα1s injection we observed a significant reduction of Cav1.1 protein as well as atrophy and fibrosis of the treated muscles. Indo-1 Ca2+ transients and Ca2+ current in voltage-clamped fibers isolated from FDB treated muscles showed that the peak Ca2+ transient elicited by short depolarizing pulses was reduced by 35% whereas the maximal conductance of the Ca2+ channels was reduced by 30%. We also found increased basal ATP release with spontaneous release events. AAV-U7delα1s treated fibers showed higher mRNA levels of the slow isoform of Troponin I and lower mRNA levels of the fast isoform of Troponin I compared with non-treated muscles. The transcriptional changes observed in these two genes after electrical stimulation were absent in AAV-U7delα1s treated fibers.These results suggest that Cav1.1 controls ATP release trough Pannexin-1 channels, activating it after low stimulation frequencies and blocking ATP release during resting. The loss of this control perturbs the normal transcriptional response to electrical activity of adult muscle fibers.ACT1111, FONDECYT 1110467, FONDAP 15010006

Following membrane depolarization S4 segments of voltage-gated cation channels translocate through the membrane resulting in the opening of the channel pore. This mechanism has been extensively studied in potassium and sodium channels yet little is known about it in voltage-gated calcium channels. Here we used de-novo ROSETTA modelling and site-directed mutagenesis to study the transition of the IVth voltage-sensing-domain (VSD) of Cav1.1 calcium channel during gating. Previously we have shown that the embryonic Cav1.1e splice variant, lacking exon 29 in the IVS3-S4 linker, has an 8-fold higher current amplitude and 30mV left-shifted voltage-sensitivity compared to the adult Cav1.1a splice variant. Modeling 4 consecutive states of the VSD suggested that the number of H-bonds formed between the arginines of IVS4 and residues in IVS3 is higher in Cav1.1e than in Cav1.1a. Among them aspartate at position 1196 (D1196) shows the strongest difference between the two splice variants. Indeed, voltage clamp recordings show that neutralizing the negative charge in S3 (D1196N) has no effect on the voltage sensitivity of Cav1.1a but confers poor voltage sensitivity to Cav1.1e. Mutations of R1210, R1217, and R1220 of Cav1.1e are currently analyzed to identify the interaction partners of D1196 during gating. Furthermore, structure modeling revealed an extracellularly exposed hydrophobic cluster formed by two leucines in the middle of IVS3-S4 loop encoded by exon 29. Point mutations which eliminate this hydrophobic cluster partially recapitulate the effects of exon 29 deletion. Together these findings suggest a model according to which the extracellular loop S3-S4 controls the orientation of S3 and S4 relative to each other. This alters charge compensation of positive charges in S4 and dramatically reduces the voltage sensitivity of CaV1.1a calcium channels. Support: FWF W1101, P23479, LFU-P7400-027-011.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that is triggered by exposure to volatile anesthetics. MH has been studied extensively in mice and pigs carrying causative mutations in the type 1 ryanodine receptor (RyR1). However, no in vivo information exists regarding how mutations in the skeletal muscle L-type Ca2+ channel (CaV1.1) precipitate MH crises. For this reason, we generated a mouse line carrying the R174W mutation. Homozygous R174W mice ambulated efficiently, reproduced and had normal lifespans. When exposed to isoflurane, homozygous R174W mice entered a hypermetabolic state ending ultimately in death. On the ultrastructural level, R174W muscle displayed limited and variable changes: some variability of the SR calsequestrin content, displacement of mitochondria in some soleus fibers of aged mice and occasional accumulation of SR stacks. On the cellular level, homozygous R174W muscle had elevated resting myoplasmic Ca2+ levels that were greatly increased upon exposure to isoflurane. Flexor digitorum brevis (FDB) fibers dissociated from homozygous R174W mice lacked L-type Ca2+ current even though intramembrane charge movements of were of similar magnitude and voltage-dependence to those recorded from wild-type fibers. Ca2+ released from the SR in response to depolarization was substantially reduced in homozygous R174W fibers suggesting a depleted SR Ca2+ store. Lipid bilayer recordings showed that the Po of RyR1s isolated from homozygous R174W mice was significantly increased at all cis Ca2+ concentrations (Feng et al., this meeting). Taken together, our results support a mechanism for MH susceptibility in which CaV1.1 R174W promotes SR Ca2+ leak without affecting EC coupling per se. This work was supported by grants from the NIH (AR055104 to KGB, AR052534 to PDA, KGB, PMH, CFA and INP) and MDA (MDA277475 to KGB). DB received a stipend from 2T32AG000279-11.