Abstract
Recently it was shown that KCNQ4-encoded voltage-dependent potassium channels (Kv7.4) play an important role in the regulation of resting vascular tone and in response to endogenous vasodilators (Hypertension, 59:877–884, 2012). However there is lack of information about Kv7.4 basic biophysical properties on the single channel level and about their regulatory mechanisms. Therefore, using standard cell-attached and inside-out configurations of the patch-clamp technique we investigated the single channel properties of Kv7.4 in HEK293 cells stably expressing the channel. We found that NPo of Kv7.4 was 0.61±0.05 (n=9, mean±SEM) in the cell-attached configuration at −50 mV (voltage applied to the patch pipette), whereas in the inside-out configuration at 0 mV NPo of Kv7.4 was 0.04±0.01 (n=9). Based on the inside-out experiments, single channel conductance of Kv7.4 was calculated as 2.31 pS. Linopirdine (10 µM), the pan-Kv7 blocker, abolished the single channel activity in both cell-attached and inside-out configuration when added to the patch pipette, but did not produce significant changes when applied to the bath solution. Bath application of PI(4,5)P2 (1 µM) to the inside-out patches or application of active Gβγ subunits (50 ng/ml) resulted in significant increase of NPo of Kv7.4 correspondingly to 0.27±0.09 (n=3) or to 0.34±0.06 (n=6) without affecting channel conductance (p<0.001 in both cases, determined by 2-way ANOVA). Moreover, bath application of gallein (100 µM), an inhibitor of Gβγ activity, reduced significantly NPo of Kv7.4 channels to 0.03±0.01 (n=3, p<0.001) in the cell-attached experiments. Apart from revealing the basic biophysical properties of single Kv7.4 channels our results show that, similar to PI(4,5)P2, Gβγ subunits play a critical role in the basal activity of voltage-dependent Kv7.4 channels.Supported by the British Heart Foundation (PG/12/63/29824) and by the Medical Research Council (MR/K019074/1).
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