Probing Mechanisms through which Drugs Affect Voltage-Sensitive Gating

Abstract

Voltage-gated ion channels, which serve as the molecular basis for electrical signaling in excitable cells, represent a valuable target for drug therapy. However, there is a void in the mechanistic understanding of drug action on voltage-dependent gating. In our recent work on PIP2 regulation of Kv7.1, we developed a methodology to isolate the effect of PIP2 on three fundamental gating processes: voltage-sensing domain (VSD) activation, pore-gate domain (PGD) opening, and PGD-VSD coupling. Using Voltage Clamp Fluorometry (VCF) to track VSD movement simultaneously with ionic current to monitor PGD opening, we were able to gauge PIP2 effects on PGD opening and VSD activation. We detected coupling by performing VCF on locked-open channel mutants, allowing us to directly observe the impact of pore-opening on VSD activation through the VSD-PGD coupling. Here we demonstrate the ability of our methodology to provide mechanistic insights into drug effects using the potent Kv7.1 channel activator ML277 (Mattmann 2012). As reported previously, 10 μM ML277 caused a maximal channel potentiation of around 4.5x initial current. We observed channel potentiation at 40 mV, at which point VSD activation is complete, indicating that ML277 acts on processes other than VSD activation. Using VCF, we see that ML277 mildly right-shifts VSD activation in WT channels. This result contradicts pore-opener behavior, which would have left-shifted the fluorescence-voltage (FV) relationship. In contrast we see a dramatic left-shift in the FV relationship of the lock-open mutant, leading to the conclusion that ML277 strengthens the coupling process. Consistently, we saw a higher apparent affinity for ML277 in Kv380 mutants, which have been shown to exhibit increased PIP2 affinity. This study shows the utility of our methodology to analyze drug effects on voltage-sensitive gating.