The overall objective of the proposed work in this developmental grant application is to further understanding of the mechanisms of ligand-gated ion channel activation, desensitization and allosteric modulator action. To this end we propose the electrophysiological characterization of newly-discovered mutants of glycine alpha1 and GABA-A alpha1 and beta2 receptor subunits. Single amino acid mutations of these subunits have been identified that result in channels that open spontaneously and desensitize in the absence of agonist. Tonic opening is revealed by a reversible strychnine- or bicuculline-induced outward current. Tonically-opening glycine and GABA-A receptors will be transiently expressed in mammalian HEK 293 cells and characterized using a fast drug application system. The use of a fast drug exchange system will allow for an accurate assessment of rates of channel opening, closing and desensitization after applying GABA-A and glycine receptor agonists or antagonists to receptors composed of wild-type or mutated subunits. Single channel outside-out patch recordings of our tonically-open channels are hypothesized to display bursts of channel opening events separated by quiescent, desensitized periods. We will then take advantage of the tonic opening and desensitization that occur spontaneously in these mutated receptors to clarify the molecular mechanisms underlying the actions of allosteric modulators. GABA and glycine receptor function is allosterically modulated by numerous classes of agents such as the barbiturates, benzodiazepines and steroidal and volatile anesthetics. Using the receptor mutants we have already created, and new mutants we propose to make, we will test the hypothesis that modulatory concentrations of these agents affect the functioning of tonically-open GABA-A and glycine receptors in the absence of agonist binding to the neurotransmitter receptor binding site, allowing us to dissociate modulator-induced stabilization of open channel states and effects on desensitization from their effects on increasing the affinity of neurotransmitter for its receptor. The work proposed will increase understanding not only of basic receptor/channel processes, but also shed insight into the molecular mechanisms of receptor modulation.
| Todorovic, Jelena; Welsh, Brian T; Bertaccini, Edward J et al. (2010) Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation. Proc Natl Acad Sci U S A 107:7987-92 |
