Abstract
Gamma-aminobutyric acid, type A (GABA(A)) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta, and one gamma subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric gamma(3) constructs. From their ability to associate with full-length alpha(1) and beta(3) subunits, we concluded that amino acid sequence gamma(3)(70-84) either directly interacts with alpha(1) or beta(3) subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to gamma(2) residues contributing to the benzodiazepine-binding site at the alpha(1)/gamma(2) interface suggested that in alpha(1)beta(3)gamma(3) receptors the sequence gamma(3)(70-84) is located at the alpha(1)/gamma(3) interface. In the absence of alpha(1) subunits this sequence might allow assembly of beta(3) with gamma(3) subunits. Other experiments indicated that sequences gamma(3)(86-95) and gamma(3)(94-107), which are homologous to previously identified sequences important for assembly of gamma(2) subunits, are also important for assembly of gamma(3) subunits. This indicates that during assembly of the GABA(A) receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.
Highlights
␥-Aminobutyric acid, type A (GABAA)[1] receptors are the major inhibitory transmitter receptors in the central nervous system and mediate fast synaptic inhibition by opening an intrinsic chloride channel (1)
For the GABAA receptor, specific amino acid sequences have been identified in this domain that seem to mediate heteromeric and homomeric assembly of  subunits (29) or assembly of ␣ with  subunits (30)
GABAA receptors formed on the surface of ␣13␥3* or ␣13␥3 transfected cells were labeled by an incubation with ␣1(1–9) antibodies, and the antibody labeled receptors were again extracted, precipitated by Immunoprecipitin, and subjected to SDS-PAGE and Western blot analysis using digoxygenized ␥3(1–35) antibodies
Summary
␥-Aminobutyric acid, type A (GABAA)[1] receptors are the major inhibitory transmitter receptors in the central nervous system and mediate fast synaptic inhibition by opening an intrinsic chloride channel (1). To identify amino acid sequences important for binding, the even shorter N-terminal ␥3 fragments ␥3(1– 84) and ␥3(1–72) were generated, and their interaction with full-length ␣1 and 3 subunits was investigated after co-transfection into HEK cells.
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