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BMC Pharmacol
2006 Jan 13;6:1. doi: 10.1186/1471-2210-6-1.
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Impact of epsilon and theta subunits on pharmacological properties of alpha3beta1 GABAA receptors expressed in Xenopus oocytes.
Ranna M
,
Sinkkonen ST
,
Möykkynen T
,
Uusi-Oukari M
,
Korpi ER
.
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Gamma-aminobutyric acid type A (GABAA) receptors provide the main inhibitory control in the brain. Their heterogeneity may make it possible to precisely target drug effects to selected neuronal populations. In situ hybridization using rat brain sections has revealed a unique expression of GABAA receptor epsilon and theta subunit transcripts in the locus coeruleus, where they are accompanied at least by alpha3, alpha2, beta1 and beta3 subunits. Here, we studied the pharmacology of the human alpha3beta1, alpha3beta1epsilon, alpha3beta1theta and alpha3beta1epsilontheta receptor subtypes expressed in Xenopus oocytes and compared them with the gamma2 subunit-containing receptors. The GABA sensitivites and effects of several positive modulators of GABAA receptors were studied in the absence and the presence of EC25 GABA using the two-electrode voltage-clamp method. We found 100-fold differences in GABA sensitivity between the receptors, alpha3beta1epsilon subtype being the most sensitive and alpha3beta1gamma2 the least sensitive. Also gaboxadol dose-response curves followed the same sensitivity rank order, with EC50 values being 72 and 411 microM for alpha3beta1epsilon and alpha3beta1gamma2 subtypes, respectively. In the presence of EC25 GABA, introduction of the epsilon subunit to the receptor complex resulted in diminished modulatory effects by etomidate, propofol, pregnanolone and flurazepam, but not by pentobarbital. Furthermore, the alpha3beta1epsilon subtype displayed picrotoxin-sensitive spontaneous activity. The theta subunit-containing receptors were efficiently potentiated by the anesthetic etomidate, suggesting that theta subunit could bring the properties of beta2 or beta3 subunits to the receptor complex. The epsilon and theta subunits bring additional features to alpha3beta1 GABAA receptors. These receptor subtypes may constitute as novel drug targets in selected brain regions, e.g., in the brainstem locus coeruleus nuclei.
Figure 1. Responses to GABA site ligands. GABA and gaboxadol sensitivities of recombinant α3β1, α3β1γ2, α3β1θ, α3β1ε and α3β1θε GABAA receptors expressed in Xenopus oocytes. A. The values for currents induced by various GABA concentrations are given as means ± standard errors (n = 8 for α3β1, n = 6 for α3β1γ2, n = 7 for α3β1θ; n = 6 for α3β1ε and n = 7 for α3β1θε receptors). GABA dose-response curves were obtained by non-linear regression fits and GABA EC50 values were calculated for individual cells, being 55 ± 6 μM for α3β1 receptors, 200 ± 38 μM for α3β1γ2 (p < 0.01, unpaired t-test from α3β1), 2.3 ± 0.5 μM for α3β1ε (p < 0.001 from α3β1) and 81 ± 18 μM for α3β1θ (p > 0.05) and 35 ± 9 μM for α3β1θε receptors. Maximal currents elicited by GABA were 1.5 ± 0.9 μA for α3β1, 2.2 ± 0.5 μA for α3β1γ2, 1.2 ± 0.3 μA for α3β1θ; 1.2 ± 1.0 μA for α3β1ε and 0.9 ± 0.4 μA for α3β1θε combinations. B. Gaboxadol-induced currents are given as means ± standard errors (n = 7 for α3β1, n = 5 for α3β1γ2, n = 4 for α3β1θ; n = 5 for α3β1ε and n = 4 for α3β1θε receptors). Gaboxadol dose-response curves were obtained by non-linear regression fits and the EC50 values were calculated for individual cells, being 139 ± 19 μM for α3β1 receptors, 411 ± 13 μM for α3β1γ2 (p < 0.05, unpaired t-test from α3β1),72 ± 5 μM for α3β1ε (p > 0.05 from α3β1), 224 ± 20 μM for α3β1θ (p > 0.05) and 165 ± 19 μM for α3β1θε receptors. Maximal currents elicited by gaboxadol were 2.5 ± 0.9 μA for α3β1, 1.2 ± 0.5 μA for α3β1γ2, 0.55 ± 0.2 μA for α3β1θ, 1.0 ± 0.4 μA for α3β1ε and 0.8 ± 0.3 μA for α3β1θε combinations.
Figure 2. Constitutive activity of ε subunit-containing receptors. The ε subunit-containing receptors display spontaneous currents that can be blocked with picrotoxin. A. A representative current trace recorded from an oocyte expressing spontaneously open α3β1ε receptors is shown. GABA (30 μM) application induced typical inward currents. Picrotoxin (Picro, 100 μM) reversibly blocked spontaneously active receptors as shown by blockade (outward current) of apparent leakage current. B. Peak amplitudes (in nA given as means ± standard errors) of GABA- and picrotoxin-induced currents in six oocytes expressing α3β1ε receptors.
Figure 3. Sensitivities to the anesthetic etomidate. Modulation of GABA-induced currents by etomidate in recombinant GABAA receptors expressed in Xenopus oocytes. A. Representative traces of etomidate (Etom, μM) modulation of GABA-induced currents (G, EC25 in μM) in different receptor subtypes. Also the effect of flurazepam at 100 μM is shown for the α3β1γ2 receptors. B. Etomidate dose-response curves in the presence of EC25 GABA (set to 100 %). The values are means ± standard errors (n = 6 for α3β1; n = 7 for α3β1γ2; n = 9 for α3β1θ; n = 11 for α3β1ε and n = 4 for α3β1θε receptors).
Figure 4. Sensitivities to the anesthetic propofol. Modulation of GABA-induced currents by propofol in recombinant GABAA receptors expressed in Xenopus oocytes. A. Representative traces of propofol (Prop, μM) modulation of GABA-induced currents (G, EC25 in μM) in different receptor subtypes. B. Propofol dose-response curves in the presence of EC25 GABA (set to 100 %). The values are means ± standard errors (n = 6 for α3β1; n = 9 for α3β1γ2; n = 6 for α3β1θ; n = 7 for α3β1ε and n = 3 for α3β1θε receptors).
Figure 5. Sensitivities to the neurosteroid pregnanolone. Modulation of GABA-induced currents by the neurosteroid pregnanolone in recombinant GABAA receptors expressed in Xenopus oocytes. A. Representative traces of pregnanolone (Preg, nM) modulation of GABA-induced currents (G, EC25 in μM) in different receptor subtypes. B. Pregnanolone dose-response curves in the presence of EC25 GABA (set to 100 %). The values are means ± standard errors (n = 6 for α3β1; n = 5 for α3β1γ2; n = 5 for α3β1θ; n = 6 for α3β1ε and n = 4 for α3β1θε receptors).
Figure 6. Sensitivities to the barbiturate pentobarbital and the benzodizepine flurazepam. Modulation of GABA-induced currents by pentobarbital and flurazepam in recombinant GABAA receptors expressed in Xenopus oocytes. A. Pentobarbital dose-response curves in the presence of EC25 GABA (set to 100 %). The values are means ± standard errors (n = 6 for α3β1; n = 9 for α3β1γ2; n = 4 for α3β1θ and n = 8 for α3β1ε receptors). B. Flurazepam dose-response curves in the presence of EC25 GABA (set to 100 %). The values are means ± standard errors (n = 6 for α3β1; n = 9 for α3β1γ2; n = 4 for α3β1θ and n = 8 for α3β1ε receptors).
Figure 7. Direct receptor activation by etomidate and propofol. Efficacy of a high concentration (300 μM) of etomidate and propofol in provoking receptor currents as compared to control EC25 GABA responses (set to 100 %). The values are means ± standard errors (n = 5 for α3β1; n = 6 for α3β1γ2; n = 5 for α3β1θ and n = 7 for α3β1ε receptors).
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