State-dependent salt bridge interaction during activation in prokaryotic cyclic nucleotide-gated channel SthK.

Abstract

The cyclic nucleotide-binding domain (CNBD) family of voltage-gated ion channels are complex molecular machines regulating membrane excitability in many cell types. While the protein structures of many CNBD channel family members have provided considerable insights into channel architecture, the mechanism of how cyclic nucleotide binding modulates channel opening is still unknown. The prokaryotic cyclic nucleotide-gated (CNG) channel, SthK, from Spirochaeta thermophila, has considerable structural and functional similarity to eukaryotic CNG channels. SthK can be readily expressed in E. coli spheroplasts and its response to ligand activation recorded with inside-out patch-clamp electrophysiology to test the functionality of various mutations. Using thermodynamic mutant cycle analysis, we have shown that there is a stabilizing interaction between arginine R125 at the end of the S4 voltage sensor helix and aspartate D261 in the B’ helix of the C-linker when the channel is in the presence of cyclic adenosine monophosphate (cAMP). Furthermore, when these residues (R125 and D261) were both mutated to cysteines, we were able to create a spontaneous disulfide bridge between these residues and lock the channel in the open, activated state, even in the absence of cAMP. These results indicate that we have identified an important state-dependent salt bridge between the C-linker domain and voltage sensor domain of this channel. Both charged residues are conserved in the eukaryotic CNGA1 channel structure, where it seems likely they play a similar role in state-dependent stabilization of the open state of these channels. Additionally, the aspartate residue is conserved across all CNG and HCN channels, further highlighting the potential significance of this C-linker residue. These new results help to better characterize the ligand-activated allosteric pathway in CNBD channels.