RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors containing either the edited or unedited forms of the α3 subunit along with β3 and γ2L. Editing substantially altered the GABA sensitivity and deactivation rate of the receptors, with the unedited form showing a lower GABA EC50 and slower decay. Comparable effects were observed with a mutation at the homologous location in the α1 subunit, suggesting a common role for this site in regulation of channel gating. Except for the response to GABA, the pharmacological properties of the receptor were unaffected by editing, with similar enhancement by a variety of modulators. Since RNA editing of the α3 subunit increases through development, our findings suggest that GABAergic neurotransmission may be more effective early in development, with greater GABA sensitivity and slower decay rates conferred by the unedited α3 subunit.
Many drugs used to treat anxiety are positive modulators of GABA A receptors, which mediate fast inhibitory neurotransmission. The GABA A receptors can be assembled from a combination of at least 16 different subunits. The receptor's subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABA A receptors is the production of subunit-selective modulators acting upon a discrete population of receptors. The anxiolytic 4-amino-7-hydroxy-2-methyl-5, 6,7,8,-tetrahydrobenzo[b]thieno [2,3-b]pyridine-3-carboxylic acid, but-2-ynyl ester (SB-205384) is widely considered to be selective for a3-containing GABA A receptors. However, it has been tested only on a1-, a2-, and a3-containing receptors. We examined the activity of SB-205384 at recombinant receptors containing the six different a subunits and found that receptors containing the a3, a5, and a6 subunits were potentiated by SB-205384, with the a6 subunit conferring the greatest responsiveness. Properties associated with chimeric a1/a6 subunits suggested that multiple structural domains influence sensitivity to SB-205384. Point mutations of residues within the extracellular N-terminal domain identified a leucine residue located in loop E of the agonist binding site as an important determinant of high sensitivity to modulation. In the a6 subunit the identity of this residue is species-dependent, with the leucine found in rat subunits but not in human. Our results indicate that SB-205384 is not an a3-selective modulator, and instead acts at several GABA A receptor isoforms. These findings have implications for the side-effect profile of this anxiolytic as well as for its use in neuronal and animal studies as a marker for contribution from a3-containing receptors.
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