Benzodiazepine modulation of partial agonist efficacy and spontaneously active GABA_A receptors supports an allosteric model of modulation

Abstract

1 Benzodiazepines (BZDs) have been used extensively for more than 40 years because of their high therapeutic index and low toxicity. Although BZDs are understood to act primarily as allosteric modulators of GABA A receptors, the mechanism of modulation is not well understood. 2 The applicability of an allosteric model with two binding sites for g-aminobutyric acid (GABA) and one for a BZD-like modulator was investigated. 3 This model predicts that BZDs should enhance the efficacy of partial agonists. 4 Consistent with this prediction, diazepam increased the efficacy of the GABA A receptor partial agonist kojic amine in chick spinal cord neurons. 5 To further test the validity of the model, the effects of diazepam, flurazepam, and zolpidem were examined using wild-type and spontaneously active mutant a1(L263S)b3g2 GABA A receptors expressed in HEK-293 cells. 6 In agreement with the predictions of the allosteric model, all three modulators acted as direct agonists for the spontaneously active receptors. 7 The results indicate that BZD-like modulators enhance the amplitude of the GABA response by stabilizing the open channel active state relative to the inactive state by less than 1 kcal, which is similar to the energy of stabilization conferred by a single hydrogen bond.

Structural Link between γ-Aminobutyric Acid Type A (GABAA) Receptor Agonist Binding Site and Inner β-Sheet Governs Channel Activation and Allosteric Drug Modulation

“…In contrast, ␤Gly1 increased GABA EC 50 26-fold and significantly reduced the Hill slope, whereas ␤Gly2, -4, or -8 increased GABA EC 50 more than 10,000-fold (Fig. 2, Table 1).…”

“…6, Table 3). All of the ␣Gly and the ␤Gly1 insertions reduced FZ potentiation of EC 4 -8 GABA-mediated current responses (50 -60%) without changing the FZ EC 50 (Fig. 6, Table 3), suggesting that these regions are important for mediating BZD efficacy.…”

“…To test whether disrupting the linker regions in the ␣-, ␤-, and ␥-subunits affected PB activation, we measured PB concentration responses from wild-type and mutant receptors. Glycine insertions in both the ␤-subunits and the ␥-subunits significantly increased PB EC 50 values by ϳ10-fold as compared with WT receptors (231 Ϯ 10 M) (Fig. 5, Table 2).…”

“…All of the mutant subunits assembled into functional receptors that responded to GABA. In general, the ␣Gly and ␥Gly insertions had minimal effects on GABA EC 50 values (Ͻ4-fold) as compared with WT (14.1 Ϯ 2.2 M), except for ␣Gly4 that had an 11-fold increase in GABA EC 50 (Fig. 2, Table 1).…”

“…Because the GABA EC 50 values were too right-shifted to be precisely determined for ␤Gly2, -4, and -8 receptors (Table 1), we approximated the -fold changes in SR-95531 K I using the The SR-95531 inhibition experiments for ␤Gly2, -4, and -8 used 30 mM GABA, which from curve-fitting estimates is below the GABA EC 50 (Table 1). Thus, for ␤Gly2 receptors, we estimate that the SR-95531 K I is between 3750 nM (using 30 mM as an upper limit for GABA EC 50 ) and 7500 nM (assuming that 30 mM is Ͻ Ͻ EC 50 ), an ϳ50 -100-fold increase in K I as compared with WT. The estimated -fold increases in SR-95531 K I for ␤Gly4 and -8 receptors were even larger (100 -400-fold).…”