Molecular Composition of Functional KCNQ Channels in Vascular Smooth Muscle Cells Based on Effects of Diclofenac

Abstract

KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, though it remains uncertain how these subunits assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics and effects of diclofenac, a known KCNQ channel activator, on human KCNQ4 and KCNQ5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells. The conductance curves of the overexpressed channels were fit by a single Boltzmann function in each case (V0.5 values: −31 mV, −44 mV, and −38 mV for KCNQ4, KCNQ5, and KCNQ4/5, respectively). Diclofenac (100 μM) increased maximum conductance of KCNQ4 channels by 38%, but inhibited KCNQ5 and KCNQ4/5 channels, reducing maximum conductance by 53% and by 32% respectively. Differences in deactivation rates and distinct voltage-dependent effects of diclofenac on channel activation and deactivation observed with each of the subunit combinations (KCNQ4, KCNQ5 and KCNQ4/5) were used as diagnostic tools to evaluate native KCNQ currents in vascular smooth muscle cells. A7r5 cells express only KCNQ5 channels endogenously and their responses to diclofenac closely resembled those of the overexpressed KCNQ5 currents. In contrast, mesenteric artery myocytes, which express both KCNQ4 and KCNQ5 channels, displayed whole-cell KCNQ currents with properties and diclofenac responses characteristic of overexpressed heteromeric KCNQ4/5 channels.