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Proline Residues Contribute to Efficient GABAp Receptor Function

Baker, Benjamin W. and Dougherty, Dennis A. and Lummis, Sarah C. R. (2020) Proline Residues Contribute to Efficient GABAp Receptor Function. ACS Chemical Neuroscience, 11 (24). pp. 4215-4222. ISSN 1948-7193.

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GABAp receptors are homomeric pentameric ligand-gated ion channels (pLGICs) and are useful for probing the molecular details of the mechanism of action in this important protein family. Here, we explore the role of proline (Pro) residues by creating mutant receptors, expressing them in HEK293 cells, and using fluorescent membrane potential sensitive dye to monitor receptor activity. The data revealed that 3 of the Pro-to-alanine substitutions resulted in nonfunctional receptors (one in the Cys-loop, one in loop A and one in the β2-β3 loop), 7 resulted in increased EC₅₀ values, and the remaining 13 resulted in receptors with properties similar to WT receptors. Further exploration of the nonfunctional mutants using expression in Xenopus laevis oocytes and whole-cell voltage-clamp electrophysiology, incorporating both canonical and noncanonical amino acids, revealed that the Pro in the Cys-loop had a preference for analogues with a high intrinsic cis bias, the Pro in loop A required a ring, and the β2-β3 loop Pro contributes to expression. The data from the Cys-loop Pro are consistent with those from other pLGICs, while those of the loop A Pro and some of the other Pros surprisingly differ. Thus, overall, the data clarify the roles of many of the Pros in the GABAp receptor and also suggest that caution must be applied in using data from one receptor to understand molecular details of all pLGICs.

Item Type:Article
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Dougherty, Dennis A.0000-0003-1464-2461
Lummis, Sarah C. R.0000-0001-9410-9805
Additional Information:© 2020 American Chemical Society. Received: July 28, 2020; Accepted: October 9, 2020; Published: November 17, 2020. We would like to thank Jenny Jeffries for expert technical assistance and Prof. Henry Lester for ongoing intellectual support. Author Contributions: S.C.R.L. and D.A.D. participated in research design. B.W.B. and S.C.R.L. conducted experiments and data analysis. D.A.D. provided essential materials. S.C.R.L. wrote the manuscript. This work was supported by the National Institute of Health grant NS-34407 (D.A.D.). The authors declare no competing financial interest.
Funding AgencyGrant Number
Subject Keywords:GABA_A receptor, acetylcholine receptor, ligand-gated ion channel, Cys-loop receptor, mutagenesis, Flexstation
Issue or Number:24
Record Number:CaltechAUTHORS:20201118-073707245
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Official Citation:Proline Residues Contribute to Efficient GABAp Receptor Function. Benjamin W. Baker, Dennis A. Dougherty, and Sarah C. R. Lummis. ACS Chemical Neuroscience 2020 11 (24), 4215-4222; DOI: 10.1021/acschemneuro.0c00483
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106715
Deposited By: Tony Diaz
Deposited On:18 Nov 2020 18:41
Last Modified:16 Dec 2020 22:10

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