Mechanism of inward rectification of neuronal nicotininc acetycholine receptors

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are wide spread in the nervous system. Ample evidence indicates that many central nAChRs are located at the nerve terminals, where they act to facilitate neurotransmitter release; however, little is known about how these receptors carry out their function. The focus of my study is to understand the mechanism(s) that underlie the function of neuronal nAChRs. Neuronal nAChRs conduct inward current at negative membrane potentials, but conduct little or no outward current at positive membrane potentials, a process known as inward rectification. Inward rectification prevents the ACh-evoked conductance increase from short-circuiting the action potential at the nerve terminal, thereby ensuring optimal depolarization of the terminal and effective neurotransmitter release. Using the outside-out single channel patch-clamp technique, I demonstrate that intracellular polyamines block neuronal nAChRs with high affinity and in a voltage dependent manner; this is true for native nAChRs expressed by sympathetic neurons as well as recombinant alpha3beta4, alpha4beta2 nAChRs expressed in Xenopus oocytes. Given the physiological concentrations of polyamines inside cells, this block can fully account for the strong macroscopic inward rectification. Furthermore, using a combined approach of site-directed mutagenesis and electrophysiology, I show that the negatively charged residues at the cytoplasmic mouth of the pore (known as the intermediate ring) mediate the interaction of intracellular polyamines with the receptor; partial removal of these residues abolishes the strong inward rectification. Interestingly, I show that the intermediate ring influences the permeation of calcium through the receptor, indicating that a molecular link exists between calcium permeability and inward rectification of neuronal nAChRs. My experiments also show that extracellular polyamines and a polyamine-related toxin, Joro spider toxin, block neuronal