期刊
ACS CHEMICAL NEUROSCIENCE
卷 11, 期 3, 页码 242-247出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschemneuro.9b00647
关键词
Cys-loop receptors; alpha 7 nAChR; nicotinic acetylcholine receptors; cysteine mutagenesis; intracellular domain; PICK1
资金
- NIH [R01DA046939]
The alpha 7 nicotinic acetylcholine receptor (alpha 7 nAChR) is involved in various intracellular signaling pathways that mediate addiction, chronic pain, and other diseases, but its intracellular domain structures remain undetermined. The presence of 17 native cysteines in alpha 7 nAChR provides opportunities for extracting structural information through site directed labeling of chemical probes in strategic locations, but it also creates uncertainties in channel function when those native cysteines must be mutated. Using site-directed mutagenesis and two-electrode voltage clamp electrophysiology measurements, we found that alpha 7 nAChR's function was well tolerated for mutations of all 13 cysteines as long as two pairs of disulfide-bond cysteines remained in the extracellular domain. Furthermore, surface plasmon resonance measurements showed that the cysteine mutations did not affect alpha 7 nAChR binding to the intracellular protein PICK1. The study suggests that a high native cysteine content does not necessarily preclude the use of single cysteine labeling for acquiring structural information on functional proteins.
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