Building GABAA Receptors for Structural Determination

Abstract

GABA is the predominant mediator of inhibitory neurotransmission in the brain, and upon release rapidly activates ionotropic GABAARs. These receptors are members of the pentameric ligand gated ion channel (pLGIC) family and are the targets of a range of clinical and endogenous allosteric modulators, including general anesthetics, alcohols and neurosteroids. Many of these compounds bind in the transmembrane domain (TMD) of GABAARs and selectively potentiate or inhibit activity, and in some cases directly gate the channel. A number of crystal structures of prokaryotic and eukaryotic pLGICs have now been solved, including the first GABAAR structure, the human β3 homopentamer. However, high resolution structures that reveal the molecular basis for allosteric modulation of GABAARs are currently unavailable. Here we report the construction, characterization and crystallization of a chimeric pLGIC, which is amenable to structural studies of GABAAR transmembrane pharmacology.We have generated a chimeric receptor of the extracellular segment from the proton-gated ion channel from Gloeobacter violaceus (GLIC) and the transmembrane segment of the anion-selective human α1 GABAA receptor. The GLIC-α1 chimera formed functional proton-gated channels when expressed in Xenopus oocytes. Proton gated currents of the chimeric channel were potentiated by positive modulatory neurosteroids and depressed by inhibitory neurosteroids, whilst GLIC is normally insensitive to these compounds. This receptor can be overexpressed in insect cells and used to generate purified and intact homopentameric receptor (as determined by native mass spectrometry). Furthermore the purified protein can be used to grow protein crystals under a range of conditions. We are currently carrying out crystallization trials to generate diffraction quality crystals of our chimera for structural determinations.