Cyclic-nucleotide-gated channels: pore topology in desensitizing E19A mutants

In cyclic-nucleotide-gated "CNG" channels, the pore-loop "P-loop" is formed by the amino acid residues R345-S371 (here called R1-S27). Residue E19 det ermines the channel's interaction with extracellular divalent cations and c ontributes to ion conduction. Neutralization of this residue with alanine i ntroduces channel desensitization. We have used serial cysteine mutagenesis to study P-loop topology in the alpha subunit of the mammalian rod CNG cha nnels containing the E19A substitution. The pore topology was tested in the closed channel state and, when cGMP was present, during and after desensit ization. With E19A substitution, the T15C, T16C, I17C and T20C mutants dese nsitized more slowly than controls. Moreover, the typical rundown produced by the I17C substitution in the wild-type "w.t." background was considerabl y reduced. Overall, with the E19A substitution, the accessibility pattern t ested by applying the thiol-specific reagents Cd2+ and MTSET from the cytop lasmic side of the plasma membrane was similar to that observed with the w. t. Moreover, P22C channels were not inhibited by Cd2+ and MTSET (which do n ot cross the lipid bilayer) applied from the inside, but were blocked by MT SEA (which permeates the plasma membrane) also applied from the inside. Thi s suggests that the residues following E19 remain accessible from the exter nal side after E19A substitution. Thus, although the residues T15 to T20 se emed to participate in the structural rearrangements producing desensitizat ion, no major P-loop remodelling occurs in desensitizing channels.