Direct Estimation of CaV1.2 Gating Parameters: Quantification of Voltage Sensor – Pore Transductions and their Modulation by FLP 64176

Calcium agonists such as FPL 64174 increase macroscopic calcium channel currents and induce substantial changes in current kinetics. Their molecular mechanism of action is currently unknown. Here we propose a technique enabling the estimation of FPL 64174 effects on rate constants of the voltage sensing machinery and pore transitions from macroscopic CaV1.2 current kinetics making use of a hybrid stochastic-deterministic optimization procedure. Current traces of wild type CaV1.2, a channel construct with neutralized segment IIS4 (IIS4N) and a pore mutant (A780T) were fitted to a circular four-state (rest, activated, open, deactivated) channel model in control and FPL 64174 (1 µM). The estimated rate constants provided novel insights in how structural elements of the voltage sensing unit and the channel pore influence the action of FPL 64174. The new approach may be applicable for the analysis of drug effects on other ion channels as well as for quantification of VS transitions and rate constants of pore gating from macroscopic current kinetics.