Project Summary Throughout the nervous system, nicotinic acetylcholine receptors (nAChRs) are tasked with facilitating rapid communication between cells by converting a chemical signal to an electrical impulse. When acetylcholine is released into the synapse, membrane bound nAChRs bind the small organic molecule and open the receptor pore in response, allowing a selected subset of extracellular ions to flow into the cell. Disruption of normal nAChR function can result in severe and debilitating neurological diseases, such as myasthenic syndromes and certain epileptic disorders. A major emphasis of our laboratory is uncovering how the three dimensional structure of nAChRs allows them to perform their physiologic task. For we assert that a basic understanding of how receptor structure endows function will allow us to more effectively intervene in the setting of nAChR- driven neurological diseases. Recently determined high-resolution nAChR structures reveal a conserved charge-charge interaction between pore-lining and peripheral α-helices that was not apparent in the first published nAChR structures. This proposal seeks to determine the function of this conserved interaction by pairing precise modifications to receptor structure with state-of-the-art single channel electrophysiology. My preliminary work shows that this transmembrane charge-charge interaction is critical for the stability of the open state of the receptor and for the uniform open channel current inherent to nAChRs. Therefore I hypothesize that a universally conserved charge-charge interaction between pore-lining and peripheral transmembrane a-helices enhances gating efficiency and facilitates uniform ion permeation in nAChRs. To test this hypothesis I will break the pore-peripheral charge-charge interaction using mutagenesis and probe the function of resultant receptors using single channel electrophysiology. First, I will determine the ion permeation characteristics of the mutant and wild-type receptors (aim 1). Then I will determine the gating energetics of wild type and mutant receptors (aim 2). Accomplishing these two aims will help provide the mechanistic framework required to understand and treat diseases driven by abnormal nAChR function.