Interaction Energies between Intracellular Regions in CNG Channel Activation

Abstract

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to an intracellular cyclic nucleotide-binding domain (CNBD). The molecular events translating the binding to the pore opening are still unknown. We investigated the role of intracellular channel regions on the activation process by constructing chimeric channels in which the N-terminus, the S4-S5 linker, the C-terminus, and the CNBD of the retinal CNGA1 subunit were systematically replaced by respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by an interaction energy according to the principles of thermodynamic double-mutant cycle analysis. We show that all four intracellular regions, including the short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits as determined from the Hill coefficients. The interaction energies are specific for pairs of regions. The interaction energy of the S4-S5 region with both the N-terminus and the C-linker was significantly different from zero at all possible combinations of intracellular regions. In contrast, at all possible combinations of intracellular regions no significant interaction energy was observed for the S4-S5 linker and the CNBD. It is concluded that in CNG channels the S4-S5 linker cooperates with both N-terminus and C-linker in the process of translating ligand binding to the pore opening.