Date of Award

Spring 2015

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Peoples, Robert W.

Second Advisor

Buchanan, James T.

Third Advisor

Gasser, Paul J.


Alcohol abuse and alcoholism are behavioral disorders involving altered synaptic transmission in the brain. The N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate-gated ion channel, has been shown to be one of the most important target sites of alcohol in the central nervous system. The NMDA receptor is formed between two obligatory GluN1 subunits and two GluN2 subunits (2A-2D). Previous studies have been mainly focused on the GluN2A subunit-containing NMDA receptor to study the molecular sites and mechanisms of alcohol action on the NMDA receptors. However, a major role for the GluN2B subunits in the action of alcohol has been pointed out recently. To test the hypothesis that alcohol interacts with specific residues in the membrane-associated (M) domains of the GluN2B subunit to modulate ion channel gating and alcohol sensitivity, we performed site-directed mutagenesis at positions in the GluN2B subunit, transfected mutant receptors into human embryonic kidney (HEK 293) cells, and did whole-cell patch-clamp recording on these cells. One position in the GluN2B subunit showed significant influence on ethanol sensitivity and ion channel functions. Moreover, the manner in which mutations at positions in the GluN2B subunit alter ethanol sensitivity differ from that of the cognate positions in the GluN2A subunit. The predicted structure of the NMDA receptor indicates a close apposition of the alcohol-sensitive positions in the M3 and M4 domains between the GluN1 and GluN2 subunits. By using both two-way ANOVA and mutant cycle analysis, significant interactions affecting ethanol inhibition and glutamate potency were observed at three pairs of positions in GluN1/GluN2B: Met818/Phe637, Phe639/Leu825, and Gly638/Met824; the last pair also interact with each other to regulate ion channel desensitization. To study whether characteristics of NMDA receptor mutations observed in a non-neuronal cell line are also observed in central nervous system (CNS) neurons, we transfected our constructed GluN1/GluN2A NMDA receptors into rat cortical neurons from postnatal day 2 or 3 by using electroporation. Because there is only few endogenous GluN2A subunits in rat cortical neurons early in development, the currents recorded from these transfected neurons are attributable to the introduced NMDA receptors. Electrophysiological recording in these transfected neurons so far yielded currents that closely resemble those observed in HEK293 cells.