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Abstract
The depressant actions of ethanol on central nervous system activity appear to be mediated by its actions on a number of important membrane associated ion channels including the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptor. Although no specific site of action for ethanol on the NMDA receptor has been found, previous studies suggest that the ethanol sensitivity of the receptor may be affected by intracellular C-terminal domains of the receptor that regulate the calcium-dependent inactivation of the receptor. In the present study, co-expression of the NR2A subunit and an NR1 subunit that lacks the alternatively spliced intracellular C1 cassette did not reduce the effects of ethanol on channel function as measured by patch-clamp electrophysiology. Full inhibition was also observed in cells expressing an NR1 subunit truncated at the end of the C0 domain (NR1(863stop)). However, the inhibitory effects of ethanol were reduced by expression of an NR1 C0 domain deletion mutant (NR1(Delta839-863)), truncation mutant (NR1(858stop)), or a triple-point mutant (Arg to Ala, Lys to Ala, and Asn to Ala at 859-861) previously shown to significantly reduce calcium-dependent inactivation. A similar reduction in the effects of ethanol on wild-type NR1/2A but not NR1/2B or NR1/2C receptors was observed after co-expression of full-length or truncated human skeletal muscle alpha-actinin-2 proteins that produce a functional knockout of the C0 domain. The effects of ethanol on hippocampal and cortical NMDA-induced currents were similarly attenuated in low calcium recording conditions, suggesting that a C0 domain-dependent process may confer additional ethanol sensitivity to NMDA receptors.
Highlights
Relevant concentrations of ethanol (0.05– 0.4 g/dl; approximately 10 –100 mM) inhibit NMDA receptors expressed in neurons and in recombinant expression systems [1,2,3,4,5,6,7]
Under the recording conditions used in this study, the majority of this desensitization is due to calcium-dependent inactivation [15, 21] because it is largely lost in cells transfected with NR1 subunits that lack a functional C0 domain (Fig. 1A, NR1⌬839 – 863, NR1858stop, and NR1-PM1) or under conditions of reduced extracellular calcium
A similar reduction in ethanol sensitivity was observed when HEK 293 cells expressing NR1-1a/2A subunits were tested under conditions of reduced extracellular calcium (Fig. 4, shaded bars). Previous studies from this laboratory showed that the effect of ethanol on NR1/2A receptors expressed in oocytes was attenuated under conditions that prevented or reduced increases in intracellular calcium upon activation of the NMDA receptor [4, 10]
Summary
Relevant concentrations of ethanol (0.05– 0.4 g/dl; approximately 10 –100 mM) inhibit NMDA receptors expressed in neurons and in recombinant expression systems [1,2,3,4,5,6,7]. Co-Expression of ␣-Actinin-2 Mimics Deletion of the C0 Domain—These results suggest that a functional C0 domain of the NR1 subunit is important in mediating a significant fraction of the ethanol inhibition of steady state NMDA currents under conditions of normal extracellular calcium.
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