Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit - PubMed (original) (raw)

Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit

Kai M Wohlfarth et al. J Neurosci. 2002.

Abstract

Attenuated behavioral sensitivity to neurosteroids has been reported for mice deficient in the GABA(A) receptor delta subunit. We therefore investigated potential subunit-specific neurosteroid pharmacology of the following GABA(A) receptor isoforms in a transient expression system: alpha1beta3gamma2L, alpha1beta3delta, alpha6beta3gamma2L, and alpha6beta3delta. Potentiation of submaximal GABA(A) receptor currents by the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was greatest for the alpha1beta3delta isoform. Whole-cell GABA concentration--response curves performed with and without low concentrations (30 nm) of THDOC revealed enhanced peak GABA(A) receptor currents for isoforms tested without affecting the GABA EC50. Alpha1beta3delta currents were enhanced the most (>150%), whereas the other isoform currents were enhanced 15-50%. At a higher concentration (1 microm), THDOC decreased peak alpha1beta3gamma2L receptor current amplitude evoked by GABA (1 mm) concentration jumps and prolonged deactivation but had little effect on the rate or extent of apparent desensitization. Thus the polarity of THDOC modulation depended on GABA concentration for alpha1beta3gamma2L GABA(A) receptors. However, the same protocol applied to alpha1beta3delta receptors resulted in peak current enhancement by THDOC of >800% and prolonged deactivation. Interestingly, THDOC induced pronounced desensitization in the minimally desensitizing alpha1beta3delta receptors. Single channel recordings obtained from alpha1beta3delta receptors indicated that THDOC increased the channel opening duration, including the introduction of an additional longer duration open state. Our results suggest that the GABA(A) receptor delta subunit confers increased sensitivity to neurosteroid modulation and that the intrinsic gating and desensitization kinetics of alpha1beta3delta GABA(A) receptors are altered by THDOC.

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Figures

Fig. 1.

Direct activation of GABAA receptors by THDOC. A, Representative currents evoked by increasing THDOC concentrations for α1β3γ2L (▪), α1β3δ (▾), α6β3γ2L (■), α6β3δ (▿), α1β3 (●), and α6β3 (○) GABAA receptor isoforms. The hatched bars indicate application of various THDOC concentrations. Note the small inflections in the currents after application of 30 μ

THDOC. B, Concentration jump using 10 μ

alphaxalone shows a rebound current more clearly because of faster solution exchange (see Materials and Methods).C, Concentration–response relations for direct activation by THDOC. Mean ± SEM current amplitudes are shown. The_left_ and right panels show α1- and α6-containing isoforms, respectively. Smaller currents were observed for δ subunit-containing isoforms. Symbols are as in_A_. See Table 1 for fitted parameters.

Fig. 2.

Modulation of submaximal GABA-evoked currents by THDOC coapplication. A, Current traces showing THDOC enhancement of EC30 GABA concentrations for α1β3γ2L (▪) and α1β3δ (▾) GABAA receptors. Hatched bars indicate THDOC application; _filled bars_indicate GABA application. B, Summary plot of THDOC enhancement of currents evoked by EC30 GABA concentration for each isoform. Data from lower THDOC concentrations are expanded in the inset for clarity. Significant potentiation was observed for THDOC concentrations of 100 n

and higher (p < 0.05).

Fig. 3.

THDOC enhanced the maximal GABAAreceptor currents without changing the GABA EC50.A_–_D, GABA concentration–response curves were obtained in the absence (●) and presence (○) of 30 n

THDOC for α1β3γ2L (A), α1β3δ (B), α6β3γ2L (C), α6β3δ (D), α1β3 (E), and α6β3 (F) isoforms. Representative maximal GABA currents without (●) and with (○) THDOC coapplication are shown in the inset of A_–_D. For the GABA plus THDOC curves, the currents were normalized to the amplitude of a 100 μ

GABA test pulse obtained from the same cell. Fitted parameters are given in Table 1.

Fig. 4.

Modulation of maximal GABA-evoked currents by THDOC coapplication. A, Increasing concentrations of THDOC were coapplied with 1 m

GABA to α1β3γ2L (▪) and α1β3δ (▾) GABAA receptors. Representative traces are shown for each isoform. Hatched bars indicate THDOC application; filled bars indicate GABA application. B, Summary of THDOC modulation, showing increasing enhancement for α1β3δ receptors and inhibition for α1β3γ2L receptors. The dashed line indicates 100% of control (1 m

GABA alone) current amplitude.Symbols indicate the mean ± SEM responses of four cells for each isoform.

Fig. 5.

Kinetics and polarity of THDOC modulation depend on subunit composition and GABA concentration. THDOC (1 μ

) was preapplied with 1 μ

GABA (A, B) and 1 m

GABA (C, D) using the concentration jump technique. Hatched bars indicate THDOC application;filled bars indicate GABA application. For the rapidly desensitizing α1β3γ2L receptors, cells were lifted from the recording dish to increase resolution of the peak currents. Direct activation was observed during the preapplication for both isoforms, with greater relative currents evoked from α1β3γ2L. Greater enhancement of 1 μ

GABA currents was observed for α1β3δ receptors, although both isoforms showed slightly increased desensitization and prolonged deactivation (A,B). With 1 m

GABA, α1β3δ receptors were enhanced substantially and pronounced desensitization was observed (C), whereas α1β3γ2L receptors were slightly inhibited (D). The control trace in D was normalized and overlaid in_gray_ to show the minimal effect on apparent desensitization. The dashed line emphasizes the decreased peak current in the presence of THDOC.

Fig. 6.

Summary of THDOC effects on peak currents, desensitization, and deactivation. A, Effect of THDOC (1 μ

) on currents evoked by 1 μ

and 1 m

GABA for α1β3δ (gray bars) and α1β3γ2L (black bars) GABAAreceptors. Values are expressed as a percentage of current evoked by GABA alone for each cell. Note the logarithmic axis. The dashed line indicates 100% of control current amplitude.B, Percentage increase in the weighted time constant of deactivation for the same conditions as in A. The_dashed line_ indicates 100% of control deactivation.C, Extent of desensitization observed with 1 m

GABA alone (white bars) or 1 m

GABA + 1 μ

THDOC (gray bars) for α1β3δ and α1β3γ2L GABAAreceptors. Data are expressed as the percentage of peak current lost during a 4 sec application of GABA or GABA + THDOC. D, The rates of desensitization during a 4 sec pulse of 1 m

GABA for α1β3δ and α1β3γ2L GABAA receptors in the absence (white bars) and presence of preapplied 1 μ

THDOC. α1β3γ2L GABAA receptor responses were fitted best by three exponentials with similar time constants and relative areas (data not shown) whether or not THDOC was present. Although the small amount of desensitization observed for α1β3δ in the presence of GABA alone was not well fitted (∗, the time constant was longer than the pulse duration), the pronounced desensitization observed in the presence of THDOC had a weighted time constant ∼2 sec. The data are from four to eight cells per condition.

Fig. 7.

THDOC enhanced single-channel open duration in α1β3δ GABAA receptor single channels. Representative α1β3δ GABAA receptor single-channel currents evoked by 1 m

GABA alone (A1), 1 m

GABA + 1

THDOC (B1), and 1

THDOC alone (C1). A portion of the top trace in each panel is expanded in the trace directly beneath it (indicated by the open bar). The_traces_ in A1 and B1 are from the same patch. The larger scale factor applies to the top traces. A2, B2, and_C2_ are the open duration histograms for all patches obtained for each condition (n = 3, 5, and 3 respectively). Superimposed lines are the fitted exponential functions describing the distributions.

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Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit - PubMed (original) (raw)