Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

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 closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of \u3b1-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

Gating of cyclic nucleotide-gated channels is voltage dependent

Cyclic nucleotide-gated channels belong to the family of voltage-gated ion channels, but pore opening requires the presence of intracellular cyclic nucleotides. In the presence of a saturating agonist, cyclic nucleotide-gated channel gating is voltage independent and it is not known why cyclic nucleotide-gated channels are voltage-insensitive despite harbouring the S4-type voltage sensor. Here we report that, in the presence of Li(+), Na(+) and K(+), the gating of wild-type cyclic nucleotide-gated A1 and native cyclic nucleotide-gated channels is voltage independent, whereas their gating is highly voltage-dependent in the presence of Rb(+), Cs(+) and organic cations. Mutagenesis experiments show that voltage sensing occurs through a voltage sensor composed of charged/polar residues in the pore and of the S4-type voltage sensor. During evolution, cyclic nucleotide-gated channels lose their voltage-sensing ability when Na(+) or K(+) permeate so that the vertebrate photoreceptor cyclic nucleotide-gated channels are open at negative voltages, a necessary condition for phototransduction