Cheung, HY; (2013) Characterization of a3-containing GABAA receptors modified by RNA editing. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Editing of ribonucleic acid (RNA) is a post-transcriptional processing mechanism that increases heterogeneity of gene products. The a3 subunit of the GABAA receptor (Gabra3) has been shown to undergo RNA editing. This results in a change from an isoleucine (I) to a methionine (M) in the third transmembrane domain of the subunit. In the work described in this thesis I have used patch-clamp recording techniques to determine the effects of this amino acid switch on the properties of recombinant GABAA receptors. I examined both macroscopic and microscopic features of GABA-evoked currents using whole-cell, cell-attached and outside-out patch-clamp recording. Although, when expressed with r3 and y subunits, both un-edited a3(I) and edited a3(M) subunits were functional, the edited subunit yielded lower current densities, suggesting a reduced surface expression. Analysis of current-voltage relationships showed clear voltage-dependence of whole- cell currents, with prominent outward rectification at low GABA concentrations. However, for neither a3r32y2L nor a3r33y2L receptors, was there any difference in rectification between a3(I)- and a3(M)-containing receptors. Results with a non-editable subunit suggested no confounding effect of endogenous editing. In cell-attached single-channel recordings both a3(I)- and a3(M)-containing receptors exhibited high intra-burst open probabilities and long burst lengths, with a trend toward longer burst lengths with a3(M). Single-channel conductance was not affected by editing, and for both a3(I)- and a3(M)-containing receptors the current-voltage relationships were essentially linear. Rapid application of GABA to outside-out patches revealed a much slower activation of a3(I)-containing receptors but no marked difference in desensitization or deactivation I discuss my findings in terms of the biology of a3-containing GABAA receptors, the origin and postulated role(s) of GABAA receptor rectification, and the likely basis of the differences between my findings and those of other groups, which may, in part, reflect differences in the behaviour and relative prevalence of ar3y and ar3 subunit assemblies.

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