Deactivation of CNGA2 Channels follows Intricate Pathways

Abstract

Cyclic nucleotide-gated (CNG) channels in olfactory neurons are heterotetrameric ligand-gated cation channels that are composed of four homologue subunits (2xCNGA2, 1xCNGA4, 1xCNGB1b). Only the CNGA2 subunits form functional homotetrameric channels that can be activated in a cooperative manner by cAMP or cGMP binding to the cyclic nucleotide-binding domains (CNBD) included in each subunit. Our aim was to kinetically further dissect the molecular mechanism leading to channel activation upon ligand binding and to channel deactivation upon ligand removal.CNGA2 channels, expressed in Xenopus oocytes, were studied in excised patches by measuring simultaneously ligand binding/unbinding and activation/deactivation by means of confocal patch-clamp fluorometry under steady-state and non-steady state conditions (182 or 277 frames per second). Concentration jumps of a fluorescent cGMP derivative (Biskup et al., Nature, 446(7134): 440-3, 2007) were applied using a fast piezoelectric system. Surprisingly, the unbinding was concentration dependent while deactivation was concentration independent. The unbinding was approximately 100 times faster from fully liganded channels in comparison with the unbinding from lowly liganded channels. The obtained data were analyzed by global fits to various types of Markovian state models. The additional information of unbinding and deactivation allowed us to refine the previously determined C4L-Model (Biskup et al., Nature, 446(7134): 440-3, 2007). To account for the very fast unbinding at saturating ligand concentrations, the C4L-Model had to be expanded: When fully liganded, the channel adopts an open state which allows, upon ligand removal, a very fast unbinding of all four ligands. In contrast, from partially liganded states this fast unbinding is occluded. Our results suggest an additional pathway for rapid ligand unbinding for the fully liganded channel.