Localization and functional properties of Kv8.2-containing voltage-gated potassium channels in primate photoreceptors

Abstract

Purpose : Mutations in the voltage-gated potassium channel subunit, K_V8.2 (KCNV2), underlie an inherited cone-rod dystrophy. However, the role of these channels in normal photoreceptor signaling is unclear. In expression systems, K_V8.2 subunits do not form functional homomeric channels, but instead combine with K_V2 subunits to alter their electrical properties. K_V8.2-containing heteromeric channels may mediate I_Kx (Beech and Barnes, 1989), a delayed-rectifer potassium current thought to counter-balance the dark current and help set the photoreceptor membrane potential. The goals of this study were to: 1) determine the protein localization and subunit interactions of retinal K_V8.2 channels and 2) to characterize the I_Kx current in primate cones.

Methods : We used subunit-specific antibodies to localize K_V8.2, K_V2.1 and K_V2.2 in macaque (n=12) and human retina (n=4). Co-immunoprecipitation and western blot assays were performed on macaque retinal lysates to examine K_V subunit interactions. Voltage-clamp recordings were made from cone inner segments in macaque retinal wholemounts. For these recordings, the cone I_Kx current was isolated by recording in a Ca²⁺-free extracellular solution that contained 1.15 mM Co²⁺ and 4 mM Cs⁺ to block other voltage-gated conductances.

Results : We found that K_V8.2 and K_V2.1 were localized to the membranes of rod and cone inner segments, whereas the Kv2.2 subunit was present only in cone inner segments. The fluorescence intensity of K_V2.2 was significantly lower in S- compared with L/M- cone photoreceptors (p < 0.05, n=4 human retinae). Co-immunoprecipitation experiments revealed interactions between K_V8.2 and both the K_V2.1 and K_V2.2 subunits (n = 3). Finally, we used voltage-clamp recordings to characterize the putative cone I_Kx current. I_Kx activated between -70 and -30 mV, with a maximum conductance of 1.16 ± 0.04 nS, half-maximal activation at -54 ± 1 mV and conductance increasing e-fold every 5.8 ± 0.3 mV (mean ± s.e.m, 11 cones from 3 retinas).

Conclusions : Our results suggest that the K_V8.2 subunit forms heteromeric channels with K_V2.1 in rods and with K_V2.1 and K_V2.2 in cones. These potassium channels are localized to the photoreceptor inner segment where they likely mediate the I_Kx current. Future studies will examine how differences in K_V channel composition impact rod and cone signaling.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.