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Updated: Jul 13, 2025

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Published on: July 13, 2013
Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization
1Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 South Grand Blvd., St. Louis, MO, 63104, USA.
Abstract:
Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.
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