Towards Revealing a Cooperative Mechanism of Camp Binding to HCN2 Cyclic Nucleotide Binding Domains at the Single-Molecule Level

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are critical for oscillatory neuronal activity in the brain and pacemaking in the heart. Although activation of these tetrameric channels can be driven solely by hyperpolarizing voltages, the binding of cyclic nucleotides to their intracellular cyclic nucleotide binding domains (CNBDs) enhances their voltage-dependence of activation through a mechanism that remains unclear. Previous studies dissecting the cooperative nature of subunit activation upon cAMP binding have relied on macroscopic methods, such as ensemble channel currents or fluorescence. Although these studies provide pivotal information in determining the gating-mechanism of HCN channels, they both afford only ensemble-averaged data, which obscures the resolution of individual binding steps. Herein, we aim to circumvent this limitation and determine the cooperative mechanism of cAMP binding to HCN2 CNBDs at the single-molecule level. By tethering an artificially-linked tetrameric CNBD construct into nanofabricated devices called zero-mode waveguides, we resolve the stepwise binding of fluorescent cAMP molecules to all four binding sites of our complex. Using hidden Markov modeling of our idealized single-molecule fluorescence time series, we begin to reveal a cooperative mechanism of cAMP binding to HCN2 CNBDs.