Functional Characterization of Gating in a Bacterial Cyclic Nucleotide-Gated Channel

Abstract

Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. The transmembrane (TM) region contains a voltage-sensing domain and a pore domain. The cytoplasmic region is comprised of a C-linker domain followed by a cyclic nucleotide-binding domain (CNBD). CNG channels assemble as a tetramer with a central pore. cAMP and cGMP bind to the CNBD and produce an allosteric conformational rearrangement that opens the pore. The rearrangement occurring at the CNBD is well characterized due in large part to structural and biochemical experiments conducted on the purified C-linker/CNBD fragment from the related hyperpolarization-activated, cyclic nucleotide-gated channel, HCN2. However, the rearrangement occurring in the rest of the channel (C-linker and TM region) is not understood. Therefore, we are interested in developing a model system for structural and functional studies of gating in full-length CNG channels. SthK is a bacterial CNG channel that has the potential to serve as an ideal model system for studying CNG channel gating. It can be expressed in E. coli and purified in large quantities, and its resting-state structure has recently been published. However, SthK is reported to exhibit a low open probability (Po) even in saturating cAMP. This property limits its usefulness in studying the allosteric opening transition. Here, we express SthK in giant E. coli spheroplasts. We patch-clamped these spheroplasts and recorded both single-channel and macroscopic currents to characterize SthK gating in a bacterial membrane. Furthermore, we introduced mutations and show that they increased the Po while preserving SthK's biochemical tractability.