GABA(A) receptor properties that shape neuronal inhibition.

Abstract

GABA_A receptors are chloride selective ligand-gated ion channels that mediate inhibitory neuronal signaling in the adult central nervous system. Native receptors are pentameric combinations of various subunits with expression patterns that are highly regulated with respect to anatomical location as well as developmental period. Although the physiological significance of such molecular diversity remains poorly understood, the striking subunit-dependence of kinetic properties and pharmacological modulation may provide a broad range of inhibition 'strategies' to the brain. The primary aim of this thesis was to determine the relevance of various GABA_A receptor properties for synaptic (non-equilibrium) and extra-synaptic (steady-state) forms of neuronal inhibition. A fibroblast expression system allowed molecular manipulations (mutations, chimeras) of receptor subunits, as well as precise kinetic measurements (concentration jump technique) of GABA_A receptor function to be achieved. This combined approach revealed several important receptor properties that may regulate the time course, efficacy, and pharmacological modulation of GABA_A receptor-mediated inhibition. Specifically, we developed a novel functional assay to demonstrate a fundamental constraint on the relationship between GABA binding and channel gating: all kinetic states from which channel opening occurs are agonist-bound; GABA unbinding is unlikely or not possible from open and desensitized states. This finding provided a conceptual framework for subsequent hypothesis testing of the specific impact of gating efficacy, desensitization and microscopic affinity on GABA_A deactivation, the major determinant of inhibitory post-synaptic current (IPSC) duration. Experimental data, supported by computer simulation results, indicated independent contributions of these three receptor properties to GABA_A receptor deactivation. These insights laid the foundation for functional evaluation of the first GABA_A receptor mutations linked to human epilepsy and sleep syndromes. Additional work on the properties and regulation of putative extra-synaptic GABA_A receptor isoforms, that are thought to mediate tonic inhibition, revealed clear subunit-dependent biophysical and pharmacological properties. Analysis of modulation by an endogenous neurosteroid indicated that these isoforms are subject to a surprisingly dynamic range of regulation. Moreover, detailed functional analysis led us to propose a novel mechanism for isoform-specific allosteric modulation that distinguishes GABA_A receptors based on differences in gating efficacy rather than primary protein structure.