Characterization of Cyclic Nucleotide Gated Channels using Atomic Force Microscope

Abstract

Cyclic nucleotide-gated (CNG) channels have a key role in the conversion of sensory information, such as light and scent, into primary electrical signals. Structurally, these channels belong to the superfamily of voltage-gated ion channels and share a significant sequence identity with K+ channels suggesting a common ancestral three-dimensional structure. However, unlike K+ channels, to date no crystal structure of CNG channel has been solved. Therefore, we decided to analyze the structure of CNG channels with Force Spectroscopy using Atomic Force Microscopy (AFM), capable of giving structural information about proteins hard to crystallize in their native environment. We expressed the GFP-CNG channel in Xenopus laevis oocytes and isolated Xenopus plasma membrane containing channels' high density (∼500/μm2). Combining AFM with total internal reflection fluorescence (TIRF) microscopy, we were able to detect CNG single-molecule fluorescence attached to membrane patches. We have then proceeded to characterize CNG channels by measuring their mechanical properties by Force Spectroscopy. These measurements allow to extract a signature of their mechanical properties and to estimate their content of α-helices, β-sheet, hydrophobic and hydrophilic domains. These measurements do not require the purification or the crystallization of the CNG protein but provide important information on conformational changes during channel gating of CNG channels.