Abstract
Cell surface expression of type A GABA receptors (GABAARs) is a critical determinant of the efficacy of inhibitory neurotransmission. Pentameric GABAARs are assembled from a large pool of subunits according to precise co-assembly rules that limit the extent of receptor structural diversity. These rules ensure that particular subunits, such as ρ1 and β3, form functional cell surface ion channels when expressed alone in heterologous systems, whereas other brain-abundant subunits, such as α and γ, are retained within intracellular compartments. Why some of the most abundant GABAAR subunits fail to form homomeric ion channels is unknown. Normally, surface expression of α and γ subunits requires co-assembly with β subunits via interactions between their N-terminal sequences in the endoplasmic reticulum. Here, using molecular biology, imaging, and electrophysiology with GABAAR chimeras, we have identified two critical residues in the transmembrane domains of α and γ subunits, which, when substituted for their ρ1 counterparts, permit cell surface expression as homomers. Consistent with this, substitution of the ρ1 transmembrane residues for the α subunit equivalents reduced surface expression and altered channel gating, highlighting their importance for GABAAR trafficking and signaling. Although not ligand-gated, the formation of α and γ homomeric ion channels at the cell surface was revealed by incorporating a mutation that imparts the functional signature of spontaneous channel activity. Our study identifies two single transmembrane residues that enable homomeric GABAAR subunit cell surface trafficking and demonstrates that α and γ subunits can form functional ion channels.
Highlights
Cell surface expression of type A GABA receptors (GABAARs) is a critical determinant of the efficacy of inhibitory neurotransmission
As expected [20, 28], ␣1BBS did not express on the surface of HEK293 cells, evident by an absence of BgTx-AF555 labeling at the cell periphery; intracellular fluorescence was observed in permeabilized cells (Fig. S1, A and B)
The 1W329A substitution caused a profound increase in the speed of current deactivation ( ϭ 0.86 Ϯ 0.1 s, n ϭ 7, p Ͻ 0.05; Fig. 5, F and H) compared with WT 1 ( ϭ 26.9 Ϯ 5.6 s, n ϭ 8) and 1W280Q, where deactivation was even slower ( ϭ 84.4 Ϯ 15.1 s, n ϭ 5, p Ͻ 0.001). These results indicate that the residues controlling homomeric receptor expression in ␣ and ␥L subunits have important trafficking and gating roles for 1 homomers depending on the substituted residue
Summary
To understand why the cell surface expression profiles for ␣ and subunits differ, we created chimeras incorporating domain switches. To determine the nature of the residues enabling cell surface expression of ␣1 subunits, Gln-241 was exchanged for alternatives with small (Gly), hydrophobic (Ala, Met), positively charged (Lys), negatively charged (Asp), polar (Asn, Ser), or aromatic (Phe, Tyr) side chains. None of these substitutions permitted cell surface expression compared with Q241W (p Ͻ 0.001), intracellular labeling was evident for all mutants (Fig. 2E and Fig. S5; p Ͼ 0.05).
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