Cyclic nucleotide-gated channels Pore topology studied through the accessibility of reporter cysteines

In voltage- and cyclic nucleotide-gated ion channels, the amino-acid loop t hat connects the S5 and S6 transmembrane domains, is a major component of d ie channel pore. It determines ion selectivity and participates in gating. In the alpha subunit of cyclic nucleotide-gated channels from bovine rod, t he pore loop is formed by tl-le residues R345-S371, here called R1-S27. The se 24 residues were mutated one by one into a cysteine. Mutant channels wer e expressed in Xenopus laevis oocytes and currents were recorded from excis ed membrane patches. The accessibility of the substituted cysteines from bo th sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium) ethyl ] methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessibl e to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the i nhibition was partial, similar to 50% when the MTS compound was applied in die absence of cGMP and 25% when it was applied in the presence of cGMP, su ggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants we re rapidly potentiated by intracellular MTSET. In T16C, a slower partial in hibition took place after the initial potentiation. Current from I17C progr essively decayed in inside-out patches. The rundown was accelerated by inwa rdly applied MTSET. The accessibility results of MTSET indicate a well-defi ned topology of the channel pore in which residues between L7 and 117 are i nwardly accessible, residue G18 and E19 form the narrowest section of the p ore, and T20, P21, P22 and V4 are outwardly accessible.