3D Structure of a Recombinant DHPR Expressed in Mouse

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.

Involvement of the Carboxy-Terminus Region of the Dihydropyridine Receptor β1a Subunit in Excitation-Contraction Coupling of Skeletal Muscle

Ca2+ release mediated by the ryanodine receptor (RyR) regulates many important cell functions including excitation-contraction (E-C) coupling in skeletal muscle, by which membrane depolarization controls the opening of RyR via the dihydropyridine receptor. Among the three RyR subtypes, RyR-1 mediates skeletal muscle E-C coupling, whereas RyR-2 and RyR-3 cannot substitute for RyR-1. We carried out expression experiments using cultured mutant skeletal myocytes not having intrinsic intracellular Ca2+ release channels to study the structure-function relationship of amino acid residues 1303-1406 in RyR-1 (D2 region). In this region the amino acid sequences are highly divergent between RyR-1 and RyR-2, and the corresponding sequence is lacking in RyR-3. Expression of RyR-1 but not of RyR-2 rescued E-C coupling in the mutant cells. Deletion of either the entire D2 region or its N-terminal half from RyR-1 preserved the function of RyR-1 as a Ca2+ release channel but resulted in the loss of E-C coupling. Substitution of the D2 region for the corresponding sequence of RyR-2 had no effect on the function of RyR-1. These results indicate that the presence of the D2 region is critical for E-C coupling in skeletal muscle, although the D2 region alone cannot determine the functional difference between RyR-1 and RyR-2.

Excitation-contraction coupling in skeletal muscle involves junctions (triads and dyads) between sarcoplasmic reticulum (SR) and transverse (T) -tubules. Two proteins of the junctional SR, ryanodine receptors (RyRs) and triadin and one protein of T tubules, dihydropyridine receptors (DHPRs) are located at these junctions. We studied the targeting of DHPRs and triadin to T-tubules and SR in skeletal muscles of dyspedic mouse embryos lacking RyR1. In normal differentiating muscle fibers DHPRs, triadin and RyRs are located in intensely immunolabeled foci that are randomly distributed across the fiber. Correlation with electron microscopy and with previous studies indicates that the foci represent the location of triads and dyads. In dyspedic fibers DHPRs and triadin antibodies stain internal foci of the two proteins; RyR antibodies are completely negative. The appearance and location of the foci in dyspedic fibers is similar to that of normal muscle, but their fluorescent intensity is weaker. The SR Ca-ATPase has more diffuse distribution than triadin in both normal and dyspedic fibers. These observations indicate that an interaction with RyRs is not necessary for the appropriate targeting of DHPRs or triadin to junctional domains of T tubules and SR respectively.

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltage-dependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating models of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

Redox modulation of diaphragm contractility: Interaction between DHPR and RyR channels

A Ca V1.1 Ca 2+ Channel Splice Variant with High Conductance and Voltage-Sensitivity Alters EC Coupling in Developing Skeletal Muscle

Skeletal muscle excitation–contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and l-type Ca2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca2+ channel is still enigmatic. Here we show that Ca2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca2+ influx is eliminated. Homozygous knock-in mice display SR Ca2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca2+ influx in mammalian skeletal muscle is an evolutionary remnant.

CaV1.1 is specifically expressed in skeletal muscle where it functions as voltage sensor of skeletal muscle excitation-contraction (EC) coupling independently of its functions as L-type calcium channel. Consequently, all known CaV1.1-related diseases are muscle diseases and the molecular and cellular disease mechanisms relate to the dual functions of CaV1.1 in this tissue. To date, four types of muscle diseases are known that can be linked to mutations in the CACNA1S gene or to splicing defects. These are hypo- and normokalemic periodic paralysis, malignant hyperthermia susceptibility, CaV1.1-related myopathies, and myotonic dystrophy type 1. In addition, the CaV1.1 function in EC coupling is perturbed in Native American myopathy, arising from mutations in the CaV1.1-associated protein STAC3. Here, we first address general considerations concerning the possible roles of CaV1.1 in disease and then discuss the state of the art regarding the pathophysiology of the CaV1.1-related skeletal muscle diseases with an emphasis on molecular disease mechanisms.

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.

NG.O.9 - Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy

Coupling between L-type Ca(2+) channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca(2+) influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca(2+) release. The L-type Ca(2+) channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca(2+) influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca(2+) spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by 401+/-15% within 10 seconds in Ca(2+)-free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/L BayK induced a rapid 245+/-12% increase in CaSpF. Nifedipine (5 micromol/L) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca(2+) channel agonist FPL-64176 (1 micromol/L) did not alter CaSpF (despite rapid and potent enhancement of Ca(2+) current, I(Ca)). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased I(Ca) only slowly (tau=18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on I(Ca) and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca(2+) channel and RyR.

This study investigated the development of Ca²(+) signaling mechanisms and their role in initiating morphogenetic cell movement in the presumptive ectoderm of Japanese newt (Cynops pyrrhogaster) during gastrulation. Histochemical staining using fluorescently labeled ryanodine and dihydropyridine probes revealed that dihydropyridine receptor (L-type Ca²(+) channels) appeared in stage 12b embryos, while ryanodine receptors were expressed in both stage 11 and 12b embryos. Transmission electron microscopy of stage 12b embryos showed abundant peripheral couplings, which are couplings of the endoplasmic reticulum and cell membrane with an approximate 12 nm gap. Caffeine increased the intracellular free Ca²(+) concentration ([Ca²(+)](i)) in presumptive ectodermal cells isolated from both stage 11 and 12b embryos, while (±)-Bay K 8644 ((±)-BayK) increased [Ca²(+)](i) in cells isolated from stage 12b embryos, but not in cells isolated from stage 11 embryos. Dantrolene and nifedipine completely inhibited increases in [Ca²(+)](i) after treatment with caffeine and (±)-BayK, respectively. Caffeine activated the motility of cells isolated from both stage 11 and 12b embryos, but (±)-BayK only activated the motility of cells isolated from stage 12b embryos. These findings suggested that formation of the Ca²(+) -induced Ca²(+) release system in presumptive ectodermal cells during gastrulation plays an important role in the initiation and execution of epibolic extension.