Studying KcsA Channel Clustering Using Single Channel Voltage-Clamp Fluorescence Imaging

Hugo McGuire; Rikard Blunck

doi:10.3389/fphys.2022.863375

Studying KcsA Channel Clustering Using Single Channel Voltage-Clamp Fluorescence Imaging

Front Physiol. 2022 Jun 3:13:863375. doi: 10.3389/fphys.2022.863375. eCollection 2022.

Authors

Hugo McGuire¹, Rikard Blunck^{1

2

3}

Affiliations

¹ Department of Physics, Université de Montréal, Montréal, QC, Canada.
² Department of Pharmacology and Physiology, Université de Montréal, Montréal, QC, Canada.
³ Interdisciplinary Research Center on Brain and Learning (CIRCA), Université de Montréal, Montréal, QC, Canada.

Abstract

Oligomerization and complex formation play a key role for many membrane proteins and has been described to influence ion channel function in both neurons and the heart. In this study, we observed clustering of single KcsA channels in planar lipid bilayer using single molecule fluorescence, while simultaneously measuring single channel currents. Clustering coincided with cooperative opening of KcsA. We demonstrate that clustering was not caused by direct protein-protein interactions or hydrophobic mismatch with the lipid environment, as suggested earlier, but was mediated via microdomains induced by the channel in the lipid matrix. We found that single channel activity of KcsA requires conically-shaped lipids in the lamellar liquid-crystalline (L_α) phase, and the need for a negative spontaneous curvature seem to lead to the deformations in the membrane that cause the clustering. The method introduced here will be applicable to follow oligomerization of a wide range of membrane proteins.

Keywords: ion channel clustering; lateral diffusion; membrane curvature; planar lipid bilayer; single molecule fluorescence.