Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

Abstract

The prevalence of aromatic residues in the ligand binding site of the GABAA receptor, as with other cys-loop ligand-gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β2Tyr97, β2Tyr157, and β2Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK-293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABAA receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β2Tyr157 and β2Tyr205 are more detrimental than β2Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process. Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABA(A) receptorSeveral tyrosine residues are located in the GABA binding pocket of the GABA(A) receptor, but specific contributions of each residue to receptor function are not fully understood. We found that mutating each tyrosine specifically slowed the GABA binding rate. This detailed kinetic characterization of binding pocket mutations will aid the description of how neurotransmitter binding leads to receptor activation. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.