Enhancement of recombinant alpha 1 beta 1 gamma 2L gamma-aminobutyric acidA receptor whole-cell currents by protein kinase C is mediated through phosphorylation of both beta 1 and gamma 2L subunits.
The gamma-aminobutyric acidA (GABA)A receptor (GABAR) beta 1 and gamma 2L 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 beta 1 and gamma 2L subtypes, the specific serine residues phosphorylated by PKC (beta 1 S409, gamma 2L S327 and S343) were changed to alanines through site-directed mutagenesis. Wild-type (alpha 1 beta 1 gamma 2L GABARs) and three mutant GABAR isoforms [alpha 1 beta 1 gamma 2L(S327A, S343A), alpha 1 beta 1(S409A) gamma 2L, and alpha 1 beta 1(S409A) gamma 2L(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 beta 1 or gamma 2L 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 after 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 beta 1 and gamma 2L subtypes enhances GABAR current and that both subtypes are required for complete PKC-mediated enhancement of alpha 1 beta 1 gamma 2L GABAR current.
Lin
Y F
YF
Department of Physiològy, University of Michigan Medical School, Ann Arbor 48109, USA.
Angelotti
T P
TP
Dudek
E M
EM
Browning
M D
MD
Macdonald
R L
RL
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D017931:DNA Primers; D007700:Kinetics; D007739:L Cells; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism*; D055503:Protein Multimerization; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D018118:Chloride Channels; D017931:DNA Primers; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D012694:Serine; D005680:gamma-Aminobutyric Acid; D011493:Protein Kinase C; D000409:Alanine