Dysgenic Myotubes Research Articles

Identifying the CaV1.1 voltage-sensing domains involved in channel gating and excitation contraction coupling.

Skeletal muscle CaV1.1 is a voltage gated calcium channel, which primarily functions as voltage sensor of excitation-contraction coupling (ECC). Upon depolarization, CaV1.1 triggers the opening of the RyR1 in the sarcoplasmic reticulum, causing the calcium release event essential for skeletal muscle contraction. Upon strong stimulation, CaV1.1 elicits a calcium current with kinetics and voltage-dependence different from those of ECC. Probably, these distinct activation properties arise from the differential roles of the four CaV1.1 voltage sensing domains (VSDs). Each VSD comprises of four transmembrane helices (S1-S4); S4 segments contain 4-5 positive charges, which interact with negative countercharges in the surrounding helices. These transient ion-pair interactions facilitate and tune the S4 motion in response to changes of the membrane potential. Using structure-guided mutagenesis with patch-clamp analysis and fluorescence calcium recordings, our group identified crucial countercharges in VSD I and IV able to regulate the channel's voltage sensitivity (I and IV) and activation kinetics (IV), without interfering with ECC. However, analogous mutations in VSDs II and III showed little or no effects on either channel gaiting or ECC, corroborating the notion that the four VSDs use different molecular mechanism for regulating their activation properties. Recently, two independent voltage-clamp fluorometry studies suggested that either VSD II or III is the main activator of ECC, based on their specific kinetics and voltage sensitivity matching those of ECC. While both studies agree that perhaps only a single VSD may activate ECC, they disagree on which one that might be. Here we address this problem using site-directed mutagenesis and chimeric CaV1.1 channels expressed in dysgenic myotubes. Constructs shifting the voltage-dependence of either ECC or channel activation reveal the specific contributions of the mutated VSDs in one or the other function of CaV1.1.

Ionic interactions between gating charges and countercharges in voltage-sensing domain I independently regulate kinetics and voltage-dependence of Cav1.1 gating.

Voltage-gated calcium channels control a variety of processes in excitable cells. However, the exact mechanisms regulating kinetics and voltage-dependence of channel activation are not fully understood. Voltage-gated activation is determined by four distinct voltage-sensing domains (VSD I-IV) coupled to a common pore. Each VSD consists of four transmembrane helices (S1-S4) with S4 containing four to five gating charges. Upon membrane depolarization, consecutive interactions of these gating charges with negatively charged countercharges are believed to facilitate an upward movement of the S4 helices, leading to the opening of the pore and activation of the channel. Previous studies linked naturally occurring mutations of the innermost gating charge R4 (R174W) and its negative countercharge (E100) in CaV1.1 to muscle disease. To investigate the contribution of VSD I and the roles of its gating- and countercharges in channel gating, we combined structure-guided site-directed mutagenesis with patch-clamp analysis in dysgenic myotubes (CaV1.1-null). As E100 in helix S2 together with D126 in helix S3 form the highly conserved charge-transfer center of VSDs, we included this second negative countercharge (D126) in our analysis. While mutation of R174A resulted in a strong right-shift of voltage dependence, charge neutralizing mutations of the countercharges E100 and D126 both lead to a left-shift of voltage-dependence and to a slowing of channel activation. The double mutant of both countercharges resulted in an additive effect on activation kinetics, but left the voltage dependence of activation unaffected compared to the single mutants. Our findings indicate that VSD I substantially contributes to the regulation of both kinetics and voltage-dependence of activation; that the same ionic interactions are critical for both properties; but that the molecular mechanisms governing the two gating properties are functionally separate.

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

The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.

Correcting the R165K substitution in the first voltage-sensor of CaV1.1 right-shifts the voltage-dependence of skeletal muscle calcium channel activation

ABSTRACTThe 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 exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a CaV1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both CaV1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of CaV1.1 channel gating.

Stac proteins associate with the critical domain for excitation-contraction coupling in the II-III loop of CaV1.1.

In skeletal muscle, residues 720-764/5 within the CaV1.1 II-III loop form a critical domain that plays an essential role in transmitting the excitation-contraction (EC) coupling Ca2+ release signal to the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum. However, the identities of proteins that interact with the loop and its critical domain and the mechanism by which the II-III loop regulates RyR1 gating remain unknown. Recent work has shown that EC coupling in skeletal muscle of fish and mice depends on the presence of Stac3, an adaptor protein that is highly expressed only in skeletal muscle. Here, by using colocalization as an indicator of molecular interactions, we show that Stac3, as well as Stac1 and Stac2 (predominantly neuronal Stac isoforms), interact with the II-III loop of CaV1.1. Further, we find that these Stac proteins promote the functional expression of CaV1.1 in tsA201 cells and support EC coupling in Stac3-null myotubes and that Stac3 is the most effective. Coexpression in tsA201 cells reveals that Stac3 interacts only with II-III loop constructs containing the majority of the CaV1.1 critical domain residues. By coexpressing Stac3 in dysgenic (CaV1.1-null) myotubes together with CaV1 constructs whose chimeric II-III loops had previously been tested for functionality, we reveal that the ability of Stac3 to interact with them parallels the ability of these constructs to mediate skeletal type EC coupling. Based on coexpression in tsA201 cells, the interaction of Stac3 with the II-III loop critical domain does not require the presence of the PKC C1 domain in Stac3, but it does require the first of the two SH3 domains. Collectively, our results indicate that activation of RyR1 Ca2+ release by CaV1.1 depends on Stac3 being bound to critical domain residues in the II-III loop.

STAC proteins associate to the IQ domain of CaV1.2 and inhibit calcium-dependent inactivation

The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (CaV) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with CaV1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in CaV1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal CaV1 channels remained unknown. Using CaV1.2/2.1 chimeras expressed in dysgenic (CaV1.1-/-) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of CaV1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of CaV1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with CaV1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the CaV1.2 IQ domain with that of CaV2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the CaV1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of CaV1.2 currents.

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.

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.

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.

Functional Analysis of CaV1.1 L-Type Calcium Channels Expressed in tsA201 Cells

Expression of modified CaV1.1 constructs in dysgenic (CaV1.1 null) myotubes has been useful in the determination of regions of the CaV1.1 that are important for skeletal muscle excitation-contraction (EC) coupling, however the use of this system to probe CaV1.1 channel gating has been problematic because dysgenic myotubes express a variety of other ion channels which conduct Ca2+ and contribute to intramembrane gating charge movement. This obstacle can be overcome by recording from tsA201 cells coexpressing CaV1.1 α1S, β1a, α2δ1 and Stac3 subunits. Our group has previously characterized, in dysgenic myotubes, a CaV1.1 mutation (R174W) which affects the innermost basic residue of the voltage-sensing S4 α-helix of Repeat I; R174W abolished activation of the L-type Ca2+ current in response to depolarization without affecting the magnitude or voltage-dependence of charge movement. Using tsA201 cells, we show that this dysfunction arises from an impaired gating transition of the RI voltage-sensor that is not detectable with short (20 ms) depolarizations. Specifically, we were unable to recruit the documented slow-moving gating charge component attributable to RI with longer (200 ms) test potentials. We next assessed the effects of corresponding mutations in Repeats II (K537W), III (R906W) and IV (K1245W) in order to determine the way in which translocation of these voltage-sensors affects channel opening. Currents and intramembrane charge movements produced by R906W were both smaller and shifted (∼10 mV) to more depolarizing test potentials. In contrast to the severe loss of function observed with R174W and the somewhat impaired gating of R906W, the K537W and K1245W mutants produced L-type currents with enormous peak amplitudes almost three times greater than wild-type CaV1.1 and with greatly impaired deactivation. Supported by Conacyt 169006 (UM), the Boettcher Foundation (RAB), and NIH AR055104, NIH AR052354 and MDA 277475 (KGB).

Characterization of the STAC3-CaV1.1 Interaction in Skeletal Muscle Excitation-Contraction Coupling

Skeletal muscle EC coupling critically depends on the physical interaction between the voltage-gated calcium channel CaV1.1 (DHPR), acting as voltage sensor, and the SR calcium release channel (RyR1). The skeletal muscle-specific DHPR components essential for EC coupling are the CaV1.1 II-III loop and the auxiliary β1a subunit. Recently, STAC3 (src homology 3 and cysteine rich domain 3) has been identified as an additional essential EC coupling protein. Further investigations indicated that STAC3 functions in membrane trafficking of CaV1.1 and linked STAC3 to a rare muscle disease Native American Myopathy (NAM). In contrast STAC3 is not expressed in the heart and thus appears not to participate in cardiac muscle EC coupling. These findings raise the question as to whether STAC3 binds exclusively to the skeletal muscle DHPR, and whether its interaction with the EC coupling complex is stable or dynamic. Here, we applied fluorescence recovery after photobleaching (FRAP) in dysgenic (CaV1.1-null) myotubes reconstituted with CaV1.1 and GFP-tagged STAC3 to demonstrate that STAC3 forms stable complexes with the skeletal muscle DHPR. Furthermore, we examined the subcellular localization of STAC3 and of the other two members of the STAC family of adaptor proteins, STAC1 and STAC2, in dysgenic myotubes reconstituted with either CaV1.1 or CaV1.2. Together these experiments demonstrate that the three members of the STAC family display differential interactions with the skeletal muscle and cardiac DHPRs. These functional differences allow the use of chimeric proteins to identify the molecular domains involved in the association of STAC3 with the skeletal muscle EC coupling apparatus. This work is supported by Austrian Research Fund FWF (P23479, P27031).

Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus.

CaV1.1 acts as both the voltage sensor that triggers excitation-contraction coupling in skeletal muscle and as an L-type Ca(2+) channel. It has been proposed that, after its posttranslational cleavage, the distal C terminus of CaV1.1 remains noncovalently associated with proximal CaV1.1, and that tethering of protein kinase A to the distal C terminus is required for depolarization-induced potentiation of L-type Ca(2+) current in skeletal muscle. Here, we report that association of the distal C terminus with proximal CaV1.1 cannot be detected by either immunoprecipitation of mouse skeletal muscle or by colocalized fluorescence after expression in adult skeletal muscle fibers of a CaV1.1 construct labeled with yellow fluorescent protein (YFP) and cyan fluorescent protein on the N and C termini, respectively. We found that L-type Ca(2+) channel activity was similar after expression of constructs that either did (YFP-CaV1.11860) or did not (YFP-CaV1.11666) contain coding sequence for the distal C-terminal domain in dysgenic myotubes null for endogenous CaV1.1. Furthermore, in response to strong (up to 90 mV) or long-lasting prepulses (up to 200 ms), tail current amplitudes and decay times were equally increased in dysgenic myotubes expressing either YFP-CaV1.11860 or YFP-CaV1.11666, suggesting that the distal C-terminal domain was not required for depolarization-induced potentiation. Thus, our experiments do not support the existence of either biochemical or functional interactions between proximal CaV1.1 and the distal C terminus.

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

CaV1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult CaV1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant CaV1.1a, the embryonic CaV1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike CaV1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in CaV1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (CaV1.1-null) myotubes reconstituted with either CaV1.1a or CaV1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric CaV1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in CaV1.1a had abolished the calcium currents, substitution of the II-III loop in CaV1.1e did not significantly reduce current amplitudes. This indicates that CaV1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in CaV1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, CaV1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, CaV1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant CaV1.1e in the skeletal muscles of myotonic dystrophy patients.

Multiple Regions Inhibit Expression of Cav1.1 Ca2+ Channels in Non-Muscle Cells

Compared to the cardiac CaV1.2, the skeletal CaV1.1 Ca2+ channel has been very difficult to express in non-muscle cells. To identify domains of CaV1.1 that inhibit such expression, we have co-transfected tsA201 cells with β1a and chimeric constructs of CaV1.1 and CaV1.2 tagged with YFP. A tandem construct of CaV1.1 linked to CaV1.2 expressed robustly in tsA201 cells, suggesting that either CaV1.2 masks a sequence of CaV1.1 which causes ER retention in non-muscle cells, or that CaV1.1 lacks a signal important for ER export. Replacement of individual transmembrane repeats of CaV1.1 (in non-tandem constructs) with the corresponding repeat of CaV1.2 was ineffective at increasing functional expression as indicated by slowly activating, L-type Ca2+ current and/or membrane-bound charge movement. However, simultaneous replacement of all four CaV1.1 repeats with those of CaV1.2, while retaining CaV1.1 sequence for the cytoplasmic domains (construct “CSk9”), resulted in increased expression, although not to the level of CaV1.2. By contrast, expression levels of CSk9 and CaV1.2 were equivalent in dysgenic myotubes. Thus, both the transmembrane and cytoplasmic domains appear to play a role in suppressing expression of CaV1.1 in non-muscle cells. To investigate which cytoplasmic domains might be particularly important at suppressing expression, we tested CaV1.2 constructs in which the I-II loop, II-III loop, I-II and II-III loops, or C-terminus were replaced with CaV1.1 sequence and found expression not to differ for any of these constructs from that of the entirely CaV1.2 construct. We are currently testing constructs of CaV1.2 with other combinations of cytoplasmic domains replaced by CaV1.1.

Differential Stability of CaVβ2a and CaVβ3 in a CaV1.2 Calcium Channel Complex

In voltage-gated calcium channels, auxiliary CaVβ subunits function in membrane targeting of the channel and modulation of its gating properties. Structural studies have revealed that the high affinity CaVα1/β interaction relies on a conserved CaVβ-binding sequence in the I-II loop of the CaVα1 subunit, called the α-interaction domain (AID). We have previously shown that in skeletal muscle triads the β1a subunit forms stable complexes with both CaV1.1 and CaV1.2, while β2a forms dynamic complexes (Campiglio et al., JCS 2013). To further test the reversibility of the CaV1.2/β2a interaction, here we injected an AID peptide into Xenopus oocytes expressing CaV1.2/β2a/α2δ-1 complexes and examined the effect of the competing peptide on current inactivation. Unexpectedly, the AID peptide had no effect on the currents of wildtype CaV1.2/β2a/α2δ-1 channel complexes. However, when a CaV1.2 AID residue known to decrease the interaction with β2a by 1000-fold was mutated (Y437A), injection of AID peptide changed the inactivation properties of the channels similar to properties of channels expressed without a β subunit. These results suggest that under our experimental conditions binding of the wild-type CaV1.2/β2a pair is too strong for peptide competition. In contrast, in wild-type CaV1.2 channel complexes containing the β3 subunit injection of the AID peptide caused a strong effect on inactivation properties, indicating efficient competition. Consistent with a lower stability of CaV1.2/β3 complexes compared to CaV1.2/β2a, β3 also showed a significantly reduced extent of co-clustering with CaV1.2 in the triads of dysgenic myotubes. Ongoing FRAP experiments will reveal whether diminished co-clustering of β3 correlates with higher mobility and dynamic β subunit exchange in a native calcium channel signaling complex. Funding: FWF P23479 and W1101 (BEF), NIH R01 HL080050 (DLM).

The Cytoplasmic Foot of RyR1 forms a Stable Homotetrameric Structure Despite Lacking the Membrane-Spanning C-Terminal Domains

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane engages in bi-directional interactions with the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) such that an “orthograde” signal from the DHPR triggers SR Ca2+ release via RyR1, and a retrograde signal from RyR1 enhances the L-type Ca2+ current via the DHPR. RyR1 (5,037 residues) assembles as a homotetramer in which the C-termini form the ion conducting pore across the SR membrane, with the remainder of the protein forming a “foot” structure which spans between the SR and plasma membranes. In an accompanying abstract (Polster et al., this meeting) we showed that a construct (YFP-RyR11:4300) which lacks the C-terminal residues was diffusely distributed in dysgenic myotubes (DHPR null), but that in dyspedic myotubes (RyR1-null but containing DHPRs) it targeted junctionally and retrogradely enhanced L-type Ca2+ current. Here we have examined the oligormerization of YFP-RyR11:4300. Measurement of FRAP for YFP-RyR11:4300 in dysgenic myotubes yielded a diffusion coefficient of 2.17x10−8 cm2/sec, which, based on previous measurements of protein diffusion in muscle, is compatible with YFP-RyR11:4300 existing as a tetramer. We also used SDS PAGE (4-15% gradient) and immunoblotting to compare YFP-RyR11:4300 (expressed in tsA-201 cells) and native RyR1. Without prior cross-linking, YFP-RyR11:4300 displayed a smaller apparent MW than wt RyR1; after glutaraldehyde cross-linking, both proteins migrated as single bands of much higher apparent MW, with YFP-RyR11:4300 again displaying a slightly greater mobility, consistent with it's being a tetramer.

The Cytoplasmic Foot of RyR1 without the Membrane Spanning Domain Targets Junctionally and Retrogradely Enhances DHPR L-Type Ca2+ Currents

In skeletal muscle, RyR1 (5,037 residues) forms a homo-tetrameric Ca2+-release channel in the sarcoplasmic reticulum (SR), mediates excitation-contraction coupling in response to an orthograde signal from the DHPR in the plasma membrane, and retrogradely enhances L-type Ca2+ current via the DHPR. The RyR1 C-terminus forms the Ca2+ channel pore across the SR membrane and is believed to be important for inter-subunit interactions, whereas the bulk (∼85%) of the protein (the so-called foot) bridges the junctional, myoplasmic gap between the SR and plasma membranes. Here, we have examined the ability of the foot domain (residues 1-4300; RyR11:4300) to target junctionally and interact with the DHPR by expression of a cDNA encoding YFP-RyR11:4300. In dysgenic (α1S-null) myotubes which lack DHPRs, YFP-RyR11:4300 was diffusely distributed and, on the basis of photobleaching, freely mobile within the cytoplasm, consistent with the loss of membrane anchoring. However, after expression in dyspedic (RyR1 null) myotubes (which have DHPRs), much of YFP-RyR11:4300 was immobile within fluorescent foci near the myotube surface, suggestive of junctional targeting and binding to DHPRs. Junctional targeting was confirmed by partial co-localization of YFP-RyR11:4300 and CFP-labeled α1S after co-expression in dyspedic myotubes. Strikingly, YFP-RyR11:4300 was able to retrogradely enhance peak Ca2+ current in dyspedic myotubes from 1.6 pA/pF (control) to 6.7 pA/pF, similar to that after expression of full-length RyR1 (7.4 pA/pF). Thus, the isolated, cytoplasmic foot of RyR1 retains the ability to target junctionally and to interact functionally with the DHPR.

Effects of Substituting Tryptophan for Basic Residues in the S4 Voltage-Sensing Helices of CaV1.1

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).

Stable incorporation vs. dynamic exchange of β subunits in a native calcium channel complex

Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of β relative to the α1 subunit, altered the specific dynamic properties of the β subunits. In contrast, a single residue substitution in the α interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the β subunit isoforms.

Dynamics of Homologous and Heterologous Calcium Channel α1 and β Subunits in the Skeletal Muscle Triad

Skeletal muscle EC-coupling requires direct interactions of the dihydropyridine receptor (DHPR) with the ryanodine receptor (RyR1) and its organization in tetrads opposite RyR1. Accordingly, tetrad formation and skeletal muscle EC-coupling fail in myotubes expressing the cardiac α1C subunit or lacking the β1a subunit. This indicates that tissue-specific protein-protein interactions between α1S, β1a and RyR1 are important for the structure and function of the skeletal muscle EC-coupling apparatus. Here we apply fluorescence recovery after photobleaching (FRAP) in dysgenic (α1S-/-) myotubes reconstituted with GFP-tagged DHPR subunits to probe the importance of these interactions in stabilizing them in the triad. Consistent with its anchoring in the triad, the α1S subunit is essentially immobile. Surprisingly, also the cardiac α1C subunit, which does not interact with RyR1, is immobile. Thus, the II-III loop - RyR1 interaction is not the determing factor for stabilizing α1S in the triad. When coexpressed with α1S, the β1a subunit showed a higher fluorescence recovery rate than the α1 subunits, suggesting that it can be dynamically exchanged in the complex. Again, the recovery did not increase when β1a was coexpressed with α1C, indicating that its putative interaction with RyR1 does not contribute to the stability of β1a in the complex. Previously we showed that all βisoforms can associate with α1S and α1C in the triads. When the non-muscle β2a or β4b subunits were expressed with α1S in dysgenic myotubes their recovery after photobleaching was significantly higher than that of β1a, demonstrating that the homologous β subunit binds the channel complex with higher affinity than heterologous β subunits. Funding: FWF P20059, W1101, T443-B18.

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.