Regulatory domains of GABAA receptors

GABAA receptors are the principal mediators of rapid, synaptic inhibition within the mammalian central nervous system. These receptors are hetero-oligomeric proteins that can be assembled from alpha(l-6), beta(l-3), gamma(l-3), delta &egr;, pi and &thgr; subunit classes (Mehta & Ticku, 1999; Bonnert et al , 1999). Each subunit comprises a putative extracellular N- terminus, four putative transmembrane domains (TM), a large intracellular domain linking TM3 and TM4, and an extracellular C-terminus. The present study investigated these domains to identify molecular determinants affecting modulation of GABA A receptor function.;The present study into regulatory domains of the GABAA receptor revealed important novel N-terminal residues for Zn2+ inhibition and a novel non-specific interaction of PTK inhibitors with the GABA A receptor. In addition, a method for probing ion channel properties using histidine was devised and the stoichiometry of a GABAA receptor complex was predicted using the binomial theorem.;An N-terminal histidine (H107) was implicated in mediating lower affinity Zn2+ binding on the ?3 subunit and also a possible role in homomeric receptor assembly/protein folding. Interestingly, mutation of the aligned residue on the alpha1 subunit (H109) strongly influenced GABA but not Zn2+ potency. Substitution of alpha1H141 located in the cysteine loop region of the N-terminal domain reduced Zn 2+ sensitivity of heteromeric GABAA receptors. An electrophysiological approach based on the relative insensitivity to Zn2+ generated by a TM2 mutation (H267A) was used to predict a tetrameric stoichiometry for beta3 subunit homomeric GABAA receptors. In addition, the substituted histidine accessibility method (SHAM) was developed to probe TM2 by individually substituting putative channel lining residues with histidines in homomeric beta3 H267A GABAA receptors. It was inferred that Zn2+ is able to penetrate the anion channel at least as far as residue 263. Finally, an initial investigation of residues in the large intracellular TM3-TM4 domain responsible for tyrosine phosphorylation of alpha1beta1gamma2S receptors discovered that external application of protein tyrosine kinase (PTK) inhibitors directly modulate GABAA receptors independently of tyrosine kinases.