Publications by authors named "Benjamin W Baker"

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Proline Residues Contribute to Efficient GABA Receptor Function.

ACS Chem Neurosci 2020 12 17;11(24):4215-4222. Epub 2020 Nov 17.

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, United Kingdom.

GABA 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 EC values, and the remaining 13 resulted in receptors with properties similar to WT receptors. Further exploration of the nonfunctional mutants using expression in 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 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 GABA receptor and also suggest that caution must be applied in using data from one receptor to understand molecular details of all pLGICs.
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http://dx.doi.org/10.1021/acschemneuro.0c00483DOI Listing
December 2020