Skeletal Muscle Dihydropyridine Receptor Research Articles

The distal C terminus of the dihydropyridine receptor β1a subunit is essential for tetrad formation in skeletal muscle

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation-contraction coupling.

Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding.

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.

The Ca2+ influx through the mammalian skeletal muscle dihydropyridine receptor is irrelevant for muscle performance

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.

The mammalian skeletal muscle DHPR has larger Ca2+ conductance and is phylogenetically ancient to the early ray-finned fish sterlet (Acipenser ruthenus)

The L-type Ca2+ channel or dihydropyridine receptor (DHPR) in vertebrate skeletal muscle is responsible for sensing sarcolemmal depolarizations and transducing this signal to the sarcoplasmic Ca2+ release channel RyR1 via conformational coupling to initiate muscle contraction. During this excitation-contraction (EC) coupling process there is a slow Ca2+ current through the mammalian DHPR which is fully missing in euteleost fishes. In contrast to ancestral evolutionary stages where skeletal muscle EC coupling is still depended on Ca2+-induced Ca2+-release (CICR), it is possible that the DHPR Ca2+ conductivity during mammalian (conformational) EC coupling was retained as an evolutionary remnant (vestigiality). Here, we wanted to test the hypothesis that due to the lack of evolutionary pressure in post-CICR species skeletal muscle DHPR Ca2+ conductivity gradually reduced as evolution progressed. Interestingly, we identified that the DHPR of the early ray-finned fish sterlet (Acipenser ruthenus) is phylogenetically positioned above the mammalian rabbit DHPR which retained robust Ca2+ conductivity, but below the euteleost zebrafish DHPR which completely lost Ca2+ conductivity. Remarkably, our results revealed that sterlet DHPR still retained the Ca2+ conductivity but currents are significantly reduced compared to rabbit. This decrease is due to lower DHPR membrane expression similar to zebrafish, as well as due to reduced channel open probability (Po). In both these fish species the lower DHPR expression density is partially compensated by higher efficacy of DHPR-RyR1 coupling. The complete loss of Po in zebrafish and other euteleost species was presumably based on the teleost specific 3rd round of genome duplication (Ts3R). Ts3R headed into the appearance of two skeletal muscle DHPR isoforms which finally, together with the radiation of the euteleost clade, fully lost the Po.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit

The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling.

3D Structure of the Dihydropyridine Receptor of Skeletal Muscle.

Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) mediates the quasi-instantaneous conversion from T-tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR). The DHPR has several key modules required for EC coupling: the voltage sensors and II-III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II-III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

FRET-Based Structural Analysis of the Skeletal Muscle DHPR using Biarsenical Labeling

The α1s skeletal muscle dihydropyridine receptor (DHPR) subunit triggers muscle contraction during excitation contraction (EC) coupling. The intracellular loops of this voltage-gated calcium channel have diverse functions, though the spatial organization of these loops relative to each other and to other DHPR subunits, is poorly understood. We employed a FRET-based assay to determine the proximity of these intracellular loops relative to the N-terminus of the α1s DHPR subunit and then determined how these proximities changed upon binding of the β1a DHPR subunit. Recombinant α1s DHPRs were expressed in HEK293T cells and then FRET was measured from YFP fused to the DHPR N-terminus to the biarsenical reagent ReAsH targeted to a tetracysteine (Tc) tag inserted into each of the DHPR intracellular loops. Energy transfer was highest to ReAsH bound to a Tc tag placed adjacent to the N-terminally fused YFP (E∼40-50%) whereas low FRET (E∼10%) was observed for a negative control construct lacking a Tc tag. Placement of Tc tags in each of the DHPR intracellular loops resulted in intermediate FRET efficiencies whereas insignificant energy transfer was observed between the DHPR N- and C-termini. Addition of purified recombinant DHPR β1a subunit resulted in significant enhancement of energy transfer between the N-terminal YFP and ReAsH targeted to the DHPR α1s II-III loop, a major EC coupling determinant. Our data demonstrate that FRET measurements using biarsenical labeling reagents offer an effective approach to study DHPR structure since disruption of native conformation is minimal. In addition, these studies should allow for subsequent measurements of changes in DHPR structure that occur during EC coupling. Supported by NIH grants R01AR059126 (to JDF) and R03AR066359 (to CFP).

Amino Acid Residues 489–503 of Dihydropyridine Receptor (DHPR) β1a Subunit Are Critical for Structural Communication between the Skeletal Muscle DHPR Complex and Type 1 Ryanodine Receptor

The β1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca(2+) release channel (RyR1). Progressive truncation of the β1a C terminus showed that deletion of amino acid residues Gln(489) to Trp(503) resulted in a loss of depolarization-induced Ca(2+) release, a severe reduction of L-type Ca(2+) currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu(496), Leu(500), and Trp(503), which are thought to mediate direct β1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence (489)QVQVLTSLRRNLSFW(503) of β1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu(496)-Leu(500)-Trp(503) within the β1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling.

Role of sarcoplasmic reticulum junctional proteins in skeletal muscle strength

Background Skeletal muscle constitutes approximately 40% of body mass, and age-induced decrease of muscle strength impinge on daily activities and on normal social life in the elderly. Loss of muscle strength has been recognised as a debilitating and life threatening condition also in cachexia in cancer patients and in clinical conditions associated with prolonged bed rest. Skeletal muscle dihydropyridine receptors (Cav1.1) act as Ca2+ channels and voltage sensors to initiate muscle contraction by activating ryanodine receptors, the Ca2+ release channels of the sarcoplasmic reticulum. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Cav1.1 and the sarcoplasmic reticulum Ca2+ storage protein calsequestrin (CASQ1). We hypothesized that JP45 and CASQ1 form a signalling pathway which modulates Cav1.1 channel activity.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels.

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels.

The DHPR Calcium Current in Mammalian Skeletal Muscle: Physiological Necessity or Tolerated Evolutionary Remnant?

In contrast to cardiac excitation-contraction coupling (ECC), skeletal-muscle ECC is based on Ca2+-influx-independent inter-channel communications between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1). The role of the small Ca2+ influx through the DHPR in mammalian skeletal muscle, which is not (immediately) required for ECC, is still enigmatic. Previously, we discovered that zebrafish, as well as all higher teleost fish, lack DHPR Ca2+ conductivity in skeletal muscle (Schredelseker et al., PNAS, 2010). Point mutation N617D in pore loop II of zebrafish DHPRα1S-b explained non-conductivity in fast muscle. To investigate the fascinating skeletal muscle DHPR Ca2+-conductivity / non-conductivity phenomenon we generated a non-conducting-DHPR knock-in mouse (n.c.DHPR) by introducing the N→D mutation into gene CACNA1S. Interestingly, homozygous n.c.DHPR mice are viable, fertile, visually indistinguishable, and identical in body-weight development to wild-type (WT) siblings. Myotubes isolated from newborn n.c.DHPR mice display complete lack of DHPR Ca2+ influx without altered ECC. No difference in locomotor activity (home cage activity), motor coordination (rotarod, beam walking), and muscle strength (endurance test, wire hang test) is observed in 3-7 months-old homozygous n.c.DHPR mice compared to WT. Identical results were obtained from forced frequency and fatigue tests on isolated EDL (fast twitch) and soleus (slow twitch) muscle fibers. As soon feasible, tests will be repeated on aged (18 months-old) mice to test for putative age-related accumulative effects on muscle performance, in order to understand if DHPR Ca2+ influx in mammalian skeletal muscle is a physiological necessity or just a tolerated evolutionary remnant of the ancestral pure Ca2+-influx dependent ECC of early chordates and phylogenetic branches below.Supported: FWF P-23229-B09; DK-W1101-B12.

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca(2+) channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α(1s) or Ca(V)1.1, α(2), β(1a), δ1, and γ), we created transgenic mice expressing a recombinant β(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α(2)-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.

Fluorescence Resonance Energy Transfer (FRET) Indicates That Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit

The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca(2+) release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the α(1S) and β(1a) subunits of the DHPR are essential for EC coupling. Within α(1S), cytoplasmic domains of importance include the I-II loop (to which β(1a) binds), the II-III and III-IV loops, and the C terminus. However, the spatial relationship of these domains to one another has not been established. Here, we have taken the approach of measuring FRET between fluorescent proteins inserted into pairs of α(1S) cytoplasmic domains. Expression of these constructs in dyspedic (RyR1 null) and dysgenic (α(1S) null) myotubes was used to test for function and targeting to plasma membrane/SR junctions and to test whether the presence of RyR1 caused altered FRET. We found that in the absence of RyR1, measureable FRET occurred between the N terminus and C terminus (residue 1636), and between the II-III loop (residue 626) and both the N and C termini; the I-II loop (residue 406) showed weak FRET with the II-III loop but not with the N terminus. Association with RyR1 caused II-III loop FRET to decrease with the C terminus and increase with the N terminus and caused I-II loop FRET to increase with both the II-III loop and N terminus. Overall, RyR1 appears to cause a substantial reorientation of the cytoplasmic α(1S) domains consistent with their becoming more closely packed.

Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.

FRET Reveals Substantial Reorientation of the Cytoplasmic Interface of the Skeletal Muscle DHPR in the Presence of RyR1

In skeletal muscle, the dihydropyridine receptor (DHPR) in the t-tubular membrane serves as Ca2+channel and as voltage sensor for excitation-contraction (EC) coupling, triggering Ca2+release via a physical/conformational coupling to the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. The particulars of the structural and functional links between these two proteins are widely unknown. The putative intracellular portions of the DHPR a1S subunit, the N-terminus, C-terminus, and the loops connecting the four homologous repeats (I-IV), play important roles in the communication with the RyR1. Examples are the β-subunit recruiting function of the I-II loop, the bi-directional signaling function of the II-III loop with the RyR1 during EC-coupling, the influence of the III-IV loop on RyR1 mediated Ca2+delivery, and the a1S C-terminus. These channel parts are believed to either directly or indirectly interact with the RyR1, and the close spatial proximity between the two channels at t-tubule/SR ‘junctions’ constitutes the structural prerequisite for this linkage. The present work provides for the first time a structural insight into the arrangement of the crucial molecular components of the DHPR-RyR1 interaction, by using measurements of fluorescence resonance energy transfer (FRET), conducted within the cellular environment of living myotubes. Upon expression, the degree of FRET was determined for different combinations of labeled cytoplasmic a1S domains, using a sensitized emission FRET variant. Confocal fluorescence microscopy was applied to check for correct expression and function of the constructs upon expression in dyspedic (RyR1 null) and dysgenic (a1S null) myotubes. The presence of RyR1 significantly altered the intramolecular energy transfer for almost every double tagged a1S construct. These measurements reveal that virtually the complete cytoplasmic a1S architecture is significantly rearranged by the presence of the RyR1.

Junctophilin 1 and 2 Proteins Interact with the L-type Ca2+ Channel Dihydropyridine Receptors (DHPRs) in Skeletal Muscle

Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent studies have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle, JP1 and JP2 are part of a complex that, in addition to ryanodine receptor 1 (RyR1), includes caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knocked down for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type Ca2+ current accompanied by a reduced number of DHPRs at the plasma membrane, whereas there was no substantial alteration in Ca2+ release from the sterol regulatory element-binding protein. Altogether, these results suggest that JP1 and JP2 can facilitate the assembly of DHPR with other proteins of the excitation-contraction coupling machinery.

Orthograde dihydropyridine receptor signal regulates ryanodine receptor passive leak

The skeletal muscle dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) are known to engage a form of conformation coupling essential for muscle contraction in response to depolarization, referred to as excitation-contraction coupling. Here we use WT and Ca(V)1.1 null (dysgenic) myotubes to provide evidence for an unexplored RyR1-DHPR interaction that regulates the transition of the RyR1 between gating and leak states. Using double-barreled Ca(2+)-selective microelectrodes, we demonstrate that the lack of Ca(V)1.1 expression was associated with an increased myoplasmic resting [Ca(2+)] ([Ca(2+)](rest)), increased resting sarcolemmal Ca(2+) entry, and decreased sarcoplasmic reticulum (SR) Ca(2+) loading. Pharmacological control of the RyR1 leak state, using bastadin 5, reverted the three parameters to WT levels. The fact that Ca(2+) sparks are not more frequent in dysgenic than in WT myotubes adds support to the hypothesis that the leak state is a conformation distinct from gating RyR1s. We conclude from these data that this orthograde DHPR-to-RyR1 signal inhibits the transition of gated RyR1s into the leak state. Further, it suggests that the DHPR-uncoupled RyR1 population in WT muscle has a higher propensity to be in the leak conformation. RyR1 leak functions are to keep [Ca(2+)](rest) and the SR Ca(2+) content in the physiological range and thus maintain normal intracellular Ca(2+) homeostasis.