Structural and functional analysis of Apo-states of the model Cys-loop receptor GluCl from Caenorhabditis elegans

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Neurotransmission at chemical synapses is modulated in many different ways by the action of neurotransmitters on receptors. Cys-loop receptors are a large family of pentameric ligand-gated ion channels that mediate fast neurotransmission. Despite sharing a common architecture they can evoke both excitatory and inhibitory effects depending on their ion selectivity. Due to this wide-spread function they are involved in various neurological diseases and serve as targets for many clinical drugs and substances of drug abuse.Despite great advances on the understanding of the structure and function of these receptors, the details of their activation and gating mechanism are still poorly understood. A recently-published structure of the anion-selective Cys-loop receptor GluCl from Caenorhabditis elegans with the allosteric agonist ivermectin bound provided important initial insights into the mechanisms of the receptor. Yet, for a complete understanding, additional structural information from different conformational states of the protein is required. All current structures exhibit a ligand-bound conformation of the receptor. Knowledge especially about the apo state of the protein will greatly improve the understanding of the structural changes upon receptor activation. I therefore want to obtain high-resolution structural information of the model protein GluCl in different conformational states.Preliminary results indicate that lipids might play an important role in the activation of the receptor. One focus of this proposal will therefore be to investigate the functional and structural role of lipids in the context of receptor activation. Because the partial allosteric agonist ivermectin binds within the transmembrane region of the receptor, structural information about the interaction with natural modulators like lipids might also help to develop new therapeutics. Additionally I want to find out how gating of the channel pore is accomplished.For this purpose large amounts of wild-type as well as mutant protein made by site-directed mutagenesis will be expressed heterologously in insect cells and purified to homogeneity by affinity and size-exclusion chromatography. Homogeneity and stability of the protein will be monitored by fluorescence-detection size exclusion chromatography. Functionality of the protein will be assessed by radioligand binding in scintillation proximity assay, two-electrode voltage clamp electrophysiology on Xenopus laevis oocytes and ion flux into reconstituted proteoliposomes. Once sufficient amounts of homogeneous and active protein are available, 3D crystallization trials of the mutants in high-lipid conditions will be set up to solve the protein structure by X-ray diffraction.