Neurotransmitter receptors of the Cys-loop superfamily mediate fast synaptic transmission throughout the central and peripheral nervous systems. Their essential task is to transduce binding of neurotransmitter into opening of an intrinsic ion channel, yet the mechanism by which this transduction is achieved remains unknown. This proposal aims to delineate two fundamental facets of the transduction mechanism in homomeric Cys-loop receptors: (1) the number of agonist binding sites and binding-pore coupling regions required for activation by the agonist and (2) the structural basis for coupling agonist binding to channel gating. The proposed studies take advantage of the relative structural simplicity of homomeric receptors, employ a novel method for generating receptors with prescribed numbers of intact binding sites and coupling regions, and build on our recent demonstration that a network of interdependent loops functionally couples ligand binding and pore domains. The experiments combine expression of chimeric and non-chimeric receptors in mammalian cells, measurements of single channel current amplitudes and dwell times, measurements of ionic currents following rapid application of agonist, site-directed mutagenesis, and structural modeling of receptors. The approaches developed will allow the transduction mechanism to be addressed in all types of Cys-loop receptors, while the overall insights will advance our understanding of how post-synaptic receptors function in health, disease and in the presence of therapeutic drugs. Lay description: Cys-loop receptors relay signals from cell to cell throughout the nervous system, and are implicated in a wide variety of diseases, such as myasthenia, hyperekplexia, epilepsy, and nicotine addiction. They are also targets for drugs used clinically, such as muscle relaxants, anxiolytics, and anticonvulsants. Knowledge of how Cys-loop receptors operate at the molecular level is essential to developing therapeutic strategies and drugs with fewer side effects.
