Methods for recognition of γ-aminobutyric acidA receptor subtypes in situ
| Method | Requirements |
|---|---|
| 1. High-resolution labeling methods | Spatial separation of receptor |
| a. Immunofluorescence | subtypes must be adequate |
| b. Immunocytochemical reaction | |
| c. Freeze-fracture/antibody labeling | |
| d. Antibody labeling in postembedding electromicroscopy1-a | |
| 2. Single cell RT-PCR,1-bcombined with patch-clamp recording | a. Only one subtype is present |
| b. Large cells are required | |
| c. All the receptor mRNAs present must give rise to the assembled receptor1-c | |
| 3. Use of an absolutely subtype-specific drug (e.g., furosemide, for α6β2/3 γ2)1-d | a. Specific for one defined composition; cases will be rare |
| b. Patch-clamping must be applicable, or the drug must be labeled, for in situ binding |
↵1-a For example, using sized gold particles (Nusseret al., 1996).
↵1-b RT-PCR, reverse transcriptase-polymerase chain reaction.
↵1-c Examples to the contrary are given by Williamson and Pritchett (1994).
↵1-d This specificity for this drug has been reported by Korpiet al. (1995), but application at the microscopic level of this or any other subtype-specific ligand has not been reported yet. Furosemide as a noncompetitive antagonist selects α4β3 γ2 receptors as well as α6β2/3 γ2 receptors, but is 14-fold less active at the former (Wafford et al., 1996).