A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4 Cytoplasmic Loop Is Required for GABAA Receptor Assembly

Abstract

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary ␥-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.The Cys-loop superfamily of ligand-gated ion channels, which includes ␥-aminobutyric acid type A (GABA A ) 2 and type C (GABA C ), nicotinic acetylcholine, glycine, and 5-hydroxytryptamine type 3 receptors, mediates fast synaptic transmission in the nervous system. Mutations that alter Cys-loop receptor surface density by affecting receptor biogenesis have been associated with idiopathic generalized epilepsies (1-4), congenital myasthenic syndromes (5), and psychiatric disorders (6). Unfortunately because the structural and cellular determinants of receptor biogenesis are poorly understood, development of effective treatment strategies remains a significant challenge.A wealth of evidence suggests that Cys-loop receptors are assembled as heteropentamers from a large repertoire of neuronal subunits (7-10). Subunits share a similar topology that includes an extracellular N-terminal domain, four transmembrane domains, three loops including a large cytoplasmic loop, and a variable length extracellular C-terminal tail (7, 11). Receptor assembly is thought to occur in the endoplasmic reticulum (ER) following glycosylation and folding of de novo synthesized subunits (12-15). Assembly is closely monitored by ER quality control machinery, and consequently subunits that fail to assemble properly are retained and degraded (15)(16)(17). Although N-terminal motifs are known to be important for subunit assembly (18,19), recent studies in nicotinic acetylcholine receptors (nAChRs) suggest that C-terminal motifs may also play a role (20).GABA A receptors are the most abundant Cys-loop receptor in the mammalian brain and are responsible for the majority of fast inhibitory neurotransmission. Like other Cys-loop superfamily receptors, they are pentamers assembled from combinations of 16 subunit subtypes ...

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

“…Glycosylation of the Third Glycosylation Site, Asn-173, Was Important for Stability of Singly Expressed Subunits-When expressed in the absence of partnering subunits, ␣1 and ␤2 subunits were retained in the ER and subsequently degraded by ER-associated degradation due to failure to assemble into homopentamers (12,31). Furthermore, mutations of these subunits that affect biogenic steps earlier than heteropentameric assembly enhanced ER-associated degradation, resulting in a total mutant subunit level that was lower than that of singly expressed native subunits (32,33).…”

“…Conversely, mutation of the second glycosylation site, Asn-104, which showed no effects on singly expressed subunit stability, significantly decreased binary receptor surface levels. Pentameric coassembly of ␣1 and ␤2 subunits can mutually increase total levels of both subunits (12,31). It is possible that ␣1 subunits functioned as chaperones by coassembling with ␤2(N173Q) subunits and increasing their stability.…”

“…Brefeldin A Treatment-Brefeldin A has been used to block ER forward trafficking and, thus, subsequent surface expression of fully assembled GABA A receptors (12). HEK293T cells were treated with 0.5 g/ml brefeldin A (Sigma) 6 h after transfection and then harvested 24 h after transfection.…”

“…The FLAG epitope, DYKDDDDK, was introduced between the eighth and ninth amino acids of the ␣1 subunit polypeptide or between the fourth and fifth amino acids of the ␤2 subunit polypeptide (numbered excluding the signal peptide). The epitope insertion at these positions has been demonstrated to cause no significant changes in electrophysiological properties or surface levels of ␣1␤2 receptors (2,12). The QuikChange site-directed mutagenesis kit (Stratagene) was used to make subunits with segmental deletions or point mutations.…”

“…Surface Expression Measurement Using Flow CytometryMeasurement of surface expression of GABA A receptor subunits using flow cytometry has been described previously (12). Briefly, transfected HEK293T cells were removed from the dishes by trypsinization and then resuspended in FACS buffer (phosphate-buffered saline (PBS) supplemented with 2% FBS and 0.05% sodium azide).…”