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 determines the channel's interaction with extracellular divalent cations and contributes to ion conduction. Neutralization of this residue with alanine introduces channel desensitization. We have used serial cysteine mutagenesis to study P-loop topology in the alpha subunit of the mammalian rod CNG channels containing the E19A substitution. The pore topology was tested in the closed channel state and, when cGMP was present, during and after desensitization. With E19A substitution, the T15C, T16C, I17C and T20C mutants desensitized more slowly than controls. Moreover, the typical rundown produced by the I17C substitution in the wild-type "w.t." background was considerably reduced. Overall, with the E19A substitution, the accessibility pattern tested by applying the thiol-specific reagents Cd2+ and MTSET from the cytoplasmic 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 not cross the lipid bilayer) applied from the inside, but were blocked by MTSEA (which permeates the plasma membrane) also applied from the inside. This suggests that the residues following E19 remain accessible from the external side after E19A substitution. Thus, although the residues T15 to T20 seemed to participate in the structural rearrangements producing desensitization, no major P-loop remodelling occurs in desensitizing channels.
Roncaglia
P
P
International School for Advanced Studies, and Istituto Nazionale di Fisica della Materia, Trieste, Italy.
Becchetti
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D003545:Cysteine / Q000235:genetics; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D017434:Protein Structure, Tertiary; D014982:Xenopus laevis
D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D002104:Cadmium; D006152:Cyclic GMP; D003545:Cysteine