Functional Characterization and Optimization of a Bacterial Cyclic Nucleotide-gated Channel

Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. They open in response to direct binding of cyclic nucleotide (cAMP or cGMP) to a cytoplasmic region of the channel. However, the conformational rearrangements occurring upon binding to produce pore opening ( gating) are not well understood. SthK is a bacterial CNG channel that has the potential to serve as an ideal model for structure-function studies of gating but is currently limited by its toxicity, native cysteines, and low open probability (). Here, we expressed SthK in giant spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the in cAMP is higher than has been previously published and that cGMP acts as a weak partial SthK agonist. Additionally, we determined that SthK expression is toxic to because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels.