McDonald, Bernard John;
(1998)
Studies on the phosphorylation of GABAA receptor β subunits.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
GABAA receptor function has been shown to be modulated by protein serine/threonine and tyrosine kinases due to direct subunit phosphorylation. Phosphorylation sites within receptor subunits have been identified and their roles in receptor regulation have been determined. PKC phosphorylates S409 in the β1 subunit as well as S327 in the γ2S and both S327 and S343 in the γ2L subunit. Phosphorylation of any of these residues reduces GABA-induced currents through receptors containing α1, β1 and γ2 subunits. PKA only phosphorylates S409 of the β1 subunit in these receptors, reducing GABA-induced currents. This effect is abolished by mutation of S409 to alanine. Regulation of neuronal GABAA receptors is more complex, currents can be reduced, unaffected or enhanced by activation of PKA, depending on the neuronal preparation studied. The role of β subunit identity on receptor regulation was investigated as a possible mechanism for such differences in PKA regulation of receptor function. Expression of subunit intracellular domains as GST-fusion proteins allowed identification of sites phosphorylated in vitro by protein serine/threonine kinases PKA, PKG, PKC and CamKII. This study extends earlier work on β1 and γ2 subunits and indicates that β2 and β3 subunits are phosphorylated at conserved sites (corresponding to β1-S409) by PKA, PKG, PKC and CamKII. Interestingly, PKC also phosphorylated S408 and CamKII phosphorylated S383 in the β3 subunit. The absence of S408 from both β1 and β2 subunits indicates potential for β subunit-dependent differential regulation of receptor function. Purified GST-fusion proteins containing subunit intracellular domains were used to immunise rabbits and produce subunit-specific polyclonal antibodies. Antibodies were purified which specifically recognise β1 and β3 subunits by western blotting, immuno-cytochemistry and immunoprecipitation. These antibodies allowed study of receptor subunit phosphorylation in a heterologous expression system. Receptor expression in HEK293 cells was used for biochemical and electrophysiological studies of β2 and β3 subunit phosphorylation by PKA. PKA did not phosphorylate the β2 subunit in these cells, accordingly PKA activation did not alter the function of receptors containing this subunit. The β3 subunit was highly phosphorylated by PKA on S408 and S409 and activation of PKA caused enhancement of GABA-induced currents through receptors containing this subunit. This enhancement was found to be critically dependent on both serine residues being phosphorylated within the same subunit. These results indicate a role for β subunit identity in the differential effects of PKA on GABAA receptor function in different neuronal cell types. This thesis describes biochemical and electrophysiological studies of GABAA receptor regulation by protein phosphorylation. Using varied approaches, differential phosphorylation and regulation of receptor subtypes was investigated. The results presented identify a novel mechanism for the differential regulation of GABAA receptor subtypes in the brain. Furthermore, this work has resulted in the production of antibody reagents which should prove important in the investigation of GABAA receptor phosphorylation in the brain.
Type: | Thesis (Doctoral) |
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Qualification: | Ph.D |
Title: | Studies on the phosphorylation of GABAA receptor β subunits |
Open access status: | An open access version is available from UCL Discovery |
Language: | English |
Additional information: | Thesis digitised by ProQuest. |
Keywords: | Biological sciences; GABA receptors |
URI: | https://discovery.ucl.ac.uk/id/eprint/10103150 |
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