Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Abstract

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

Keywords: Ca2+ entry; CaV1.2; allostery; fluorometry; gating mechanism.

Fig. 1.

Ca_V membrane topology, putative structure, and S4 helix homology. (A) Ca_V channel-forming α₁ subunits consist of four concatenated repeats, each encompassing one voltage sensor domain (VSD) and a quarter of the central pore domain (PD) (1). Stars indicate the positions of fluorophore labeling. (B) The atomic structure of an Na_V channel (Protein Data Bank ID code 4EKW; top view) (56) shown as a structural representation for the Ca_V α₁ subunit. (C) The α₁ subunit asymmetrically associates with auxiliary β, α₂δ, and calmodulin (CaM) subunits (–16). (D) Sequence alignment of VSD helix S4 from each of four Ca_V1.2 repeats and the archetypal homotetrameric Shaker K⁺ channel. Conserved, positively charged Arg or Lys is in blue. Residues substituted by Cys for fluorescent labeling are marked: F231 (VSD I), L614 (VSD II), V994 (VSD II), and S1324 (VSD IV).

Fig. 2.

Optical tracking of the conformational rearrangements of each human Ca_V1.2 VSD. (A) Membrane current (I_m) from oocytes expressing Ca_V1.2 (α_1C/α₂δ/β₃) channels in extracellular 2 mM Ba²⁺. The traces acquired at pulses from −90 to −90, −40, 0, and 30 mV are shown superimposed above the simultaneously acquired fluorescence traces. The channels were labeled at helix S4 of VSDs I, III, IV (2-((5(6)-Tetramethyl-rhodamine)carboxylamino)ethyl methanethiosulfonate, MTS-TAMRA), or II (tetramethylrhodamine-5'-maleimide, TMRM). Increasing the test potential resulted in local VSD conformational rearrangements resolved as fluorescence deflections (F). (B) No voltage-dependent F is observed in labeled oocytes expressing WT Ca_V1.2 without engineered Cys.

Fig. 3.

Each VSD exhibits distinct voltage dependence and kinetics. (A) Mean normalized conductance (G; black ▼) and charge movement (Q; white right-pointing triangle) from WT channels and F from VSDs I (blue ●), II (red ▲), III (green ◆), and IV (yellow ■). The curves are fits to single or (for G) the sum of two Boltzmann distributions (parameters in SI Appendix, Tables S1 and S3). Error bars indicate ± SEM. (B) Representative membrane current (gray) from WT channels for a −90- → 20-mV pulse, with superimposed F reported from VSD I (τ₁ = 2.6 ms, 59%; τ₂ = 8.1 ms), II (τ₁ = 1.1 ms, 98%; τ₂ = 20 ms), III (τ₁ = 0.88 ms, 68%; τ₂ = 9.2 ms), and IV (τ = 17 ms). The black dashed lines are exponential functions with the reported time constants. Mean kinetic parameters are in SI Appendix, Table S2. The sequence of activation for the Ca_V1.2 VSDs (half-time to maximum, t_0.5) is VSD II (1.0 ms), III (1.4 ms), I (2.9 ms), and IV (11 ms). Ionic currents from Cys mutant channels superimposed to their respective fluorescence traces are in presented in SI Appendix, Fig. S2B.

Fig. 4.

Ca_V structure-based kinetic models to account for optical and electrophysiological data. All schemes assume that each VSD activates independently, resulting in 16 shut states (top tiers). Resting VSDs are in white. Active VSD I is in blue, active VSD II is in red, active VSD III is in green, and active VSD IV is in orange. In scheme I, all VSDs are required to activate before channel opening. SI Appendix, Fig. S3 shows data fitting and model parameters. In scheme II, the activations of VSDs I–III are obligatory for pore opening (SI Appendix, Fig. S4). In scheme III, the activations of VSDs II and III are obligatory for pore opening (SI Appendix, Fig. S5). Scheme IV is an allosteric gating mechanism where VSD activation is not obligatory for pore opening, which in principle, allows opening transitions from any shut state. The activation of each VSD contributes energy W toward stabilizing the open state. A simplified version of scheme IV is shown to the right of scheme IV (Fig. 5 and SI Appendix, Fig. S6).

Fig. 5.

An allosteric model designates VSDs II and III as the drivers for the opening of α_1C/β₃/α₂δ Ca_V1.2 channels. (A) Mean normalized total charge displacement (Q; white right-pointing triangle), ionic conductance (G; black ▼), and F reported from VSDs I (blue ●), II (red ▲), III (green ◆), and IV (yellow ■) with superimposed predictions of scheme IV (curves). (B) Fluorescence traces recorded from each VSD normalized to the steady-state probability of activation (scheme IV predictions are in black). The timescale for VSD IV traces is 75 ms. (C) Membrane current from WT channels (maroon) for the same pulses as in B with superimposed scheme IV predictions (black). Activation of VSDs II and III significantly biases the open state (W₂ ∼ W₃ ∼ −50 meV), and therefore, channel opening occurs at physiologically relevant potentials. VSD I makes a smaller contribution (W₁ = −16 meV), whereas VSD IV is effectively not involved in Ca_V1.2 opening (W₄ < 1 meV). Model parameters are in SI Appendix, Fig. S6B.