Expression and purification of a functional heteromeric GABAA receptor for structural studies
Fig 5
Removal of glycosylation sites on the N-terminus reduces α1/β2 expression in oocytes.
(A) Sequence alignment of the N-terminus for GABAA and GluClα subunits identifying residues for deletion (red line) and predicted sites for glycosylation (*). The red bar represents the first predicted α-helix in the extracellular domain. A blue box outlines the predicted signal peptide. GluClαcryst is the sequence used to obtain the crystal structure. (B) Sequence alignment of the C-terminus for GABAA and GluClα subunits identifying residues for deletion (red line). (C) Removal of an 11-residue tail from the α1 subunit in the GFPuv-α1-LT/β2-LT construct (Fig 4) does not change receptor behavior. (D) Deletion of the N-terminus (ΔN) in either α1 or β2 subunit reduces receptor expression in the α1-EGFP/β2 construct. (E) Site directed mutagenesis of predicted glycosylation sites reduces α1-EGFP/β2 receptor expression. Absolute fluorescence intensities are shown on the same scale. FSEC traces shown in (D) and (E) were obtained from the same batch of oocytes.