Abstract
Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the open state, but S3 in the closed state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.
Highlights
Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides
We demonstrate how single-molecule force spectroscopy (SMFS) can be used to examine the gating of CNGA1 channels that are overexpressed in membranes from X. laevis oocytes[21,40,41]; the plasma membrane of these oocytes contains few native membrane proteins[41,42,43,44]
We identify F–D curves using bioinformatics analysis and by engineering proteins that are composed of CNGA1 channels linked at their C-termini to an SMFS marker, that is, a protein with a known unfolding pattern that act as a fingerprint
Summary
Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. We show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of a-helix folding This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels. Ion channels are membrane proteins that play a major functional role and they are grouped in superfamilies[15,16]: the superfamily of voltage-gated ion channels comprises Na þ , K þ and Ca2 þ channels, whose gating (transitions between the open and closed conformation) depends on the membrane voltage This superfamily includes cyclic nucleotide-gated (CNG) channels[17,18,19,20,21] that are voltage dependent[21] but are opened by the binding of cyclic nucleotides to the cyclic nucleotide-binding (CNB) domain[17,19,20]. The full-length channel has never been crystallized and the conformational changes that are associated with gating are poorly understood
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