Extended Data Fig. 2: Sequence alignment of GABAA receptor α1, β3 and γ2 subunits, biochemical characterization and binding assays.
From: Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer
a, Alignment of wild-type GABAA receptor subunit sequences, in which 1 represents the first residue of the mature protein. α-helices (grey cylinders), β-strands (black arrows) and associated loops are indicated. Glycosylation sites are indicated by a blue pentagon and the associated subunit residue is highlighted in blue. Residues identified as coordinating PIP2 binding are highlighted in yellow and are indicated by yellow hexagons. The alignment graphic was prepared on the ESPript 3.0 server (http://espript.ibcp.fr/ESPript/ESPript/). b, Structure of a single α1 subunit. c, Western blot analysis of cell lysates from LMNG-solubilized control HEK293 cells and α1β3γ2L GABAA receptor cells, and purified α1β3γ2L GABAA receptors in nanodiscs. The arrowhead denotes the band corresponding to the full-length GABAA receptor subunits, which migrates as a species of about 51–55 kDa. With the exception of the α1 subunit (which displays a small degree of proteolysis following reconstitution, denoted by an asterisk), GABAA receptor subunits do not display apparent proteolysis during solubilization, purification and reconstitution. Western blots were repeated twice independently with similar results. d, GABA enhanced displaceable [3H]flunitrazepam binding to purified receptors in a concentration-dependent manner in the presence or absence of Mb38. Points represent individual samples from two separate experiments.
