Single-molecule observation of binding cooperativity in non-activated pacemaker channels

Abstract

Cooperativity in HCN2 pacemaker channel activation is a key determinant of channel function, including its regulation by cyclic-nucleotides as ligands. Here we investigate the binding of fluorescently labeled cAMP to resting HCN2 channels in their native membrane environment. For this GFP-tagged HCN2 channels are stably and inducibly expressed in HEK293 cells, from which supported native membranes are stripped onto plasma-cleaned coverslips. Binding to endogenous cAMP-affinities is blocked with a custom inhibitor protocol.