Key points are not available for this paper at this time.
GABAA receptors are critical mediators of fast synaptic inhibition in the brain, and the predominant receptor subtype in the central nervous system is believed to be a pentamer composed of α, β, and γ subunits. Previous studies on recombinant receptors have shown that protein kinase C (PKC) and PKA directly phosphorylate intracellular serine residues within the receptor β subunit and modulate receptor function. However, the relevance of this regulation for neuronal receptors remains poorly characterized. To address this critical issue, we have studied phosphorylation and functional modulation of GABAAreceptors in cultured cortical neurons. Here we show that the neuronal β3 subunit is basally phosphorylated on serine residues by a PKC-dependent pathway. PKC inhibitors abolish basal phosphorylation, increasing receptor activity, whereas activators of PKC enhance β3 phosphorylation with a concomitant decrease in receptor activity. PKA activators were shown to increase the phosphorylation of the β3 subunit only in the presence of PKC inhibitors. We also show that the main sites of phosphorylation within the neuronal β3 subunit are likely to include Ser-408 and Ser-409, residues that are important for the functional modulation of β3-containing recombinant receptors. Furthermore, PKC activation did not change the total number of GABAA receptors in the plasma membrane, suggesting that the effects of PKC activation are on the gating or conductance of the channel. Together, these results illustrate that cell-signaling pathways that activate PKC may have profound effects on the efficacy of synaptic inhibition by directly modulating GABAA receptor function. GABAA receptors are critical mediators of fast synaptic inhibition in the brain, and the predominant receptor subtype in the central nervous system is believed to be a pentamer composed of α, β, and γ subunits. Previous studies on recombinant receptors have shown that protein kinase C (PKC) and PKA directly phosphorylate intracellular serine residues within the receptor β subunit and modulate receptor function. However, the relevance of this regulation for neuronal receptors remains poorly characterized. To address this critical issue, we have studied phosphorylation and functional modulation of GABAAreceptors in cultured cortical neurons. Here we show that the neuronal β3 subunit is basally phosphorylated on serine residues by a PKC-dependent pathway. PKC inhibitors abolish basal phosphorylation, increasing receptor activity, whereas activators of PKC enhance β3 phosphorylation with a concomitant decrease in receptor activity. PKA activators were shown to increase the phosphorylation of the β3 subunit only in the presence of PKC inhibitors. We also show that the main sites of phosphorylation within the neuronal β3 subunit are likely to include Ser-408 and Ser-409, residues that are important for the functional modulation of β3-containing recombinant receptors. Furthermore, PKC activation did not change the total number of GABAA receptors in the plasma membrane, suggesting that the effects of PKC activation are on the gating or conductance of the channel. Together, these results illustrate that cell-signaling pathways that activate PKC may have profound effects on the efficacy of synaptic inhibition by directly modulating GABAA receptor function. γ-aminobutyric acid type A protein kinase C phorbol dibutyrate cAMP-dependent kinase Ca2+/calmodulin-dependent kinase II enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis GABAA1receptors are key mediators of fast synaptic inhibition in the brain. These receptors are pentameric hetero-oligomers that can be assembled from 6 subunit classes with multiple members: α(1–6), β(1–3), γ(1–3), δ, ε, and θ subunits (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar, 3Davies P.A. Hanna M.C. Hales T.G. Kirkness E.F. Nature. 1997; 385: 820-823Crossref PubMed Scopus (368) Google Scholar, 4Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Brown N. Wafford K.A. Whiting P.J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: 9891-9896Crossref PubMed Scopus (280) Google Scholar). Heterologous co-expression of receptor α, β, and γ subunits has been shown to reproduce many of the physiological and pharmacological properties of neuronal GABAA receptors (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar, 3Davies P.A. Hanna M.C. Hales T.G. Kirkness E.F. Nature. 1997; 385: 820-823Crossref PubMed Scopus (368) Google Scholar, 4Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Brown N. Wafford K.A. Whiting P.J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: 9891-9896Crossref PubMed Scopus (280) Google Scholar).There is considerable interest in understanding the endogenous mechanisms used by neurons to regulate GABAA receptor function. Much emphasis has focused on the role of receptor phosphorylation because the observation that purified receptor preparations are phosphorylated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (5Kirkness E.F. Bovenkerk C.F. Ueda T. Turner A.J. Biochem. J. 1989; 259: 613-616Crossref PubMed Scopus (84) Google Scholar, 6Browning M.D. Bureau M. Dudek E.M. Olsen R.W. Proc. Natl. Acad. Sci.(U. S. A.). 1990; 87: 1315-1318Crossref PubMed Scopus (146) Google Scholar, 7Browning M.D. Endo S. Smith G.B. Dudek E.M. Olsen R.W. Neurochem. Res. 1993; 18: 95-100Crossref PubMed Scopus (34) Google Scholar). Studies on recombinant receptors have revealed that receptor β and γ subunits are substrates for a number of protein kinases. Specifically, the β subunits are phosphorylated on a conserved serine residue (Ser-409 or Ser-410) by PKC, PKA, Ca2+/Calmodulindependent protein kinase (CamKII), and cGMP-dependent protein kinase (8Moss S.J. Doherty C.A. Huganir R.L. J. Biol. Chem. 1992; 267: 14470-14476Abstract Full Text PDF PubMed Google Scholar, 9Moss S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (243) Google Scholar, 10Krishek Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. 1997; PubMed Scopus Google Scholar). Ser-408 is also a PKA in the β3 subunit S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). GABAA receptors are also substrates of the kinase and within the subunit S.J. Smart T.G. Nature. 1995; PubMed Scopus Google Scholar, C.F. McKernan R.M. Whiting S.J. Smith G.B. Olsen R.W. Res. Res. 1995; PubMed Scopus Google Scholar). of recombinant receptors by PKA or PKC functional effects from to on the of the the of the sites phosphorylated and the of kinase activation S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar, S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, T.P. Dudek E.M. M.D. R.L. Google Scholar, T.G. 1997; PubMed Scopus Google Scholar). role of protein kinase in modulating neuronal GABAAreceptors has been studied with S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, T.G. 1997; PubMed Scopus Google Scholar). these studies have revealed that receptor can be by that the of PKC or However, in the of the phosphorylation of GABAA receptor subunits these in receptor remains address this we have used and to the regulation of GABAA receptor by protein kinase in cultured cortical neurons. results that the β3 subunit is basally phosphorylated by a PKC-dependent pathway. PKC inhibitors abolish this basal phosphorylation with increase in activity. activators of PKC enhance the of β3 phosphorylation to a decrease in activity. We show that this modulation of is by in of receptor of PKA only increase the of β3 phosphorylation in the presence of PKC of a protein and the results that pathways that the of PKC may have profound effects on synaptic inhibition by directly modulating the of receptor receptors are mediators of fast synaptic inhibition in the brain, important (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar). is of central to the regulation of the functional properties of these receptors in Studies on recombinant receptors have that receptor phosphorylation of key conserved serine residues within the β and subunits is a of GABAA receptor PKA and PKC activation S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar, S.J. Smart T.G. Nature. 1995; PubMed Scopus Google Scholar, T.P. Dudek E.M. M.D. R.L. Google Scholar). activation or inhibition can have effects on GABAA receptor in neurons S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, J. Neurosci. 1997; 17: PubMed Google Scholar, M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar, J. 1999; PubMed Scopus Google the of the pathways that receptor phosphorylation in the this we have studied the regulation of GABAA receptors by protein in cortical and phosphorylation with for the subunits cortical neurons. a of we the β3 basal phosphorylation of the β3 subunit to be on a PKC-dependent in these neurons. These with the of recombinant have revealed that the intracellular of the β3 subunit is phosphorylated on by PKC S.J. 1997; PubMed Scopus Google Scholar). basal phosphorylation of the β3 subunit by PKC is also in with the of GABAA receptors with the of PKC and in cortical neurons N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google Scholar). of these with GABAAreceptors is for the basal phosphorylation of the β3 subunit in this Furthermore, activation of PKC in cortical neurons by the of to the β3 subunit N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google with a increase in the of GABAA receptor phosphorylation shown β3 subunit is also a PKA and the sites of phosphorylation in have been Ser-408 and S.J. 1997; PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). of the β3 subunit in cortical neurons by a also only in the presence of PKC inhibitors. observation a GABAA receptors and pathways in neurons. to is that phosphorylation phosphorylation, the of PKC or to the intracellular of the β3 subunit N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google directly the sites of phosphorylation within the β3 subunit to in recombinant we PKA and PKC phosphorylate Ser-408 and within the intracellular of the β3 subunit a protein or in S.J. 1997; PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). of the β3 subunit phosphorylated by PKC or PKA in neurons were to the of the β3 subunit in These results that in the phosphorylation sites within the β3 subunit are likely to include Ser-408 and These residues have been in recombinant receptors and a critical role in functional modulation of GABAA receptors by kinase S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (243) Google Scholar, 10Krishek Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google the functional of modulating the of β3 subunit phosphorylation in were of PKC with C a increase in a decrease in these results show that in receptor with in β3 subunit However, to that these functional effects are by phosphorylation of the β3 subunit the of in GABAA receptor phosphorylation sites have been by did not to be the in the of β3 subunit phosphorylation on increase in and the in decrease in of However, that cortical neurons a of GABAA receptor this may from receptor with receptor that are by PKC M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google revealed that PKC the with effects on in cortical neurons. is with studies on recombinant receptors composed of and PKC sites within the and subunits Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar). the of β3 PKC activation receptors phosphorylation of in β3 and in J. and T. in PKC can also in of receptors in of receptor phosphorylation Smart T.G. S.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To PKC in cortical neurons on receptor were by and to PKC activators be receptor of in a decrease of in be is of neurons to in the and on a decrease in is that this the of because of a of neurons. in the effects of PKC on the of recombinant and neuronal receptors may the of GABAA receptors to the by in with M. B. Neurosci. PubMed Scopus Google Scholar, S.J. Olsen R.W. Nature. 1999; PubMed Scopus Google Scholar). has also been to GABAA receptor in a number of neuronal preparations and neurons Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. Whiting 1992; Google Scholar). PKC can enhance GABAA receptor in and neurons M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar). these effects of PKC activation from of GABAA receptor or the activation of pathways by PKC remains to be of PKC in modulating GABAA receptor has been by of the γ or PKC in in S.J. S. Proc. Natl. Acad. S. A.). 1995; PubMed Scopus Google Scholar, T. Neurosci. 1999; PubMed Scopus Google we have that GABAA receptor and phosphorylation can be by PKC in neurons. and that activate PKC pathways may have profound effects on synaptic inhibition modulation of GABAA receptor subunit GABAA1receptors are key mediators of fast synaptic inhibition in the brain. These receptors are pentameric hetero-oligomers that can be assembled from 6 subunit classes with multiple members: α(1–6), β(1–3), γ(1–3), δ, ε, and θ subunits (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar, 3Davies P.A. Hanna M.C. Hales T.G. Kirkness E.F. Nature. 1997; 385: 820-823Crossref PubMed Scopus (368) Google Scholar, 4Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Brown N. Wafford K.A. Whiting P.J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: 9891-9896Crossref PubMed Scopus (280) Google Scholar). Heterologous co-expression of receptor α, β, and γ subunits has been shown to reproduce many of the physiological and pharmacological properties of neuronal GABAA receptors (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar, 3Davies P.A. Hanna M.C. Hales T.G. Kirkness E.F. Nature. 1997; 385: 820-823Crossref PubMed Scopus (368) Google Scholar, 4Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Brown N. Wafford K.A. Whiting P.J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: 9891-9896Crossref PubMed Scopus (280) Google Scholar). is considerable interest in understanding the endogenous mechanisms used by neurons to regulate GABAA receptor function. Much emphasis has focused on the role of receptor phosphorylation because the observation that purified receptor preparations are phosphorylated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (5Kirkness E.F. Bovenkerk C.F. Ueda T. Turner A.J. Biochem. J. 1989; 259: 613-616Crossref PubMed Scopus (84) Google Scholar, 6Browning M.D. Bureau M. Dudek E.M. Olsen R.W. Proc. Natl. Acad. Sci.(U. S. A.). 1990; 87: 1315-1318Crossref PubMed Scopus (146) Google Scholar, 7Browning M.D. Endo S. Smith G.B. Dudek E.M. Olsen R.W. Neurochem. Res. 1993; 18: 95-100Crossref PubMed Scopus (34) Google Scholar). Studies on recombinant receptors have revealed that receptor β and γ subunits are substrates for a number of protein kinases. Specifically, the β subunits are phosphorylated on a conserved serine residue (Ser-409 or Ser-410) by PKC, PKA, Ca2+/Calmodulindependent protein kinase (CamKII), and cGMP-dependent protein kinase (8Moss S.J. Doherty C.A. Huganir R.L. J. Biol. Chem. 1992; 267: 14470-14476Abstract Full Text PDF PubMed Google Scholar, 9Moss S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (243) Google Scholar, 10Krishek Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. 1997; PubMed Scopus Google Scholar). Ser-408 is also a PKA in the β3 subunit S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). GABAA receptors are also substrates of the kinase and within the subunit S.J. Smart T.G. Nature. 1995; PubMed Scopus Google Scholar, C.F. McKernan R.M. Whiting S.J. Smith G.B. Olsen R.W. Res. Res. 1995; PubMed Scopus Google Scholar). of recombinant receptors by PKA or PKC functional effects from to on the of the the of the sites phosphorylated and the of kinase activation S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar, S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, T.P. Dudek E.M. M.D. R.L. Google Scholar, T.G. 1997; PubMed Scopus Google Scholar). role of protein kinase in modulating neuronal GABAAreceptors has been studied with S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, T.G. 1997; PubMed Scopus Google Scholar). these studies have revealed that receptor can be by that the of PKC or However, in the of the phosphorylation of GABAA receptor subunits these in receptor remains To address this we have used and to the regulation of GABAA receptor by protein kinase in cultured cortical neurons. results that the β3 subunit is basally phosphorylated by a PKC-dependent pathway. PKC inhibitors abolish this basal phosphorylation with increase in activity. activators of PKC enhance the of β3 phosphorylation to a decrease in activity. We show that this modulation of is by in of receptor of PKA only increase the of β3 phosphorylation in the presence of PKC of a protein and the results that pathways that the of PKC may have profound effects on synaptic inhibition by directly modulating the of receptor receptors are mediators of fast synaptic inhibition in the brain, important (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar). is of central to the regulation of the functional properties of these receptors in Studies on recombinant receptors have that receptor phosphorylation of key conserved serine residues within the β and subunits is a of GABAA receptor PKA and PKC activation S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar, S.J. Smart T.G. Nature. 1995; PubMed Scopus Google Scholar, T.P. Dudek E.M. M.D. R.L. Google Scholar). activation or inhibition can have effects on GABAA receptor in neurons S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, J. Neurosci. 1997; 17: PubMed Google Scholar, M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar, J. 1999; PubMed Scopus Google the of the pathways that receptor phosphorylation in the this we have studied the regulation of GABAA receptors by protein in cortical and phosphorylation with for the subunits cortical neurons. a of we the β3 basal phosphorylation of the β3 subunit to be on a PKC-dependent in these neurons. These with the of recombinant have revealed that the intracellular of the β3 subunit is phosphorylated on by PKC S.J. 1997; PubMed Scopus Google Scholar). basal phosphorylation of the β3 subunit by PKC is also in with the of GABAA receptors with the of PKC and in cortical neurons N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google Scholar). of these with GABAAreceptors is for the basal phosphorylation of the β3 subunit in this Furthermore, activation of PKC in cortical neurons by the of to the β3 subunit N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google with a increase in the of GABAA receptor phosphorylation shown β3 subunit is also a PKA and the sites of phosphorylation in have been Ser-408 and S.J. 1997; PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). of the β3 subunit in cortical neurons by a also only in the presence of PKC inhibitors. observation a GABAA receptors and pathways in neurons. to is that phosphorylation phosphorylation, the of PKC or to the intracellular of the β3 subunit N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google directly the sites of phosphorylation within the β3 subunit to in recombinant we PKA and PKC phosphorylate Ser-408 and within the intracellular of the β3 subunit a protein or in S.J. 1997; PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). of the β3 subunit phosphorylated by PKC or PKA in neurons were to the of the β3 subunit in These results that in the phosphorylation sites within the β3 subunit are likely to include Ser-408 and These residues have been in recombinant receptors and a critical role in functional modulation of GABAA receptors by kinase S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (243) Google Scholar, 10Krishek Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google the functional of modulating the of β3 subunit phosphorylation in were of PKC with C a increase in a decrease in these results show that in receptor with in β3 subunit However, to that these functional effects are by phosphorylation of the β3 subunit the of in GABAA receptor phosphorylation sites have been by did not to be the in the of β3 subunit phosphorylation on increase in and the in decrease in of However, that cortical neurons a of GABAA receptor this may from receptor with receptor that are by PKC M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google revealed that PKC the with effects on in cortical neurons. is with studies on recombinant receptors composed of and PKC sites within the and subunits Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar). the of β3 PKC activation receptors phosphorylation of in β3 and in J. and T. in PKC can also in of receptors in of receptor phosphorylation Smart T.G. S.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To PKC in cortical neurons on receptor were by and to PKC activators be receptor of in a decrease of in be is of neurons to in the and on a decrease in is that this the of because of a of neurons. in the effects of PKC on the of recombinant and neuronal receptors may the of GABAA receptors to the by in with M. B. Neurosci. PubMed Scopus Google Scholar, S.J. Olsen R.W. Nature. 1999; PubMed Scopus Google Scholar). has also been to GABAA receptor in a number of neuronal preparations and neurons Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. Whiting 1992; Google Scholar). PKC can enhance GABAA receptor in and neurons M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar). these effects of PKC activation from of GABAA receptor or the activation of pathways by PKC remains to be of PKC in modulating GABAA receptor has been by of the γ or PKC in in S.J. S. Proc. Natl. Acad. S. A.). 1995; PubMed Scopus Google Scholar, T. Neurosci. 1999; PubMed Scopus Google we have that GABAA receptor and phosphorylation can be by PKC in neurons. and that activate PKC pathways may have profound effects on synaptic inhibition modulation of GABAA receptor subunit GABAA receptors are mediators of fast synaptic inhibition in the brain, important (1Macdonald R.L. Olsen R.W. Annu. Rev. Neurosci. 1994; 17: 569-602Crossref PubMed Scopus (1781) Google Scholar, 2Rabow L.E. Russek S.J. Farb D.H. Synapse. 1995; 21: 189-274Crossref PubMed Scopus (457) Google Scholar). is of central to the regulation of the functional properties of these receptors in Studies on recombinant receptors have that receptor phosphorylation of key conserved serine residues within the β and subunits is a of GABAA receptor PKA and PKC activation S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar, S.J. Smart T.G. Nature. 1995; PubMed Scopus Google Scholar, T.P. Dudek E.M. M.D. R.L. Google Scholar). activation or inhibition can have effects on GABAA receptor in neurons S.J. Smart T.G. Rev. Neurosci. Scopus Google Scholar, J. Neurosci. 1997; 17: PubMed Google Scholar, M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar, J. 1999; PubMed Scopus Google the of the pathways that receptor phosphorylation in the To this we have studied the regulation of GABAA receptors by protein in cortical and phosphorylation with for the subunits cortical neurons. a of we the β3 basal phosphorylation of the β3 subunit to be on a PKC-dependent in these neurons. These with the of recombinant have revealed that the intracellular of the β3 subunit is phosphorylated on by PKC S.J. 1997; PubMed Scopus Google Scholar). basal phosphorylation of the β3 subunit by PKC is also in with the of GABAA receptors with the of PKC and in cortical neurons N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google Scholar). of these with GABAAreceptors is for the basal phosphorylation of the β3 subunit in this Furthermore, activation of PKC in cortical neurons by the of to the β3 subunit N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google with a increase in the of GABAA receptor phosphorylation shown β3 subunit is also a PKA and the sites of phosphorylation in have been Ser-408 and S.J. 1997; PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). of the β3 subunit in cortical neurons by a also only in the presence of PKC inhibitors. observation a GABAA receptors and pathways in neurons. to is that phosphorylation phosphorylation, the of PKC or to the intracellular of the β3 subunit N. J. Olsen R.W. P.J. S.J. J. Neurosci. 1999; PubMed Google Scholar). To directly the sites of phosphorylation within the β3 subunit to in recombinant we PKA and PKC phosphorylate Ser-408 and within the intracellular of the β3 subunit a protein or in S.J. 1997; PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). of the β3 subunit phosphorylated by PKC or PKA in neurons were to the of the β3 subunit in These results that in the phosphorylation sites within the β3 subunit are likely to include Ser-408 and These residues have been in recombinant receptors and a critical role in functional modulation of GABAA receptors by kinase S.J. Smart T.G. Blackstone C.D. Huganir R.L. Science. 1992; 257: 661-665Crossref PubMed Scopus (243) Google Scholar, 10Krishek Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. Smart T.G. Neurosci. PubMed Scopus Google Scholar). To the functional of modulating the of β3 subunit phosphorylation in were of PKC with C a increase in a decrease in these results show that in receptor with in β3 subunit However, to that these functional effects are by phosphorylation of the β3 subunit the of in GABAA receptor phosphorylation sites have been by did not to be the in the of β3 subunit phosphorylation on increase in and the in decrease in of However, that cortical neurons a of GABAA receptor this may from receptor with receptor that are by PKC M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar). revealed that PKC the with effects on in cortical neurons. is with studies on recombinant receptors composed of and PKC sites within the and subunits Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar). the of β3 PKC activation receptors phosphorylation of in β3 and in J. and T. in PKC can also in of receptors in of receptor phosphorylation Smart T.G. S.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To PKC in cortical neurons on receptor were by and to PKC activators be receptor of in a decrease of in be is of neurons to in the and on a decrease in is that this the of because of a of neurons. in the effects of PKC on the of recombinant and neuronal receptors may the of GABAA receptors to the by in with M. B. Neurosci. PubMed Scopus Google Scholar, S.J. Olsen R.W. Nature. 1999; PubMed Scopus Google Scholar). has also been to GABAA receptor in a number of neuronal preparations and neurons Blackstone C.D. Huganir R.L. S.J. Smart T.G. 1994; Full Text PDF PubMed Scopus Google Scholar, S.J. Whiting 1992; Google Scholar). PKC can enhance GABAA receptor in and neurons M. J. Proc. Natl. Acad. Sci.(U. S. A.). 1999; 96: PubMed Scopus Google Scholar, M. M. J. Neurosci. 1999; PubMed Google Scholar). these effects of PKC activation from of GABAA receptor or the activation of pathways by PKC remains to be of PKC in modulating GABAA receptor has been by of the γ or PKC in in S.J. S. Proc. Natl. Acad. S. A.). 1995; PubMed Scopus Google Scholar, T. Neurosci. 1999; PubMed Scopus Google Scholar). we have that GABAA receptor and phosphorylation can be by PKC in neurons. and that activate PKC pathways may have profound effects on synaptic inhibition modulation of GABAA receptor subunit We of the for critical of the
Brandon et al. (Fri,) studied this question.