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Characteristics of concatemeric GABA(A) receptors containing ?4/? subunits expressed in Xenopus oocytes.

ABSTRACT: GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, ?4 and ? subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant ? subunits can be difficult to express in heterologous systems.We engineered concatemeric (fused) subunits to ensure ? and ?4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (?1 +?2 +?2L), and with free-subunit ?4/? receptors, expressed in Xenopus oocytes.?-?2 -?4 +?2-?4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of ?4/?-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a ?-selective positive modulator), furosemide, and Zn(2+) . We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, ?-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations.The neurosteroid structure-activity profile for ?4/?-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as ?1/?2/?2-containing receptors.

SUBMITTER: Shu HJ

PROVIDER: S-EPMC3413859 | biostudies-other | 2012 Apr

REPOSITORIES: biostudies-other

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Characteristics of concatemeric GABA(A) receptors containing α4/δ subunits expressed in Xenopus oocytes.

Shu Hong-Jin HJ Bracamontes John J Taylor Amanda A Wu Kyle K Eaton Megan M MM Akk Gustav G Manion Brad B Evers Alex S AS Krishnan Kathiresan K Covey Douglas F DF Zorumski Charles F CF Steinbach Joe Henry JH Mennerick Steven S

British journal of pharmacology 20120401 7

<h4>Background and purpose</h4>GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems.<h4>Expe ...[more]

PMID: 21950777

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Project description:1. We have investigated the effect of diltiazem and its newly synthesized derivative (+,-)-trans-3-acetoxy-8-chloro-2,3-dihydro-5[2-diisopropylamine)ethyl]-2-(4-methoxyphenyl)-1,5-benzothiazepin-4-(5H)-ona hydrochloride (JAC-65) on several recombinant human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. 2. At 3 microM, both drugs have little effect on the maximal currents evoked by brief pulses of acetylcholine (ACh) in five subtypes of nAChRs (alpha7, alpha3beta2, alpha4beta2, alpha3beta4, and alpha4beta4), showing little selectivity among subtypes. 3. However, both drugs accelerate the decay of the ionic currents evoked upon continuous stimulation of ACh, being this effect larger with JAC-65, and in beta4*-nAChRs. Such an effect was dependent on the concentrations of both the drug and of the agonist used, and showed the characteristics of a non-competitive antagonism. 4. We have further investigated the effect of both drugs when combined with submicromolar concentrations of nicotine, such as those present in plasma of cigarette smokers, and found that JAC-65, but not diltiazem, is able to greatly enhance the desensitizing effect of these low concentrations of nicotine, specially in beta4*-nAChRs. 5. Experiments in alpha4beta4-nAChRs failed to show voltage dependence of the action of JAC-65. Moreover, recovery from desensitization followed the same time course regardless of the presence of the drug, suggesting that the main mechanism of action of JAC-65 does not involve open channel block. 6. In summary, both drugs, diltiazem and JAC-65, seem to act through a non-competitive mechanism, accelerating the decay of the ionic currents, being JAC-65 more effective than diltiazem at the concentrations used in beta4*-nAChRs. Thus, the differences between both benzothiazepines when measuring various parameters suggest that their mechanisms of action could be slightly different. This would require further investigation.

Dataset Information

Characteristics of concatemeric GABA(A) receptors containing ?4/? subunits expressed in Xenopus oocytes.

Publications

Characteristics of concatemeric GABA(A) receptors containing α4/δ subunits expressed in Xenopus oocytes.

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