LAUSR

An important regulatory mechanism of excitation-evoked Ca2+ influx is voltage-dependent, or pre-pulse, facilitation (VDF): a memory property of some CaV channels, whereby channel activation is enhanced following a pre-conditioning pulse of sufficient duration and amplitude. While VDF can greatly increase Ca2+ influx and confer memory of excitation, the molecular determinants for its voltage- and time-dependent properties are unclear. We posited that VDF emerges from the activity of one, or more, of the four homologous, but non-identical, voltage-sensing domains (VSDs) of the pore-forming α1 subunit. Using voltage clamp fluorometry, we optically tracked the activation of each VSD in human CaV1.2 (α1C) channels expressed in Xenopus oocytes in association with β3 subunits, voltage-clamped by the cut-open oocyte method, in the absence or presence of facilitating pre-pulses (80 mV, 200 ms). Ba2+ was used as the charge carrier to exclude Ca2+-dependent processes. Pre-pulses increased the activation of VSD-I and -II at 20 mV by ∼60% and accelerated VSD-I activation kinetics by ∼10-fold. VSD-III activation was shifted to more hyperpolarized potentials by ∼20mV and doubled in effective valence. In non-facilitated α1C/β3 channels, VSD-II and -III are weakly-coupled to channel opening (Savalli et al., 2016 J Gen Physiol); pre-pulse-induced facilitation of their activation suggests that VDF engages these VSDs with the channel gate. This premise is supported by the finding that association of the α2δ-1 subunit (which mimics facilitation; Platano et al., 2000 Biophys J) strongly couples VSD-II and -III to the channel gate (Pantazis et al., 2014 PNAS). Since VSD-I (i) is also potentiated by pre-pulses, but (ii) is weakly-coupled to channel opening in both non-facilitated α1C/β3 and “constitutively facilitated” α1C/β3/α2δ channels, our work highlights it as a plausible candidate for the structural determinant of VDF.