Optically-Resolved Conformational Rearrangements of the Voltage-Sensing Domains of the Human CaV1.2 Channel

Abstract

Voltage-gated Ca2+ channels (CaV) consist of four tandem transmembrane repeats (I-IV), each including two pore-forming helices and four segments that make up a voltage-sensing domain (VSD). To understand voltage-dependent gating in human CaV1.2 channels, we have optically tracked the activation of the S4 helices of repeats I, III and IV by site-directed fluorescent labeling of introduced Cysteines with thiol-reactive probes. The channels were co-expressed with their modulatory subunits β3 and α2δ in Xenopus oocytes. Ionic current and fluorescence emission were simultaneously recorded using the cut-open oocyte voltage clamp fluorometry technique. Prior voltage-clamp, oocytes were injected with 100 nl 80 mM BAPTA.4K, to prevent the activation of endogenous Ca2+-gated Cl− channels. The extracellular solution contained 2 mM Ba2+ or 10 mM Ca2+ as charge carrier. Gating currents were recorded by replacing Ca2+ or Ba2+ with Co2+. 0.1 mM ouabain was added to abolish Na+/K+ ATPase non-linear charge movement. Voltage-dependent fluorescence changes (ΔF) were reported by fluorophores attached to substituted Cysteines at the extracelular end of the S4 segment in repeats I, III and IV, tracking local, voltage-dependent conformational rearrangements. The voltage dependence of observed fluorescence deflections preceded ionic activation, reporting repeat-specific VSD transitions taking place during shut states of the channel. Prolonged sojourns at depolarized potentials (+50 mV) shifted the voltage-dependence of reported ΔF to more hyperpolarized potentials by >50mV, recapitulating previously-characterized gating current properties. In summary, we report the first optical characterization of VSD conformational changes in a human voltage-gated Ca2+ channel, revealing repeat-specific voltage- and time-dependent properties.