This Research Scientist Award is based upon current work being performed to understand the molecular basis of ethanol's specificity of action on ligand-gated ion channels. This proposal, tests the hypothesis that the selectivity of ethanol to affect responses to NMDA and GABA from some, but not all, neurons is due to the presence of specific NMDA and GABAA receptor subtypes. The model approach proposed to study the action of ethanol on NMDA and GABAA receptors is an update of progress made on R01s AA09122 and AA10025, grants defining the molecular mechanism(s) responsible for ethanol's specificity on specific receptor subtypes. In respect to ethanol's action on GABAA receptor subtypes, we have demonstrated zolpidem predicts one GABAA receptor subtype sensitive to ethanol. Likewise, we have shown a close relationship between ifenprodil and ethanol antagonism of NMDA responses. In this proposal, each cultured and dissociated neurons cell will be pharmacologically characterized with patch-clamp electrophysiology, the cytoplasm will be extracted from the cell, and mRNAs for GABAA and NMDA receptor subunits within each individual neuron will be identified with RT/PCR.
In Specific Aim I, work will initially focus on the hypothesis that the alpha1beta2gamma2 subunit combination is one GABAA receptor subtype sensitive to ethanol and zolpidem. Due to present uncertainty, particular emphasis is being placed on which gamma2 variants in neurons with this subunit combination are sensitive to ethanol. A second portion of work on GABAA receptors relates to defining the molecular mechanism by which ethanol's action is dependent upon secondary neural inputs to a cell. Finally, we will determine if diazepam-insensitive receptors, containing alpha4 or alpha6 subunits, are sensitive to ethanol.
Specific Aim II will identify the mRNAs for NMDA receptor subunits within individual neurons sensitive to ethanol antagonism of NMDA. Current results suggest that some, but not all, NMDA receptor subtypes sensitive to ifenprodil are sensitive to ethanol. Since ifenprodil binds to NMDA receptors purportedly containing the NMDAR-2b subunit, it can assumed that the receptor subtype sensitive to ethanol will contain this NMDA receptor subunit, as well as an appropriate NMDAR-1 variant. Studies are underway to confirm this hypothesis. The model proposed to define the molecular basis of ethanol's action to affect GABAA and NMDA receptor subtypes, when applied to all ligand- gated ion channels, will provide a means to identify neuroanatomical sites at which ethanol has the greatest probability of affecting brain function. Further, based upon structural analysis of ethanol's action on protein-protein inter-actions for the receptors, results may ultimately allow us to predict other protein-protein interactions influenced by ethanol.
| Yang, X; Criswell, H E; Breese, G R (1999) Action of ethanol on responses to nicotine from cerebellar Purkinje neurons: relationship to methyllycaconitine (MLA) inhibition of nicotine responses. Neurochem Int 35:185-94 |
