A conserved Cys-loop receptor aspartate residue in the M3-M4 cytoplasmic loop is required for GABAA receptor assembly.
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 gamma-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.
Lo
Wen-yi
WY
Program in Neuroscience, Vanderbilt University, Nashville, Tennessee 37232, USA.
Botzolakis
Emmanuel J
EJ
Tang
Xin
X
Macdonald
Robert L
RL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D009435:Synaptic Transmission
D011963:Receptors, GABA-A; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; D003545:Cysteine