Structural Analysis of the Voltage-Dependent Calcium Channel β Subunit Functional Core and Its Complex with the α1 Interaction Domain

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Homozygous zebrafish of the mutant relaxed (red ts25 ) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarizationinduced but not caffeine-induced Ca 2؉ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the ␣1S subunit is normal, whereas the ␤1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the ␤1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a ␤1a-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the ␣1S subunit in the absence of ␤1a. Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an Ϸ2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the ␤1a subunit does not prevent triad targeting of the DHPR ␣1S subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitationcontraction coupling in ␤1-null model organisms.calcium channels ͉ excitation-contraction coupling ͉ tetrads ͉ zebrafish E xcitation-contraction (EC) coupling is understood as the signal transduction process connecting membrane depolarization to the contraction of muscle cells. This process is initiated by the concerted action of two Ca 2ϩ channels, the plasmalemmal voltage-gated dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptor (RyR). In junctions of the SR with the plasma membrane (peripheral couplings) or with the transverse tubules (triads), membrane depolarization is sensed by the DHPR, which then triggers RyR opening and Ca 2ϩ release from the SR. In skeletal muscle cells, this signaltransduction is independent of Ca 2ϩ influx through the DHPR (1) but depends on protein-protein interaction between the DHPR and the RyR1 (2, 3). This physical coupling requires the coordinated arrangement of DHPRs and RyR1s in the junctions. In skeletal muscle triads and peripheral couplings, groups of four DHPRs (tetrads) are arranged in orthogonal arrays matching the opposing RyR1 arrays (4). Formation of DHPR tetrads requires the presence of RyR1.The skeletal muscle DHPR complex is composed of the voltage-sensing and pore-forming ␣ 1S subunit and the auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ (5). Targeted deletions of the ␣ 2 ␦-1 and ␥ subunits do not critically interfere with EC coupling function (6, 7). In contrast, ␣ 1S and ␤ 1a subunit null-mutant mice display a lack of EC coupling and, thus, lethal muscle paralysis (8, 9). Although failure o...

Section: Resultsmentioning

confidence: 99%

Section: ) Statistical Significance Was Determined By Using the ⌬Cmentioning

confidence: 99%

The β _1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle

Schredelseker

Biase

Obermair

et al. 2005

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164

“…Cav ␤ subunits are cytoplasmic proteins that strongly regulate Cav channels through direct interaction with pore-forming ␣1 subunits (14)(15)(16)(17). The ␤ subunits are also critical for assembly of the channel complex (18), correct plasma membrane targeting (19,20), and stimulation of channel activity (21).…”

Section: Depolarization In Cav Channel Opening In T Cells T Cell Recmentioning

confidence: 99%

Critical role for the β regulatory subunits of Cav channels in T lymphocyte function

Badou¹,

Jha²,

Matza³

et al. 2006

147

Calcium ion is a universal signaling intermediate, which is known to control various biological processes. In excitable cells, voltagegated calcium channels (Cav) are the major route of calcium entry and regulate multiple functions such as contraction, neurotransmitter release, and gene transcription. Here we show that T lymphocytes, which are nonexcitable cells, express both regulatory ␤ and pore-forming Cav1 ␣1 subunits of Cav channels, and we provide genetic evidence for a critical role of the Cav ␤3 and Cav ␤4 regulatory subunits in T lymphocyte function. Cav ␤-deficient T lymphocytes fail to acquire normal functions, and they display impairment in the T cell receptor-mediated calcium response, nuclear factor of activated T cells activation, and cytokine production. In addition, unlike in excitable cells, our data suggest a minimal physiological role for depolarization in Cav channel opening in T cells. T cell receptor stimulation induces only a small depolarization of T cells, and artificial depolarization of T cells using KCl does not lead to calcium entry. These observations suggest that the Cav channels expressed by T cells have adopted novel regulation͞gating mechanisms.C alcium ion plays critical and specific roles in various T cell functions, including activation, differentiation, proliferation, and cytokine production (1, 2). In T lymphocytes, ligation of the T cell receptor (TCR) by antigen leads to the release of calcium from intracellular stores, triggering the calcium release-activated calcium current (3), and a potential candidate for calcium release-activated calcium current channel was recently reported (4-6). But the complexity of the calcium response in T cells suggests the expression of more than one plasma membrane calcium channel. Although it is established that, in excitable cells, Cav channels constitute the major route of calcium entry (7), the functional presence of the Cav1 channels in T lymphocytes has been suggested (8-13) but has remained controversial because of the lack of a reliable and specific loss-of-function approach.Cav ␤ subunits are cytoplasmic proteins that strongly regulate Cav channels through direct interaction with pore-forming ␣1 subunits (14-17). The ␤ subunits are also critical for assembly of the channel complex (18), correct plasma membrane targeting (19,20), and stimulation of channel activity (21). A number of potential ␣1-␤ combinations are likely to form a Cav channel complex (22), and, among these, the ␤4 and ␤3 subunits are key subunits that associate with Cav1 channels (23-25).A spontaneous mutation named lethargic, which arose in the mouse inbred strain BALB͞cGn in 1962, is recognizable in homozygous mice at 2 weeks of age by the onset of ataxia, seizures, and lethargic behavior (26,27). These mice also exhibit a generalized immunological disorder including defective cell-mediated immune responses (28). It has been reported, using a positional cloning approach, that this syndrome was the result of a mutation of the Cav ␤4 subunit gene (29). This mutatio...

“…2 and 3). Both effects depend critically on the binding of Ca v β to the α interacting domain (AID) in the cytoplasmic loop (referred to as the I-II loop) connecting the first two homologous repeats of Ca v α 1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Gating regulation by Ca v β also needs a continuous α-helix between the AID and the S6 segment of the first repeat (IS6) of Ca v α 1 (3,8,9,12).…”

mentioning

confidence: 99%

Direct inhibition of P/Q-type voltage-gated Ca ²⁺ channels by Gem does not require a direct Gem/Ca _v β interaction

Fan

Buraei

Luo

et al. 2010

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca 2+ channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca 2+ channel β subunits (Ca v β), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca 2+ channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca v β but not the Gem/Ca v β interaction, and that Gem coimmunoprecipitates with the P/Q channel α 1 subunit (Ca v α 1 ) in a Ca v β-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca v α 1 critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca v α 1 abolishes Gem inhibition of P/Q channels and confers Ca v β-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca v α 1 that binds Ca v β). Our results challenge the prevailing view regarding the role of Ca v β in RGK inhibition of high voltage-activated Ca 2+ channels and prompt a paradigm in which Gem directly binds and inhibits Ca v β-primed Ca v α 1 on the plasma membrane.