Β1a Subunit Research Articles

Germline de novo variant F747S extends the phenotypic spectrum of CACNA1D Ca2+ channelopathies.

Germline gain-of-function missense variants in the pore-forming Cav1.3 α1-subunit (CACNA1D gene) confer high risk for a severe neurodevelopmental disorder with or without endocrine symptoms. Here, we report a 4-week-old new-born with the novel de novo missense variant F747S with a so far not described prominent jittering phenotype in addition to symptoms previously reported for CACNA1D mutations including developmental delay, elevated aldosterone level and transient hypoglycemia. We confirmed the pathogenicity of this variant in whole-cell patch-clamp experiments with wild-type and F747S mutant channels heterologously expressed together with α2δ1 and cytosolic β3 or membrane-bound β2a subunits. Mutation F747S caused the quantitatively largest shift in the voltage dependence of activation (-28mV) reported so far for CACNA1D germline mutations. It also shifted inactivation to more negative voltages, slowed the time course of current inactivation and slowed current deactivation upon repolarization with both co-expressed β-subunits. In silico modelling and molecular docking, simulations revealed that this gain-of-function phenotype can be explained by formation of a novel inter-domain hydrogen bond between mutant residues S747 (IIS6) with N1145 (IIIS6) stabilizing selectively the activated open channel state. F747S displayed 2-6-fold increased sensitivity for the L-type Ca2+ channel blocker isradipine compared to wild type. Our data confirm the pathogenicity of the F747S variant with very strong gain-of-function gating changes, which may contribute to the novel jittering phenotype. Increased sensitivity for isradipine suggests this drug for potential symptomatic off-label treatment for carriers of this mutation.

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.

CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.

Which CaV1.1 Voltage Sensor(s) Activate RYR1?

The skeletal muscle L-type Ca­V channel is composed by the pore-forming α1s (CaV1.1) in association with α2δ-1, β1a and γ1 subunits in 1:1:1:1 stoichiometry. This large protein complex makes physical contact with Ryanodine (RyR1) channel located in the sarcoplasmic reticulum (SR), conferring voltage-dependence to the Ca2+ release channel, thus coupling electrical excitation with contraction. CaV1.1 channels possess four homologous but non-identical voltage sensing domains (VSDs), which undergo voltage-dependent rearrangements: we propose that one VSD is a stronger candidate than all others to be the sensor responsible for RyR1 voltage-dependent activation. We reconstituted human CaV1.1 complexes (α1s, α2δ-1, β1a, γ1 and stac3) in Xenopus oocytes. We used the voltage clamp fluorometry technique to simultaneously record Ca2+ currents (2mM external Ca2+) and the structural rearrangements of the four individual VSDs. We found that the voltage-dependent activation curves of the four VSDs are spread from −150 to +80mV, all preceding pore opening, with VSD-I and VSD-IV operating at more hyperpolarized potentials (VSD-I Vhalf=-35.6±2.2mV, z=1.0±0.01e0, VSD-IV Vhalf=-64.9±2.7mV, z=0.74±0.02e0) than VSD-II and VSD-III (VSD-II Vhalf=7.2±2.8mV, z=1.6±0.1e0, VSD-III Vhalf=-23.9±3mV, z=1.6±0.04e0). Each VSD exhibited distinct time-dependent properties, with VSD-I and VSD-II displaying slow activation kinetics (VSD-I τ=39.8±2ms, VSD-II τ=132±16ms, at 20mV), while VSD-III and VSD-IV activated rapidly (VSD-III τ=1.8±0.2ms, VSD-IV τ=2±1ms, at 20mV). The operation of the four voltage sensors was then investigated under a physiologically-relevant action potential (AP) clamp (skeletal muscle AP). Within the short AP clamp (∼5ms), no clear structural rearrangements of the slowest sensors (VSD-I and VSD-II) could be observed, while VSD-III and VSD-IV were readily activated. Based on i) kinetics, ii) voltage-dependence, iii) proximity with intracellular II-III loop (known to be a critical element in EC coupling), VSD-III is a strong candidate as sensor conferring voltage-dependent properties to RyR1 and the SR Ca2+ release.

The Ca2+ channel subunit CaV β2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

β-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca2+ channels (CaV β2a) and its SH3 domain (β2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV β2a or β2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV β2a in the presence of exogenous α1-interaction domain. β2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following β2a-SH3 injection. Furthermore, β2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV β2a and β2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

RGK Proteins Preferentially Inhibit Fast-Inactivating Voltage-Gated Calcium Channels: Implications for Human Disease

Voltage-gated calcium channels (VGCC) are critical for nerve, heart, and skeletal muscle function, and their mutations can cause neurological and cardiovascular disease. Some of these mutations alter channel inactivation, leading to aberrant Ca2+ influx into cells. For example, Timothy syndrome (TS) is characterized by severe cardiomyopathy, and is often accompanied by autism spectrum disorder. The abnormalities arise from a point mutation in the L-type VGCC that slows channel inactivation. On the other hand, some types of migraine and epilepsy are associated with point mutations that speed the inactivation of P/Q-type VGCC. RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that strongly inhibit L-, N-, P/Q- and R-type VGCC. They are difficult to study because their overexpression nearly completely abolishes VGCC currents. Thus, we studied RGK-mediated inhibition using two-electrode voltage clamp (TEVC) in Xenopus oocytes, where we titrated the amount of injected RNA to achieve a more physiological level of VGCC inhibition of ∼50%. Under these conditions, we discovered that mutations that speed inactivation, such as those that cause familial hemiplegic migraine, turn channels hypersensitive to Gem inhibition. Remarkably, when we challenged the non-inactivating TS channel with Gem, we found that it was insensitive to inhibition. Furthermore, expressing VGCC with the β2a subunit, a Ca2+ channel auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition.This could potentially occur if Gem were to trap channels in the inactivated state, thereby inhibiting inactivating channels, while sparing non-inactivating channels. Our additional findings corroborate this since channel recovery from inactivation is much slower in the presence of Gem.These results shed light on a new type of VGCC regulation. In addition, our results point to RGKs as potential players in calcium channelopathies.

Stac3 enhances expression of human CaV1.1 in Xenopus oocytes and reveals gating pore currents in HypoPP mutant channels.

Mutations of CaV1.1, the pore-forming subunit of the L-type Ca2+ channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). However, functional assessment of HypoPP mutant channels has been hampered by difficulties in achieving sufficient plasma membrane expression in cells that are not of muscle origin. In this study, we show that coexpression of Stac3 dramatically increases the expression of human CaV1.1 (plus α2-δ1b and β1a subunits) at the plasma membrane of Xenopus laevis oocytes. In voltage-clamp studies with the cut-open oocyte clamp, we observe ionic currents on the order of 1 μA and gating charge displacements of ∼0.5-1 nC. Importantly, this high expression level is sufficient to ascertain whether HypoPP mutant channels are leaky because of missense mutations at arginine residues in S4 segments of the voltage sensor domains. We show that R528H and R528G in S4 of domain II both support gating pore currents, but unlike other R/H HypoPP mutations, R528H does not conduct protons. Stac3-enhanced membrane expression of CaV1.1 in oocytes increases the throughput for functional studies of disease-associated mutations and is a new platform for investigating the voltage-dependent properties of CaV1.1 without the complexity of the transverse tubule network in skeletal muscle.

Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β1a subunit and assess its role on Ca2+ metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β1a subunit. Expression of the variant subunit in β1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β1a-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca2+ conductance, current kinetics, or sarcoplasmic reticulum Ca2+ load were observed. Measurement of resting free Ca2+ and Na+ concentrations shows that both cations were significantly elevated in β1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+ entry through Na+/Ca2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant β1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

Calmodulin and ATP support activity of the Cav1.2 channel through dynamic interactions with the channel.

Key points Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode.α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail.ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels.These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation.CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent. Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+‐dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C‐terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co‐expressed with β2a and α2δ subunits in HEK293 cells. In the inside‐out mode, α1C and α1C∆ showed minimal open‐probabilities in a basic internal solution (run‐down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3–5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7–9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C‐terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl‐terminal tail is removed.

Core skeletal muscle ryanodine receptor calcium release complex.

The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum (SR) calcium store. The protein complex is essential for skeletal excitation-contraction (EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins. The proteins essential for EC-coupling include the DHPR α1S subunit in the transverse tubule membrane, the DHPR β1a subunit in the cytosol and the RyR1 ion channel in the SR membrane. The other core proteins are triadin and junctin and calsequestrin, associated mainly with SR. These SR proteins are not essential for survival but exert structural and functional influences that modify the gain of EC-coupling and maintain normal muscle function. This review summarises our current knowledge of the individual protein/protein interactions within the core complex and their overall contribution to EC-coupling. We highlight significant areas that provide a continuing challenge for the field. Additional important components of the Ca2+ release complex, such as FKBP12, calmodulin, S100A1 and Stac3 are identified and reviewed elsewhere.

Stac3 has a direct role in skeletal muscle-type excitation–contraction coupling that is disrupted by a myopathy-causing mutation

In skeletal muscle, conformational coupling between CaV1.1 in the plasma membrane and type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) is thought to underlie both excitation-contraction (EC) coupling Ca(2+) release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca(2+) current via CaV1.1. Recent work has shown that EC coupling fails in muscle from mice and fish null for the protein Stac3 (SH3 and cysteine-rich domain 3) but did not establish the functional role of Stac3 in the CaV1.1-RyR1 interaction. We investigated this using both tsA201 cells and Stac3 KO myotubes. While confirming in tsA201 cells that Stac3 could support surface expression of CaV1.1 (coexpressed with its auxiliary β1a and α2-δ1 subunits) and the generation of large Ca(2+) currents, we found that without Stac3 the auxiliary γ1 subunit also supported membrane expression of CaV1.1/β1a/α2-δ1, but that this combination generated only tiny Ca(2+) currents. In Stac3 KO myotubes, there was reduced, but still substantial CaV1.1 in the plasma membrane. However, the CaV1.1 remaining in Stac3 KO myotubes did not generate appreciable Ca(2+) currents or EC coupling Ca(2+) release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca(2+) currents and EC coupling Ca(2+) release, whereas expression of Stac3W280S (containing the Native American myopathy mutation) partially restored Ca(2+) currents but only marginally restored EC coupling. We conclude that membrane trafficking of CaV1.1 is facilitated by, but does not require, Stac3, and that Stac3 is directly involved in conformational coupling between CaV1.1 and RyR1.

Abstract 435: The Mitochondrial Calcium Uniporter is Necessary for Cardiac Energetic Signaling During Chronic Stress

The mitochondrial calcium uniporter (MCU) is a multicomponent channel that is the primary mechanism for mitochondrial Ca 2+ uptake ( m Ca 2+ ). We previously reported that the MCU is required for energetic signaling to meet contractile demand during the ‘fight or flight’ response. In addition, we showed that deletion of the pore-forming component ( Mcu gene) protected against mitochondria permeability transition pore (MPTP) opening and ischemia-reperfusion injury. However, results from our study and others questioned the physiological relevance of MCU-mediated Ca 2+ uptake during chronic stress states featuring sustained intracellular Ca 2+ load ( i Ca 2+ ). To address this, we deleted Mcu from cardiomyocytes in adult mice ( Mcu cKO) and implanted osmotic pumps to deliver the β adrenergic agonist isoproterenol (iso, 70 mg/kg/day for 14d). In contrast to controls, Mcu cKO mice lacked contractile responsiveness to chronic βAR stimulation with evidence of LV dysfunction and failure by d14 ( Fig 1 ). Next, we crossed the Mcu cKO with mice overexpressing the β2a subunit (β2a-Tg) of the L-type Ca 2+ channel (LTCC). This model displays enhanced LTCC activity and cardiac contractility, but with added stress such as iso infusion, Ca 2+ overload eventually leads to MPTP-dependent cell death and heart failure. Surprisingly, loss of Mcu in this model was lethal with all mice dying by d13 ( Fig 2 ). Baseline echocardiography revealed that loss of Mcu ablated all β2a-mediated enhancements in LV contractility and accelerated dysfunction post-iso. These findings demonstrate that MCU-mediated m Ca 2+ uptake is critical to meet energetic demand during chronic stress states featuring sustained i Ca 2+ load.

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.

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).

Persistent increases in Ca(2+) influx through Cav1.2 shortens action potential and causes Ca(2+) overload-induced afterdepolarizations and arrhythmias.

Persistent elevation of Ca(2+) influx due to prolongation of the action potential (AP), chronic activation of the β-adrenergic system and molecular remodeling occurs in stressed and diseased hearts. Increases in Ca(2+) influx are usually linked to prolonged myocyte action potentials and arrhythmias. However, the contribution of chronic enhancement of Cav1.2 activity on cardiac electrical remodeling and arrhythmogenicity has not been completely defined and is the subject of this study. Chronically increased Cav1.2 activity was produced with a cardiac specific, inducible double transgenic (DTG) mouse system overexpressing the β2a subunit of Cav (Cavβ2a). DTG myocytes had increased L-type Ca(2+) current (ICa-L), myocyte shortening, and Ca(2+) transients. DTG mice had enhanced cardiac performance, but died suddenly and prematurely. Telemetric electrocardiograms revealed shortened QT intervals in DTG mice. The action potential duration (APD) was shortened in DTG myocytes due to significant increases of potassium currents and channel abundance. However, shortened AP in DTG myocytes did not fully limit excess Ca(2+) influx and increased the peak and tail ICa-L. Enhanced ICa promoted sarcoplasmic reticulum (SR) Ca(2+) overload, diastolic Ca(2+) sparks and waves, and increased NCX activity, causing increased occurrence of early and delayed afterdepolarizations (EADs and DADs) that may contribute to premature ventricular beats and ventricular tachycardia. AV blocks that could be related to fibrosis of the AV node were also observed. Our study suggests that increasing ICa-L does not necessarily result in AP prolongation but causes SR Ca(2+) overload and fibrosis of AV node and myocardium to induce cellular arrhythmogenicity, arrhythmias, and conduction abnormalities.

Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β1a subunit.

BackgroundAlthough excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1.MethodsWe used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes.ResultsWe demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10–100 nM of the β1a subunit. Activation of RyR2 by 10 nM β1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for β1a. A reduction in activation was also observed when 10 nM β1a was added to the alternatively spliced (ASI(−)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM β1a activation observed for both RyR2 and ASI(−)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling.ConclusionsWe conclude that the β1a subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0049-3) contains supplementary material, which is available to authorized users.

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes

The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers.

In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for β(1a) has been difficult to test because β(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, β(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a β(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with β(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β(1a) subunit in skeletal-type EC coupling.

Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Cavβ1a NH2-terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation.