Abstract
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 (i.e. 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 (Po). Here, we expressed SthK in giant Escherichia coli spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the Po 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 E. coli because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout E. coli cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the Po in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels.
Highlights
Cyclic nucleotide– gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction
Cyclic nucleotide– gated (CNG)2 channels mediate signal transduction in the visual and olfactory systems [1,2,3]. They belong to the voltage-gated ion channel superfamily, CNG channels are only weakly voltage-dependent and instead open upon direct binding of cyclic nucleotide to a cytoplasmic region of the channel [4, 5]
CNG channels transmit a conformational change from the ligand-binding site in each subunit (CNBD) to an active site (Fig. 1A)
Summary
CNG channels transmit a conformational change from the ligand-binding site in each subunit (CNBD) to an active site (pore gate) (Fig. 1A). This allosteric regulation can be envisioned in a gating scheme based on Monod, Wyman, and Changeux (MWC) [10] where a concerted channel opening transition is driven by an increase in affinity for the ligand when the pore gate is open. At 0 mV, the voltage for most structural experiments of purified protein, SthK has been shown to have an ϳ10% Po in saturating cAMP, limiting studies of the conformational transitions in the C-linker and TM region [22]. We characterized the cNMP dependence and voltage dependence of SthK in a bacterial membrane, developed a method to reduce the toxicity of SthK expression, and engineered a Cys-free SthK construct with a high Po
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