Chun Jin1, John J Woodward. 1. Department of Neurosciences and Center for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Abstract
BACKGROUND: N-Methyl-D-aspartate (NMDA) receptors are glutamate-activated ion channels that are assembled from NR1 and NR2 subunits. These receptors are highly enriched in brain neurons and are considered to be an important target for the acute and chronic effects of ethanol. NR2 subunits (A-D) arise from separate genes and are expressed in a developmental and brain region-specific manner. The NR1 subunit has 8 isoforms that are generated by alternative splicing of a single gene. The heteromeric subunit makeup of the NMDA receptor determines the pharmacological and biophysical properties of the receptor and provides for functional receptor heterogeneity. Although results from previous studies suggest that NR2 subunits affect the ethanol sensitivity of NMDA receptors, the role of the NR1 subunit and its multiple splice variants is less well known. METHODS: In this study, all 8 NR1 splice variants were individually coexpressed with each NR2 subunit in human embryonic kidney 293 (HEK293) cells and tested for inhibition by ethanol using patch-clamp electrophysiology. RESULTS: All 32 subunit combinations tested gave reproducible glutamate-activated currents and all receptors were inhibited to some degree by 100 mM ethanol. The sensitivity of individual receptors to ethanol was affected by the specific NR1 splice variant expressed with receptors containing the NR1-3 and NR1-4 subunits among the least inhibited by ethanol. CONCLUSIONS: These results suggest that regional, developmental, or compensatory changes in the expression of NR1 splice variants may significantly affect ethanol inhibition of NMDA receptors.
BACKGROUND: N-Methyl-D-aspartate (NMDA) receptors are glutamate-activated ion channels that are assembled from NR1 and NR2 subunits. These receptors are highly enriched in brain neurons and are considered to be an important target for the acute and chronic effects of ethanol. NR2 subunits (A-D) arise from separate genes and are expressed in a developmental and brain region-specific manner. The NR1 subunit has 8 isoforms that are generated by alternative splicing of a single gene. The heteromeric subunit makeup of the NMDA receptor determines the pharmacological and biophysical properties of the receptor and provides for functional receptor heterogeneity. Although results from previous studies suggest that NR2 subunits affect the ethanol sensitivity of NMDA receptors, the role of the NR1 subunit and its multiple splice variants is less well known. METHODS: In this study, all 8 NR1 splice variants were individually coexpressed with each NR2 subunit in humanembryonic kidney 293 (HEK293) cells and tested for inhibition by ethanol using patch-clamp electrophysiology. RESULTS: All 32 subunit combinations tested gave reproducible glutamate-activated currents and all receptors were inhibited to some degree by 100 mM ethanol. The sensitivity of individual receptors to ethanol was affected by the specific NR1 splice variant expressed with receptors containing the NR1-3 and NR1-4 subunits among the least inhibited by ethanol. CONCLUSIONS: These results suggest that regional, developmental, or compensatory changes in the expression of NR1 splice variants may significantly affect ethanol inhibition of NMDA receptors.