GABA(A) receptor beta subunit phosphorylation and modulation determine amplitude and time course of inhibitory currents.

摘要

The GABA_A receptor complex mediates most inhibitory signaling in the adult mammalian central nervous system. GABA_A receptors are functionally and structurally diverse due to multiple combinations of subunit subtypes that comprise the pentameric subunit receptor complex. In addition to functional diversity due to molecular receptor heterogeneity, GABA_A receptor function can be modulated by exogenous and endogenous modulatory agents. Modulation of inhibition is important for neuronal plasticity, receptor crosstalk, and pathogenesis and treatment of neurological diseases.; The primary aim of this thesis was to determine the role of the GABA _A receptor β subunit in regulation of neuronal inhibition. A recombinant expression system permitted the use of specific receptor subunit subtypes and receptor mutations. We utilized a modified rapid concentration jump protocol and established single channel recording methodology to determine the rapid non-steady state kinetics of GABA elicited currents as well as steady state single channel kinetics. Additionally, our rapid concentration jump protocol provided the means minimize the nonspecific effects of extracellular kinase modulators due to the precise timing of modulator exchange relative to GABA application.; Initially, we provided descriptive information about how phosphorylation and GABA modulators altered current decay. Subsequently, we utilized multiple recording modalities and paradigms such as rapid application of brief and long GABA pulses, paired pulse experiments, whole cell population experiments, and single channel analysis to provide constraints, which permitted a mechanistic interpretation of data. By taking all our collected data into account when drawing mechanistic conclusions, we were able to develop the first GABA _A receptor model that was not only based on all collected data, but was able to accurately simulate all the recordings obtained with wild type GABA_A receptors. We then used that model to test hypotheses about the mechanism of β subunit-mediated modulation of GABA_A receptor current by protein kinase A-mediated phosphorylation, by loreclezole, and by a human disease mutation. The data here illuminate important aspects of β subunit regulation of the GABA_A receptor complex and provide a theoretical framework upon which other investigators can predict and test hypotheses of GABA_A receptor function in relation to other receptor modulators.