Single Molecule Force Spectroscopy of CNGA1 Channels “In Situ” Reveals Major Conformational Changes upon Gating

Abstract

Single Molecule Force Spectroscopy (SMFS) is a powerful tool to investigate the structural properties of proteins avoiding all crystallisation problem. SMFS can be used to study membrane proteins directly in their lipid environment, but so far only few proteins have been investigated in their natural environment, i.e. embedded in natural membranes. Here we investigate the structure and function of cyclic nucleotide gated (CNG) channels, cationic channels, mediating sensory transduction in photoreceptor and olfactory sensory neurons, which open upon binding to cyclic nucleotides. Although the cyclic nucleotide binding (CNB) domain has been localized in the C-terminal, the conformational details still remain unveiled. Here we correlate the functional opening of the channel with a conformational change that occurs in the CNB domain and in the pore region. CNGA1 channels were over-expressed in Xenopus laevis oocytes and their functionality were verified by electrophysiology. Membrane patches were subsequently deposited on mica, exposing the cytoplasmic side. Patches were identified by AFM imaging and more than 300.000 force-distance (F-d) traces were acquired for each conditions. Several engineered constructs including I27 and N2B domains were used as SMFS finger-prints. Using the WLC model and a custom developed cluster analysis approach, F-d traces in closed state, and in the open state were compared, the latter showing larger unfolding forces (about 55 pN in the closed state and about 85 pN in the open state) in the trans-membrane domains and two additional force peaks located in the cytoplasmic domain (Lc ≈ 80 nm) and in the pore region (Lc ≈ 148 nm), indicating the formation of differently folded structures. Therefore SMFS can be used to identify the conformational changes of proteins underlying gating of ionic channels in situ.