PMC

Benzodiazepine pharmacology of γ2L- and γ1-containing GABA_ARs. Averaged and normalized current traces from multiple cells expressing either α2β2γ2L or α2β2γ1 GAB_ARs before (black) and during (gray) continuous perfusion of diazepam (A) and flunitrazepam (B). The accompanying bar plots are pooled data for current decay and peak amplitude. Note that both benzodiazepines markedly slowed the decay rate of α2β2γ2L GABA_ARs (*, p < 0.05; ****, p < 0.0001), but had no significant (ns) effect on α2β2γ1 GABA_ARs. Neither drug significantly altered the peak amplitude of the currents.

γ1 subunits slow synaptic current kinetics. A, schematic representation of the subunit chimeras used to investigate γ subunit related synaptic clustering. Swapping the intracellular and TM4 domains made the chimeras. B, averaged synaptic currents recorded from heterosynapses expressing the wild-type α2β2γ2L and α2β2γ1 (reproduced from ) and chimeric α2β2γ1-γ2L and α2β2γ1-γ2L GABA_ARs. Note the similar activation and deactivation between wild-type and corresponding chimeric GABA_ARs. C, averaged values for activation and deactivation for the wild-type and chimeric GABA_ARs showing that both activation and deactivation are strongly dependent on the ID plus TM4 domains of the γ subunit.

Heterosynapses expressing GABA_ARs. A, fluorescent micrograph of an HEK293 cell expressing red fluorescent protein and neuronal GAD65-positive contacts (green) being formed on the HEK293 cell. B, confocal section of an HEK293 cell showing HA-tagged neuroligin (red) and neuronal GAD65-positive contacts (green, arrows). C, segments of whole cell recordings from HEK293 cells transfected with the indicated GABA_ARs, in co-culture with cortical neurons. Synaptic currents were of variable amplitude and frequency from cell to cell, but were consistently recorded across transfections. D, averaged synaptic currents for the four GABA_ARs as indicated. The inclusion of the α2 and γ1 subunits effectively reduced the rates of current activation and deactivation. E, pooled data for the activation (left) and deactivation (right) rates for macropatch and synaptic currents.

Distinguishing αβ and αβγ receptors. A, sample single channel recordings from patches expressing α2β2 (above) and α1β2 (below) receptors, along with the corresponding amplitude histograms. Transfecting only α and β subunits produced GABA-activated channel activity of ∼1 pA in amplitude. B and C, recordings from patches excised from cells transfected with α2, β2, γ2L or γ1 (B), or α1, β2, γ2L or γ1 (C), showing examples of αβγ (∼2 pA) and αβ (∼1 pA) channel activations in the same patches. The accompanying amplitude histograms show that αβ and αβγ channels are clearly distinguishable in terms of amplitude, and the bar graphs on the far right show the relative proportions of αβγ and αβ channel activations, averaged over 3–5 patches, for each αβγ channel transfection type.

Single channel activations and I-V. Single channel currents elicited by saturating (3 mm, above) and subsaturating (2 μm, below) concentrations of GABA for patches expressing (A) α1β2γ2L, (B) α1β2γ1, (C) α2β2γ2L, and (D) α2β2γ1 GABA_ARs. The tcrit values at 3 mm GABA, ranged between 25 and 35 ms, whereas those for activity elicited by 2 μm GABA ranged between 35 and 45 ms. Accompanying the activations are the I-V relationships of each receptor, generated from averaged data from 3 to 5 patches, along with sample currents at ±70 and ±35 mV (the open level is indicated by a red broken line). The main subunit-dependent differences are the duration of discrete active periods, especially at 2 μm GABA. Bursts of activity from α2-containing receptors remain active for 3–4-fold longer than bursts recorded from α1-containing receptors. The burst lengths for the four GABA_ARs follow the same pattern as the deactivation rates of macropatch currents.

Macropatch recordings. A and B, open pipette response (downward deflection) elicited by rapid, lateral translation of a double-barrel glass tube (θ-tube, inset). One of the barrels contained a standard extracellular solution, whereas the other contained a barrel diluted by 50% with distilled water. Open pipette responses were used to optimize agonist application onto macropatches. C, averaged sweeps of macropatch currents recorded from patches expressing, α1β2γ2L, α1β2γ1, α2β2γ2L, and α2β2γ1 GABA_ARs in response to ∼1 ms application of 3 mm GABA (arrowhead). The currents for all four GABA_ARs develop rapidly, with a 10–90% rise times of <1 ms. Current deactivation has a slower time course and shows a clear α-subunit isoform correlation, with α1-containing receptors deactivating more rapidly than those containing the α2 subunit. Averaged data for the 10–90% rise time (D) and deactivation rate (E) for the macropatch data.

GABA-gated activation mechanisms. A, shut and open dwell histograms for data obtained at 3 mm GABA. The histograms shown for all four receptors have three shut and three open components, suggesting that they are kinetically similar. B, consensus activation mechanism for activation by GABA that described the single channel activity (histograms) most accurately for the four receptors. A denotes the agonist and A₂ denotes a doubly liganded receptor, R. The superscript numbers denote the state number and the asterisk denotes open, conducting states. The letters above the double arrows that connect states are the rate constants governing the forward and backward transitions. C, simulated ensemble currents for α1β2γ2L, α1β2γ1, α2β2γ2L, and α2β2γ1 GABA_ARs using Scheme 1 with averaged rate constants (). The macropatch currents were generated by setting the channel number to 1000 and the agonist application time to 1 ms in QuB. The time constants for activation and deactivation are shown for each current. D, other postulated schemes that fit the data adequately, including Scheme 2 () and a scheme containing looped connections (Scheme 4). Note that all schemes have at least one shut-shut transition between the binding steps (red arrows) and the open states.