Discrimination between cyclic-nucleotides by cyclic-nucleotide gated ion channels.

Abstract

Cyclic-nucleotide-gated (CNG) ion channels play important roles throughout the entire nervous system, particularly in the signal transduction of the retina and olfactory system. They are regulated by the cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). These two chemically very similar molecules bind to a specialized intracellular domain called the cyclic nucleotide-binding domain (CNBD). CNG channels are key in translating the chemical signal of the second messenger molecules cAMP and cGMP into an electrical response by inducing a conformational change in the CNBD that modulates the opening of the channel gate in the pore domain allowing the flow of ions across cell membranes. Functional measurements have shown that, in SthK (a prokaryotic homolog with high sequence and structural similarities to CNG channels), cGMP barely leads to pore opening while cAMP activates the channel. Here we address the question of how the CNBD can finely discriminate between the two chemically and structurally very similar ligands. Using a combination of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), and force probe molecular dynamics simulations, we elucidate, quantitatively and at the atomic level, the mechanism of CNBD ligand discrimination in SthK. We find that cAMP binds to the SthK CNBD with a slightly stronger affinity than cGMP, and can access a deep-bound state that is inaccessible to cGMP. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation. We identify crucial residues and dynamic hydrogen bonding patterns within the binding pocket that give rise to this discrimination.