Membrane phospholipids induce moderate positive cooperativity in closed pacemaker channels.

Abstract

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels) play pivotal role in pacemaking activity in the heart and brain. Previous studies have shown the effects of phospholipids like PIP2 on voltage dependent activation of HCN channels. Similar lipid dependent effects on channel activity have also been reported for other ion channels at an ensemble level. However, there is a lack of information regarding how ligand binding to membrane proteins itself is affected by membrane phospholipids at a molecular level. Here, we probe cAMP binding to full length HCN1 channel reconstituted into nanodiscs containing soy polar lipids to mimic a membrane environment. HCN1 nanodiscs were immobilized on zero mode waveguides (ZMWs) and binding of fluorescently labelled cAMP (fcAMP) was monitored over time using total-internal-reflection-fluorescence microscopy (TIRF). Surprisingly, we observed a moderate positive cooperativity in fcAMP binding to the closed HCN1 channel. The observed bound probability distributions deviated from the expected binomial distribution from an identical and independent binding model. These results are significantly different from the cAMP binding to the closed HCN1 channel in detergent micelles.2 Our results shed new light on the contribution of membrane phospholipids to cooperativity in ligand binding to HCN channels.