Protein Kinases and Protein Recruitment at Inhibitory Synapses

Although it is well known that the postsynaptic GABAAR and GlyR anchor protein gephyrin is a substrate for serine/threonine kinase, nothing is known about the functional consequences of gephyrin phosphorylation89. However, protein phosphorylation also influences postsynaptic recruitment and functional modulation of GABAARs. Compared with excitatory neurotransmitter receptor systems, however, much less is known. PKC phosphorylation of GABAaR P and Y subunits was repeatedly shown to depress GABAAR-mediated currents90-93. In line with this, BDNF was identified to depress inhibitory postsynaptic currents through TrkB receptor activation and, further downstream, recruitment of PKC activity to inhibitory postsynaptic sites94. In hippocampal neurons, however, BDNF was found to exert a dual effect on postsynaptic GABAAR currents, which was characterized by a rapid (within 5-10 min) transitory increase in phosphorylation of the GABAAR P3 subunit, increasing the amplitudes of miniature inhibitory postsynaptic currents (mIPSC), followed by PP2A-mediated dephosphorylation, decreasing mIPSC amplitudes95. In this case, the BDNF-induced phosphorylation of the GABAAR P3 subunit not only involved the activation of PKC, but also recruited PP2A activity, which are both located downstream to the TrkB receptor-initiated signal. Apparently, phosphorylation of P subunit-containing GABAARs not only modulates the functional properties of GABAAR currents, but also differentially affects association of PKC and PP2A with these subunits, that is reduction of, and increase in, PKC and PP2A recruitment, respectively, to phosphorylated GABAAR P subunits. PKC shares its phosphorylation target residues within the GABAAR P3 subunit with a number of other kinases, including PKA. PKA activation was found to differentially influence GABAAR function according to the use of either two adjacent serine residues (S408 and S409) as phosphorylation targets, which potentiates GABA-activated responses, or the use of S409 only, which reduces GABA-elicited responses96. This scenario significantly gains complexity when considering that the protein phosphatase PP1A dephosphorylates GABAAR P3 subunits. The PP1A anchoring protein phospholipase C-related inactive protein type 1 (PRIP1) traps PP1A close to GABAAR P3 subunits. PKA phosphorylation of PRIP1 releases active PP1A from PRIP1, which then allows for dephosphorylation of P3 subunit-GABAARs. PKA activation, for example in response to dopamine D1 receptor activation97, thus contributes to the fine tuning of the GABAAR phosphorylation state, and it emerges once again that it is a complex spatial and temporal interplay between kinases and phosphatases, which determines the functional output of kinases.

In this context, it important to remind that it is the GABAAR y2 subunit, which is made responsible for postsynaptic GABAAR stabilization and which also is a phosphorylation target of a number of protein kinases98-100. Alternative splicing of GABAAR y2 transcripts results in the generation of two splice variants y2S and Y2L, the latter being generated by insertion of an eight-amino acid long sequence into the large intracellular loop. The y2S subunit contains several serine residues, which are substrates for protein kinases101. Interestingly, the insertion of the additional eight amino acids, however, adds another phosphorylation target residue to the GABAAR y2 subunit. It is this additional phosphorylation target residue (S343), which emerges as functionally relevant with respect to postsynaptic GABAAR recruitment. Consistent with this, CaMKII and PKC were identified to specifically phosphorylate the y2L subunit102, and PKC activation by phorbol ester specifically enhanced postsynaptic accumulation of the GABAAR y2L intracellular domain103. Thus, PKC was identified as positive modulator of GABAergic synapse formation100. As for kinase signaling at glutamatergic synapses, protein phosphatases also contribute to signaling at GABAergic synapses. For example, the protein phosphatase calcineurin (PP2B) leads to dephosphorylation of S327, present in both y2S and y2L loops. This is accompanied by depression of inhibitory synaptic responses104, but, in this case, it is not clear if the depression is due to reduced postsynaptic GABAAR recruitment or due to modulation of GABAAR channel function.

Altogether, this is a challenging issue because the balance between y2S and y2L GABAAR isoforms seems to be related to schizophrenia. In fact, a predominance of GABAAR y2L over y2S was found in the prefrontal cortex of humans suffering from schizophrenia105. In addition, the expression levels of the GABA-transporter GAT1 and the GABA-synthesizing enzyme GAD-67 were found to be reduced106. Therefore, the upregulation of GABAAR y2L may be considered as a compensatory mechanism to counteract the deficits in the GABA release machinery, or it might actually trigger schizophrenia since both Ca2+-sensitive (isoform y) and Ca2+-insensitive PKCs (isoform Z) were shown to be overactive in schizophrenics107, presumably as a consequence of enhanced membrane-anchoring of these PKCs through binding of the receptor for activated C-kinase 1 (RACK1)108. Altogether, modulation of channel function and postsynaptic GABAAR recruitment depends on (1) the subunit that is phosphorylated, (2) the phosphorylation target residue within each of the subunits, and (3) the relative phosphorylation status of target proteins, resulting from a complex interplay between several protein kinases and phosphatases.

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