Structure and function of the GabaA receptor

Using site-directed mutagenesis and whole-cell patch clamping, this study investigated the structure of the GABAa receptor in relation to its function. The baculovirus expression system was used to express human ocißi subunits in Sfi cells. The GABAa receptor is widely distributed throughout the central nervous system and is the site of action for many anaesthetic drugs. The mechanisms that underlie receptor activation by GABA and modulation by drugs are not well understood and are, therefore, the focus of this study. The role of highly conserved residues in the first and second membranespanning regions was investigated. Site-directed mutations were made in either the otj subunit, the ßi subunit or both to examine possible subunit-specific effects on receptor function. Assembly of receptors in the plasma membrane was monitored by immunofluoresence using flow cytometry. The binding of radiolabelled muscimol, a high affinity GABA analogue, to receptors in permeabilised cells was used to determine whether subunits had formed heteromeric receptors. Changes in the characteristics of the whole-cell current in response to GABA showed the functional effects of the mutations. Possible changes in receptor pharmacology were also investigated. All mutations carried out showed subunit specificity in their effects indicating that subunits contribute unequally to receptor function. Most mutated receptors showed changes in desensitisation that varied depending upon the position of the residue and the substitution made. The response to GABA was abolished when a threonine residue at the 13' position in M2 was mutated to alanine in the ß subunit. The apparent affinity for GABA was decreased when a leucine residue at the 9' position in M2 was mutated to either phenylalanine or tyrosine in only the ß subunit. These effects demonstrate the importance of the M2 region of the ß subunit in transmitting conformational changes involved in GABA-activation. Potentiation by pentobarbital was abolished when the T12'Q mutation was in the ß subunit implicating a discrete site in the action of pentobarbital. The response to GABA was reduced when tyrosine at position 231 in the M l region was mutated to leucine in the a subunit. This indicates a role for M l in GABA-activation that is a subunit specific. The findings in this study demonstrate roles for residues in M l and M2 in coupling between agonist binding and channel activation and indicate separate mechanisms of action for GABA and the potentiating effect of pentobarbital. Publications and Abstracts Structural Determinants of Desensitisation and Allosteric Regulation by Pentobarbitone of the GABAa Receptor. B Birnir, ML Tierney, JE Dalziel, GB Cox and PW Gage. Journal o f Membrane Biology, Vol. 155, Jan 1997. Coupling Between Agonist Binding and Channel Activation Involves a Highly Conserved Leucine Residue in the M2 Region of otißi GABAa Receptors. JE Dalziel, B Birnir, GB Cox and PW Gage. Biophysical Journal Abstracts, Vol.74, No.3, Feb 1998. Size, Hydrophobicity and Chemical Reactivity of a Highly Conserved Leucine Residue in the M2 Region Affect GABAa otißi Ion Channel Function. JE Dalziel, B Birnir, GB Cox and PW Gage. Proceedings o f the Australian Physiological and Pharmacological Society, Vol.27, No.2, Dec 1996. Site-directed Mutations in the GABAa Receptor Produce Subunit Specific Changes in Ion Channel Function. JE Dalziel, B Birnir, ML Tierney, GB Cox and PW Gage. Proceedings o f the Australian Society for Biochemistry and Molecular Biology. Vol. 28,1996. Single Site Mutations in the Second Transmembrane Region in the GABAa Receptor Alter Desensitisation. JE Dalziel, B Birnir, ML Tierney, GB Cox, PW Gage. Proceedings o f the Australian Physiological and Pharmacological Society, Vol.26, No.2, Sept 1995.