Direct Evidence For Diazepam Modulation of GABAA Receptor Microscopic Affinity

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Abstract

Alteration of agonist affinity is a potential mechanism for pharmacological modulation of ligandgated receptor channel function. The time course for receptor activation and current onset is determined by the combined rates for two kinetic transitions that underlie the protein conformations for binding agonist and channel gating. Using ultrafast ligand exchange techniques, we distinguish between these previously difficult to separate events and demonstrate their independent pharmacological modulation. Diazepam, which increases apparent affinity of γ-aminobutyric acid (GABA) to GABAA receptors, was used to examine its effects on GABA binding and ion channel gating of expressed α2β1γ2 receptors from excised outside-out patches of acutely transfected HEK 293 cells. Diazepam increased rates of current onset evoked by low concentrations (<1 mM) but not at saturating GABA concentrations. Furthermore, rates of current decay were not affected during brief applications of GABA, and thus, demonstrated a diazepam specific effect on ligand binding affinity and not channel gating kinetics. However, current decay during and following prolonged GABA applications were altered by diazepam in a fashion similar to that for higher concentrations of GABA which also increased receptor desensitization. These findings and analysis by computer modeling indicated that diazepam likely enhances GABA receptor currents primarily by accelerating GABA association to its receptor at the first agonist binding site. These results provide the first direct physiological evidence for pharmacological modulation of microscopic binding affinity of GABA receptors. Copyright © 1996 Elsevier Science Ltd

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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