ALTERED LIGAND SPECIFICITY BY PROTONATION IN THE LIGAND-BINDING DOMAIN OF CYCLIC NUCLEOTIDE-GATED CHANNELS

Cyclic nucleotide-gated (CNG) ion channels are the critical mediators between the second messengers of sensory transduction and the cell's m embrane potential. The photoreceptor CNG channels are activated by the direct binding of cGMP but can also be activated to a much lesser ext ent by cAMP. In rod CNG channels expressed in Xenopus oocytes, we demo nstrate two types of potentiation by protons. One type potentiated cGM P-bound and cAMP-bound channels to the same extent, while another pote ntiated only cAMP-bound channels. Both types of potentiation could be described by a mechanism in which protons bound primarily to the chann el open configuration. The potentiation specific to cAMP-bound channel s could be accounted for by protonation of aspartic acid 604 (D604). I t is the unfavorable electrostatic interaction between the carboxylate of D604 and the purine ring of cAMP that accounts for the normally po or activation of the channels by cAMP. Protonation at this site remove d the unfavorable interaction and allowed cAMP to act as nearly a full agonist. Protonation of a second amino acid, H468, contributed to the nucleotide-nonspecific potentiation and is likely to be an element of the channel gating assembly. Protons potentiate native rod channels l ess than channels formed from subunit 1. In heteromultimeric channels formed by coexpressing subunit 1 with subunit 2, we found a similar at tenuation of potentiation. The absence of protonatable amino acids in subunit 2 at positions corresponding to H468 and D604 can explain the reduced effects of pH on native channels.