Does Bimodal Agonism in Cyclic Nucleotide-Gated (CNG) Channels Preserve the Classical Binding Site and Pore Structure?

Abstract

Binding of cGMP or cAMP to cyclic nucleotide-gated (CNG) channels typically increases open probability, and negative agonism (ligand-dependent suppression of opening) is rarely reported. The catfish CNGA2 subtype exhibits an unusual “bimodal agonism”: steady-state open probability increases with initial cGMP binding events (“pro” action) but decreases with subsequent cGMP binding events (“con” action). Con action is specific for cGMP and does not occur for cAMP. We investigated whether con action could be explained without postulating a non-canonical secondary binding site or an atypical non-conductive pore structure. We formulated a simple allosteric model postulating a switch in binding site affinity and coupling behaviour triggered by a critical ligand occupancy, and applied this model to three derivatives of catfish CNGA2 differing only in CNB fold sequence and exhibiting different maximum open probabilities. Dose-response relations of all three channels for cGMP (bimodal) and cAMP (pro action only) were well-explained by global model-fitting with shared closed-state dissociation constants, consistent with conservation of state-independent cyclic phosphate contacts. The effect of a mutation (V457S) outside the canonical binding site was well explained as enhanced pro action coupling and weakened con action coupling; an alternative model where V457S left con action coupling unchanged but affected only binding (a non-canonical site) produced inferior fits to data. The closed state induced by con action exhibited strong binding affinity for the pore-blocker tetracaine, resembling the classical closed channel formed during pro action at low open probabilities. Our findings support a model where V457 indirectly influences the orientation of ligand contacts in the canonical binding site; con action is produced when destabilization of cGMP contacts unique to the high-occupancy open state reverses the pore conformational change associated with pro action gate-opening.