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Understanding the molecular gates of KirBac3.1

S. El-Ajouz,1 A.P. Hill,1 J.M. Gulbis2 and J.I. Vandenberg,1 1Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia and 2The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.

Potassium currents across cell membranes are an essential part of electrical signalling in all cells. KirBac channels are prokaryotic homologs to mammalian Kir channels and superposition of the recently solved structures show that the fold and key features are almost indistinguishable. Potassium currents are proposed to be switched on and off using two molecular gates in the permeation pathway, in the inner helix bundle crossing and in the selectivity filter. Previous studies on KirBac3.1 channels used flux assays to highlight activatory mutants that open these molecular gates. However, there is a lack of single channel data from KirBac3.1 channels and gating is not clearly understood.

Here, we investigated single channel currents from excised patches of Chinese hamster ovary (CHO) cells expressing recombinant KirBac3.1 channels. We showed that the single channel currents of the wild-type channel have two types of gating, a slow gating mode and a high frequency flicker mode. Based on structural information and previously studied mutants we identified mutants that target the slow gating or the high frequency flicker mode. Initial experiments show that the mutant S129R, which is located near the inner helix bundle crossing, affected the slow gating and the mutant F88L, which is located near the selectivity filter reduced the frequency flicker mode of the inward currents. These results provide evidence that there is a molecular gate at the selectivity filter, which is important in the flicker gating seen in KirBac potassium channels, and a molecular gate at the helix bundle crossing that is important for slow gating.