The properties of cysteine mutants in the pore region of cyclic-nucleotide-gated channels.
The properties of the pore of cyclic-nucleotide-gated channels are thought to depend mostly on the structure of the "P loop" which connects the S5 and S6 transmembrane segments. We applied scanning cysteine mutagenesis to the P loop of the alpha subunit of cGMP-gated channels from bovine rod, i.e. to the amino acid residues K346 to S371 (here named K2-S27). Mutant channels were expressed in Xenopus laevis oocytes and currents recorded from inside-out patches, in symmetrical sodium and in the absence of divalent cations. Cysteine mutations had minor effects on the affinity for cGMP and the selectivity to monovalent alkali cations. However, in mutants within the segment W9-T16, open probability was strongly reduced. It was less than 0.2 in saturating cGMP compared to 0.8 in wild-type channels. Moreover, W9C and L12C mutants were outward rectifiers, while T16C was an inward rectifier. These mutant channels showed a time dependence in the development of the steady-state current. Currents from I17C channels progressively decayed in inside-out patches. This rundown was prevented by 1 mM dithiothreitol on the cytoplasmic side of the plasma membrane, suggesting that I17 is an intracellular residue. On the other hand, mutants within the segment T20-S27 had normal open probabilities, around 0.8 in saturating cGMP, but altered single-channel conductance.
Becchetti
A
A
Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy. becchett@sissa.it
Gamel
K
K
D000818:Animals; D002414:Cations, Monovalent / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000008:administration & dosage / Q000494:pharmacology*; D003545:Cysteine / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects*; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011994:Recombinant Proteins; D014982:Xenopus laevis
D002414:Cations, Monovalent; D007473:Ion Channels; D011994:Recombinant Proteins; D006152:Cyclic GMP; D003545:Cysteine