The N-methyl-D-aspartate NMDA) receptor has been clearly established as a key site of ethanol action, but the precise mechanism(s) by which ethanol exerts its effects remain unclear. Current research has identified two seemingly distinct phases of acute ethanol inhibition: an """"""""early"""""""" (sub-second) phase in which channel function is directly inhibited by ethanol, and a """"""""late"""""""" (minutes) phase in which induction of intracellular effector proteins modulates surface expression and intracellular trafficking of NMDARs. Two residues, phenylalanine 639 in TM3 of GluN1 and alanine 825 in TM4 of GluN2A, have been shown to be critically involved in mediating early phase inhibition of channel function by ethanol. In addition to these sites, work by our laboratory has shown that mutation of select residues in the Pre-TM4 region of GluN2B can drastically alter agonist potency while leaving ethanol sensitivity unchanged, implying ethanol acts subsequent to initial agonist-induced conformational changes. These data support recent models of NMDA receptor gating in which rearrangement of transmembrane linker regions (including the Pre-TM4 region) contributes significantly to channel gating, and that such topographical rearrangement helps define ethanol action on channel function. While early phase inhibition occurs regardless of subunit composition, late phase modulation of NMDA currents by ethanol appears to preferentially affect GluN2B-containing NMDARs in a brain region dependent manner via the induction of intracellular signaling cascades. A number of these cascades have been relatively well characterized, but it remains unclear if early phase inhibition of GluN2B-containing NMDARs defines the magnitude of late phase responses. In the proposed project, we will use whole-cell and single channel electrophysiological methods to probe the functional interactions of TM3/Pre-TM4/TM4 regions in defining early phase ethanol sensitivity. We will then introduce ethanol resistant receptors in GluN2B-knockout tissue via viral-transduction and using biochemical and electrophysiological methods, determine how early phase alcohol inhibition dictates late phase responses. The training opportunities afforded by this grant will not only contribute significantly to understanding the fundamental actions of alcohol, but as well greatly facilitate my long-term goal of becoming a successful independent investigator.
NMDA receptors are critical for a number of higher order cognitive functions, and have been shown to be a primary site of action for the widely abused drug alcohol. While a significant body of research has dichotomized ethanol action on NMDARs into two distinct phases, fast and slow, it remains unclear if the two phases are mechanistically linked. The proposed project will study these two phases of action and determine if there is a causal link between the two;the results of which will greatly enhance our understanding of the fundamental actions of alcohol and facilitate the development of novel therapeutic methods for alcohol abuse disorders.
|Hughes, Benjamin A; Woodward, John J (2016) Disruption of S2-M4 linker coupling reveals novel subunit-specific contributions to N-methyl-d-aspartate receptor function and ethanol sensitivity. Neuropharmacology 105:96-105|