Steady-state, single-channel gating of GABA(A) receptors (GABARs ) is complex. Simpler gating may dominate when triggered by rapid GABA transients present during fast inhibitory synaptic transmission and is critical to understanding the time course of fast IPSCs. We studied the single-channel activity of expressed alpha1beta1gamma2 GABARs in outside-out patches from human embryonic kidney 293 cells triggered by rapidly applied GABA (10-2000 microm) pulses (2-300 msec). Activation was analyzed with the time to first channel opening after GABA presentation, or first latency (FL). FL distributions are monoexponential at low GABA concentrations and biexponential above 30 microm GABA. The fast rate increases supralinearly to a plateau of approximately 1100 sec(-1), the apparent activation rate. The slow rate and amplitude are insensitive to GABA concentration. The results argue that doubly liganded receptors can rapidly desensitize before opening. Gating after the first opening was quantified with analysis of open probability conditioned on the first opening (P(o/o)). P(o/o) functions are biexponential, dominated by a fast component, and insensitive to GABA concentration. This suggests that open channels convert primarily to fast but also to slow desensitized states. Furthermore, dual modes of fast desensitization may influence IPSC amplitude and thereby synaptic efficacy. The findings provided for the construction of a mathematical gating model that accounts for FL and P(o/o) functions. In addition, the model predicts the time course of macroscopic current responses thought to mimic IPSCs. The results provide new insights into dominant gating that is likely operational during fast GABAergic synaptic transmission.

The γ‐subunit in recombinant γ‐aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA‐triggered Cl− currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non‐competitive to competitive. To investigate underlying receptor function we studied Zn2+ effects on macroscopic and single‐channel currents of recombinant α1β2 and α1β2γ2 receptors expressed heterologously in HEK‐293 cells using the patch‐clamp technique and rapid solution changes. Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 μM)‐triggered currents of α1β2 receptors in a concentration‐dependent manner (inhibition equation parameters: concentration at half‐amplitude (IC50) = 0.94 μM; slope related to Hill coefficient, S= 0.7) that was unaffected by GABA concentration. The γ2 subunit (α1β2γ2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50= 51 μM, S= 0.86); increased GABA concentration (100 μM) antagonized inhibition by reducing apparent affinity (IC50= 322 μM, S= 0.79). Zn2+ slowed macroscopic gating of α1β2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For α1β2γ2 receptors, Zn2+ accelerated a fast component of apparent desensitization. Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of α1β2 receptors, but had little effect on currents from α1β2γ2 receptors. Steady‐state fluctuation analysis of macroscopic α1β2γ2 currents (n= 5) resulted in control (2 μM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 ± 5.6 and 1.41 ± 0.15 ms, means ± s.e.m.). Zn2+ (200 μM) reduced the total power almost sixfold and accelerated the slow (23 ± 2.8 ms, P < 0.05) without altering the fast (1.40 ± 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 ± 0.55; Zn2+, 4.9 ± 0.37, P < 0.05). Zn2+ (500 μM) depression of previously activated current amplitudes (% control) for α1β2γ2 receptors was independent of GABA concentration (5 μM, 13.2 ± 0.72 %; 100 μM, 12.2 ± 2.9 %, P < 0.8, n= 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated α1β2 receptors with current depression more than fourfold less sensitive (5 μM GABA, IC50= 3.8 μM, S= 0.84) relative to that in constant Zn2+. The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non‐conducting states, site affinity is state‐dependent, and the features of state dependence are determined by the γ‐subunit.

Millimolar concentrations of the barbiturate pentobarbital (PB) activate γ-aminobutyric acid (GABA) type A receptors (GABARs) and cause blockade reported by a paradoxical current increase or “tail” upon washout. To explore the mechanism of blockade, we investigated PB-triggered currents of recombinant α1β2γ2S GABARs in whole cells and outside-out membrane patches using rapid perfusion. Whole cell currents showed characteristic bell-shaped concentration dependence where high concentrations triggered tail currents with peak amplitudes similar to those during PB application. Tail current time courses could not be described by multi-exponential functions at high concentrations (≥3,000 μM). Deactivation time course decayed over seconds and was slowed by increasing PB concentration and application time. In contrast, macropatch tail currents manifested eightfold greater relative amplitude, were described by multi-exponential functions, and had millisecond rise times; deactivation occurred over fractions of seconds and was insensitive to PB concentration and application time. A parsimonious gating model was constructed that accounts for macropatch results (“patch” model). Lipophilic drug molecules migrate slowly through cells due to avid partitioning into lipophilic subcellular compartments. Inclusion of such a pharmacokinetic compartment into the patch model introduced a slow kinetic component in the extracellular exchange time course, thereby providing recapitulation of divergent whole cell results. GABA co-application potentiated PB blockade. Overall, the results indicate that block is produced by PB concentrations sixfold lower than for activation involving at least three inhibitory PB binding sites, suggest a role of blocked channels in GABA-triggered activity at therapeutic PB concentrations, and raise an important technical question regarding the effective rate of exchange during rapid perfusion of whole cells with PB.

Benzodiazepines induce a series of clinical effects by modulating subtypes of γ -aminobutyric acid type A receptors in the central nervous system. The brain concentration-time profiles of diazepam that correspond to these effects are unknown, but can be estimated with physiologically based pharmacokinetic (PBPK) modeling. In this study, a PBPK model for the 1,4-benzodiazepines diazepam and nordiazepam was developed from plasma concentration-time courses with PK-Sim software to predict brain concentrations. The PBPK model simulations accurately parallel plasma concentrations from both an internal model training data set and an external data set for both intravenous and peroral diazepam administrations. It was determined that the unbound interstitial brain concentration-time profiles correlated with diazepam pharmacodynamic end points. With a 30-mg intravenous diazepam dose, the peak unbound interstitial brain concentration from this model is 160 nM at 2 minutes and 28.9 nM at 120 minutes. Peak potentiation of recombinant γ -aminobutyric acid type A receptors composed of α1β2γ 2s, α2β2γ 2s, and α5β2γ 2s subunit combinations that are involved in diazepam clinical endpoints is 108%, 139%, and 186%, respectively, with this intravenous dose. With 10-mg peroral administrations of diazepam delivered every 24 hours, steady-state peak and trough unbound interstitial brain diazepam concentrations are 22.3 ± 7.5 and 9.3 ± 3.5 nM. Nordiazepam unbound interstitial brain concentration is 36.1 nM at equilibrium with this diazepam dosing schedule. Pharmacodynamic models coupled to the diazepam unbound interstitial brain concentrations from the PBPK analysis account for electroencephalographic drug effect, change in 13-to 30-Hz electroencephalographic activity, amnesia incidence, and sedation score time courses from human subjects.