| |
[摘要]：A GABA(A) receptor beta 3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three beta 3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of beta 3 subunits. The mutation had three major effects on GABA(A) receptors. First, coexpression of beta 3(G32R) subunits with alpha 1 or alpha 3 and gamma 2L subunits in HEK293T cells reduced surface expression of gamma 2L subunits and increased surface expression of beta 3 subunits, suggesting a partial shift from ternary alpha beta 3 gamma 2L receptors to binary alpha beta 3 and homomeric beta 3 receptors. Second, beta 3(G32R) subunits were more likely than beta 3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, alpha 1 beta 3(G32R)gamma 2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because gamma 2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that alpha 1 beta 3(G32R)gamma 2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the beta 3 subunit N-terminal alpha-helix in receptor assembly and gating. |
|
