Direct Evidence For Diazepam Modulation of GABAA Receptor Microscopic Affinity
Section snippets
Cell culture and transfection
Transformed human embryonic kidney (HEK) cells 293 (ATCC CRL 1573) were plated onto 35 mm culture dishes in MEM (Sigma, with glutamine and glucose added) and 10% fetal calf serum and maintained in 5% CO2. Cells were transfected using standard Ca2PO4 precipitation techniques (Chen and Okayama, 1987) with plasmid (pCis2; Pritchett et al., 1988) containing either human α2β1 which were blunt ended back to back on the same promoter and γ2S (3:2 ratio, 3 μg total cDNA). Electrophysiological
RESULTS
Using ultrafast ligand exchange, GABA alone or in the presence of diazepam, was applied to excised outside-out multi-channel patches from α2β1γ2 expressing acutely transfected HEK 293 cells. Current responses from the α2β1γ2 receptors were reproducible between cells and transfection lots which allowed for comparison of currents responses between patches (Lavoie et al., unpublished observations). In the same patch, serial switching (<1 ms application, 1 s period, 10 applications, each ligand)
DISCUSSION
Diazepam modulated GABA-evoked currents by producing: (1) an increase in peak current; (2) accelerated rates of current onset at non-saturating GABA concentrations; (3) accelerated rate of current decay or desensitization during GABA application; (4) no change in the decay or deactivation of currents after brief pulse applications of GABA; and (5) prolongation of deactivation currents after step applications of GABA without changing the exponential time constants for deactivation.
The
Acknowledgements
Supported by a University of Utah Graduate Fellowship Award to AML, and by NIH NS31519, a Mallinckrodt Scholar Award and a Huntsman Cancer Institute award to RET. The authors thank Drs N.L. Harrison and D. Pritchett for gracious use of plasmids and J.J. Tingey and D. Grimes for their assistance in cell transfection.
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