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. 2002 Aug;136(7):965-74.
doi: 10.1038/sj.bjp.0704795.

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors

N Brown  1 , J Kerby, T P Bonnert, P J Whiting, K A Wafford
Affiliations

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors

N Brown et al. Br J Pharmacol. 2002 Aug.

Abstract

1: The pharmacology of the stable cell line expressing human alpha(4)beta(3)delta GABA(A) receptor was investigated using whole-cell patch-clamp techniques. 2: alpha(4)beta(3)delta receptors exhibited increased sensitivity to GABA when compared to alpha(4)beta(3)gamma(2) receptors, with EC(50)'s of 0.50 (0.46, 0.53) microM and 2.6 (2.5, 2.6) microM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher efficacy at alpha(4)beta(3)delta receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. 3: The delta subunit conferred slow desensitization to GABA, with rate constants of 4.8+/-0.5 s for alpha(4)beta(3)delta and 2.5+/-0.2 s for alpha(4)beta(3)gamma(2). However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. 4: alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2) demonstrated equal sensitivity to inhibition by the cation zinc (2-3 microM IC(50)). However, alpha(4)beta(3)delta receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC(50) for GABA antagonists SR-95531 and picrotoxin, was similar for alpha(4)beta(3)delta and alpha(4)beta(3)gamma(2). Likewise, inhibition was observed on both subtypes at high and low pH. 5: alpha(4)beta(3)delta receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on alpha(4)beta(3)gamma(2) cells, and the inverse agonist DMCM showed allosteric inhibition of alpha(4)beta(3)gamma(2) receptors. 6: The efficacy of neurosteroids at alpha(4)beta(3)delta receptors was greatly enhanced over that observed at alpha(4)beta(3)gamma(2) receptors. The greatest effect was observed using THDOC with 524+/-71.6% potentiation at alpha(4)beta(3)delta and 297.9+/-49.7% at alpha(4)beta(3)gamma(2) receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at alpha(4)beta(3)delta receptors versus alpha(4)beta(3)gamma(2) receptors. 7: We show that the alpha(4)beta(3)delta receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965-974

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Figures

Figure 1
Figure 1
Comparison of GABA-gated currents on L(-tk) stable cell lines expressing α4β3γ2 and α4β3δ GABAA receptors. (a) Concentration–response curves for GABA on α4β3γ2 and α4β3δ GABAA receptors. Data represent the mean±s.e.mean of eight cells in each case. (b) An example recording of inward currents in response to increasing concentrations of GABA on α4β3δ GABAA receptors.
Figure 2
Figure 2
Concentration–response curves for the GABA agonists (a) P4S and (b) THIP on L(-tk) cells expressing α4β3γ2 and α4β3δ receptors. Dashed line represents maximal response to GABA. Data are normalized to the maximum response to GABA (100 μM) on each cell and represent the mean±s.e.mean of six or more cells.
Figure 3
Figure 3
Effect of the δ subunit on receptor kinetics. Individual recordings showing the desensitization following a 10 s application of a maximally effective concentration of (a) GABA (100 μM) (b) P4S (100 μM on α4β3δ and 1 mM on α4β3γ2) and (c) THIP, (100 μM on α4β3δ and 1 mM on α4β3γ2). Data are all from individual cells and current amplitudes are indicated by the scale bars.
Figure 4
Figure 4
Current-voltage relationship to GABA for α4β3δ and α4β3γ2. Currents were evoked by ramping the cell holding potential from −70 to 60 mV using a 100 ms pulse in the presence and absence of 100 μM GABA. The difference current, obtained by subtracting control from current in the presence of GABA was normalized to the current at −70 mV, and the data from five cells averaged to produce the mean current-voltage response for each receptor. Data shown are mean and s.e.mean for each receptor subtype.
Figure 5
Figure 5
Effect of the δ subunit on sensitivity to inorganic cations. Inhibition of a GABA EC50 current from L(-tk) cells expressing α4β3γ2 and α4β3δ GABAA receptors by increasing concentrations of (a) zinc chloride and (b) lanthanum chloride. Data represent the mean±s.e.mean of at least five cells.
Figure 6
Figure 6
The effect of pH on α4β3γ2 and α4β3δ GABAA receptors. EC50 GABA currents elicited at different pH's were normalized to the EC50 current at pH 7.4. Cells were voltage-clamped at −60 mV, and data set represents mean±s.e.mean of six cells.
Figure 7
Figure 7
Effect of the δ subunit on benzodiazepine sensitivity. Modulation of control GABA EC20 responses in L(-tk) cells expressing α4β3γ2 and α4β3δ receptors by the benzodiazepines (a) flunitrazepam (b) Ro15-4513 (c) bretazenil and (d) DMCM. The data shown are the mean±s.e.mean of at least four cells.
Figure 8
Figure 8
Effect of the δ subunit on anaesthetic sensitivity. Potentiation of control GABA EC20 responses in L(-tk) cells expressing α4β3γ2 and α4β3δ receptors by (a) pentobarbitone (b) propofol and (c) etomidate. Data shown are the mean±s.e.mean of at least five cells.
Figure 9
Figure 9
Effect of the δ subunit on neurosteroid sensitivity. The modulatory effects of the neurosteroids on GABA currents inL(-tk) cells expressing α4β3γ2 and α4β3δ receptors (a) 5α-pregnan-3α-ol-20-one (b) alphaxalone (c) THDOC and (d) pregnenolone sulphate. The data reported represent the mean±s.e.mean of at least six cells.
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