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 pro...

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Bibliographic Details
Published in:The Journal of biological chemistry 2019-05, Vol.294 (18), p.7503-7515
Main Authors: Morgan, Jacob L.W., Evans, Eric G.B., Zagotta, William N.
Format: Article
Language:English
Subjects:
allosteric regulation
allostery
bacteria
channel activation
Cyclic AMP - metabolism
Cyclic GMP - metabolism
cyclic nucleotide
cyclic nucleotide gating
Cyclic Nucleotide-Gated Cation Channels - chemistry
Cyclic Nucleotide-Gated Cation Channels - metabolism
electrophysiology
Escherichia coli - metabolism
Escherichia coli Proteins - metabolism
Ion Channel Gating
Molecular Biophysics
Patch-Clamp Techniques
potassium channel
prokaryotic ion channel
Protein Conformation
Spheroplasts - metabolism
voltage-gated ion channel
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.RA119.007699