Houston, C.M.; (2005) Modulation of the GABAA receptor by CaMK-II dependent phosphorylation. Doctoral thesis , University of London.

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Abstract

Phosphorylation is an important mechanism for the modulation of ligand- gated ion channel function. CaMK-II dependent modulation of GABAa receptors was investigated through analysis of GABAA receptor mediated currents recorded by whole-cell patch clamp from non-neuronal and neuronal cell lines and from primary neurones in culture. Application of pre-activated a-CaMK-II had no effect on recombinant receptors expressed in HEK293 cells. Whole-cell currents recorded from cerebellar granule cells were significantly increased in peak amplitude on application of pre- activated a-CaMK-II. Analysis of spontaneous (s)IPSCs recorded from cerebellar granule cells in culture in the presence of pre-activated a-CaMK-II revealed an increase in sIPSC amplitude and decay time constants. Expression of recombinant GABAa receptors in the neuronal cell line NG108-15 allowed the functional effect of CaMK-II to be studied in a recombinant system. CaMK-II was able to up-regulate the function of cd/SlS and al/ftS GABAA receptors but not alfil'il receptors. This modification of function occurred through phosphorylation of Ser on the /33 subunit and through a downstream activation of a tyrosine kinase and subsequent phosphorylation of the 72S subunit at previously identified sites of tyrosine kinase phosphorylation, Tyr357 and Tyr359. Tyrosine kinase phosphorylation of 72S occurred when co-expressed with cd/31 or cd/33 but not with cd/2. Transfection of different GABAA receptor subunits into cerebellar granule cells was carried out to confirm the importance of these sites in a neuronal environment. Analysis of sIPSCs recorded from cerebellar granule cell cultures prepared from /52 subunit knockout mice revealed a-CaMK-II modification of /32 subunit-containing receptors at the synapse, resulting in an increase in sIPSC amplitude with no change in decay times. This was in contrast to the lack of any functional modulation of cd/52y2S receptors observed in NG108-15 cells. These findings are potentially important for understanding the mechanisms of CaMK-II dependent modulation of the GABAA receptor and may also have significant implications for the understanding of inhibitory synaptic plasticity.

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