Cyclic nucleotide-gated channels: intra- and extracellular accessibility to Cd2+ of substituted cysteine residues within the P-loop

In cyclic nucleotide-gated (CNG) channels from the bovine rod, the pore loop "P-loop", connecting the S5 and S6 transmembrane segments, is formed by the residues R345-S371 (here named R1-S27). It determines channel selectivity and contributes to gating. We have studied its topology, by testing the accessibility to Cd2+ of serially substituted cysteine residues. Channels were expressed in Xenopus oocytes. The accessibility of V4C, S6C, T16C, I17C, T20C, P22C and S27C from the cytoplasmic side of the plasma membrane was tested by applying 1-100 mu M Cd2+ to the inner face of inside-out patches, at negative membrane potentials. Under these conditions, the effect of Cd2+ on wild-type channels was negligible. The accessibility of the same residues from the external side of the membrane was tested by measuring CNG current inhibition persisting after wash-out of Cd2+ applied to outside-out patches. T16C and I17C channels were strongly inhibited by Cd2+ from the inside, in the presence of cGMP. The K-d for T16C block was 16 mu M. Thus the T16 and I17 residues participate directly in channel function and are accessible from the cytoplasmic side when the channels are open. In contrast, V4C, T20C and P22C residues were only inhibited when 100 mu M Cd2+ was applied externally, suggesting that V4C, T20C and P22C face the outer side of the P-loop