Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

Figure 1.. Muscle and CaV1.1 organization. A) Muscle to muscle cell perspective. (left) Morphology of a segment of a skeletal muscle fiber (right). Note the characteristic striated pattern of muscle fibers, which results from highly organized array between sarcolemma, sarcoplasmic reticulum (SR), contractile elements and cytoarchitecture of the fibers. B) Structure of the triad. The cartoon depicts the triad, a specialized membrane-organelle array formed by the T-tubule and two segments of the terminal junctional SR. The T-tubules are infoldings of the sarcolemma that propagate the action potential radially into the fiber. CaV1.1 (blue) are located at the T-tubules, working primarily as voltage sensors that initiate the early steps of EC coupling. The SR Ca2+ release channel, RyR1 (brown), is predominantly located on the junctional domain of the SR surface. Typical profiles of triads (cross-sections) contain only two rows of RyR1 associated with alternating tetrads [2]. C) Detailed architecture of the triad with a focus on CaV1.1 tetrads and RyR1 arrays as shown in (b). About half of the total RyR1s do not associate with CaV1.1, resulting in an alternating pattern of “free” and…

Figure 2.. CaV1.1 structure. Heteromultimeric protein complex of CaV1.1. α1S, β1a, α2δ-1, γ subunits, and SH3 domains of Stac3 are colored in blue, green, Orange, yellow, and purple, respectively. B) Side and upper views of the α1S subunit, each domain is shown in shades of blue. Red dots indicate Ca2+ ions. Panels A and B were prepared with Chimera [22]. Protein data bank (PDB) IDs: 5GJV (CaV1.1) and 6UY7 (Stac3). STAC3 orientation and position relative to α1 and the β subunits is unknown.

Figure 3.. Topology of the voltage sensor and selectivity filter domain of CaV1.1 α1S subunit. A) Cartoon of α1S subunit topology shows four homologous but non-identical domains, each containing six transmembrane helices (S1-S6). S1-S4 represents the voltage sensing domain (VSD, blue) while S6-S6 represents the pore-forming domain (Pore, yellow). Each domain contains an S5-S6 loop (P-Loop) buried in the pore acting as a selectivity filter. Intracellular loops connecting each domain are variable in length. The I–II and II–III loops are critical for EC coupling. Deletion of Exon 29 induces a 19 amino acid shortening of the S3-S4IV extracellular loop (red). Positively charged lysine and arginine within S4 are indicated as a “+” while countercharges within S2 and S3 are indicated as “-“. B) Selectivity filter amino acid sequences of rabbit, human, and zebra fish CaV1.1 and human CaV2.2. Critical glutamate residues used for Ca2+ selectivity are indicated in red, while zebra fish P-loop Ca2+ non-conductive mutations N617D is indicated in cyan. Selectivity filter (SF) sequence is indicated in bold and yellow, while alpha helices within P-loop are indicated in green. C) Top view of rabbit CaV1.1…

Figure 4.. Functional site-directed fluorometry of CaV1.1 S4 signals evaluated in Xenopus Oocytes and muscle fibers. A) Ionic current recording from cut-open oocytes (black) with 2 mM Ba2+ in the external and superimposed fluorometric signal from each VSDs. Note the overlap of the fluorometric and ionic signal for VSD-I. Mean voltage dependence of the fluorometric signal for each VSD from cut open oocytes voltage clamp in presence of 2 mM Ba2+ and fitted with a Boltzmann function. Note the differences in voltage dependence and slope of each VSDs fluorometric signal. C) Normalized fluorometric signal recorded from muscle fibers in response to self-propagated action potential by field stimulation and its comparison with optically measured membrane voltage (AP, yellow), action potential-evoked charge movement (Q, gray), Ca2+ transient (Ca2+, black), and estimated SR Ca2+ release flux (Rel, olive). D) Overlay of normalized fluorometric signals presented in C and kinetics quantification. Note the differences in kinetics for fluorometric signals from different VSDs. Time to peak, rise time, and time to 50% are faster for VSD-II. Panels A and B, and C, reproduced with permission from Refs. .[83]…

Figure 5.. Allosteric model for L-type Ca2+ current and RyR Ca2+ release. A) Hypothetical structural representation of a CaV1.1 tetrad coupled to RyR1 homotetramer. Four α1S subunits (blue) are opposed to a RyR homotetramer (yellow). CaV1.1 has four VSDs that alter their conformation in response to surface transmembranal voltage changes. RyR1 does not have an intrinsic voltage sensing mechanism and relay on the voltage sensing machinery of CaV1.1, via mechanical coupling, to release Ca2+. The α1S-RyR1 organization depicted here is hypothetical but based on the model suggested by Samsó et al. [92]. Side and upper views (left and right respectively) in Panel A were created with BioRender and Chimera [22], PDBs: 5GJW and 5TAL for CaV1.1 and RyR1, respectively. B) Allosteric scheme for voltage dependent CaV1.1 channel opening and RyR1 activation. Four distinct VSDs (VSDI-IV) within one CaV1.1 control CaV1.1's pore conformation from close (“C”) to open (“O”) with either all or some VSDs in active (“A”) or resting (“R”) state. In parallel, four distinct CaV1.1s, each with four VSDs (i.e. tetrads array) control RyR1 pore conformational change from close (“C”) to open (“O”) with either all or some…