The γ-aminobutyric acid(A) (GABA)(A) receptor (GABAR) β1 and γ2L subtypes have been shown to be phosphorylated in vitro by protein kinase C (PKC) [J. Biol. Chem. 267:14470-14476 (1992); Neuron 12:1081-1095 (1994)]. To determine the physiological consequences of phosphorylation of GABAR isoforms containing the β1 and γ2L subtypes, the specific serine residues phosphorylated by PKC (β1 S409, γ2L S327 and S343) were changed to alanines through site-directed mutagenesis. Wild-type (α1β1γ2L GABARs) and three mutant GABAR isoforms [α1β1γ2L(S327A, S343A), α1β1(S409A)γ2L, and α1β1(S409A)γ2L(S327A, S343A) GABARs) were expressed in mouse L929 fibroblasts through transient cotransfection. Recordings were obtained from each cell with the use of the whole-cell patch-clamp technique. The initial recording was made with the use of control intrapipette solution, and a second recording from the same cell was obtained with pipettes containing either constitutively active PKC [protein kinase M (PKM)] or control solution to obtain paired GABA concentration-response relationships. All GABAR isoforms studied had equivalent maximal GABA currents and similar GABA concentration-response profiles under the control condition. Intracellular PKM treatment increased the maximal current and EC50 value in cells expressing wild-type GABARs. However, PKM reimpalement did not significantly change these parameters in cells expressing any of the mutant GABAR isoforms, indicating that the mutation of either the β1 or γ2L subtype alone was sufficient to prevent enhancement of GABAR current by PKM. No significant changes were obtained during control reimpalement recordings of wild-type or mutant receptors. Furthermore, PKM treatment did not alter the time constants of GABA current desensitization kinetics measured from cells expressing wild- type or mutant receptors. These data thus suggest that PKC phosphorylation of the β1 and γ2L subtypes enhances GABAR current and that both subtypes are required for complete PKC-mediated enhancement of α1β1γ2L GABAR current.
|Original language||English (US)|
|Number of pages||11|
|State||Published - Jul 1996|
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