Kaitocephalin antagonism of glutamate receptors expressed in Xenopus oocytes.
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ABSTRACT:
SUBMITTER: Limon A
PROVIDER: S-EPMC2860192 | biostudies-other | 2010 Mar
REPOSITORIES: biostudies-other
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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.
| S-EPMC1573339 | biostudies-other
Project description:GABAρ1 receptors are highly expressed in bipolar neurons of the retina and to a lesser extent in several areas of the central nervous system (CNS), and dopamine and serotonin are also involved in the modulation of retinal neural transmission. Whether these biogenic amines have a direct effect on ionotropic GABA receptors was not known. Here, we report that GABAρ1 receptors, expressed in X. laevis oocytes, were negatively modulated by dopamine and serotonin and less so by octopamine and tyramine. Interestingly, these molecules did not have effects on GABA(A) receptors. 5-Carboxamido-tryptamine and apomorphine did not exert evident effects on any of the receptors. Schild plot analyses of the inhibitory actions of dopamine and serotonin on currents elicited by GABA showed slopes of 2.7 ± 0.3 and 6.1 ± 1.8, respectively, indicating a noncompetitive mechanism of inhibition. The inhibition of GABAρ1 currents was independent of the membrane potential and was insensitive to picrotoxin, a GABA receptor channel blocker and to the GABAρ-specific antagonist (1,2,5,6-tetrahydropyridine-4-yl)methyl phosphinic acid (TPMPA). Dopamine and serotonin changed the sensitivity of GABAρ1 receptors to the inhibitory actions of Zn(2+). In contrast, La(3+) potentiated the amplitude of the GABA currents generated during negative modulation by dopamine (EC(50) 146 μM) and serotonin (EC(50) 196 μM). The functional role of the direct modulation of GABAρ receptors by dopamine and serotonin remains to be elucidated; however, it may represent an important modulatory pathway in the retina, where GABAρ receptors are highly expressed and where these biogenic amines are abundant.
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Project description:AimThe continuous presence of an agonist drives its receptor into a refractory state, termed desensitization. In this study, we tested the hypothesis that a competitive antagonist, SR95531, could facilitate the recovery of ?1?2?2 GABAA receptor from functional desensitization.Methods?1?2?2 GABAA receptors were expressed in Xenopus oocytes. GABA-evoked currents were recorded using two-electrode voltage-clamp technique. Drugs were applied through perfusion.ResultsLong application of GABA (100 ?mol/L) evoked a large peak current followed by a small amplitude steady-state current (desensitization). Co-application of SR95531 during the desensitization caused a larger rebound of GABA current after removal of SR95531. Furthermore, application of SR95531 after removal of GABA increased the rate of receptor recovery from desensitization, and the recovery time constant was decreased from 59±3.2 s to 33±1.6 s. SR95531-facilitated receptor recovery from desensitization was dependent on the perfusion duration of SR95531. It was also dependent on the concentration of SR95531, and the curve fitting with Hill equation revealed two potency components, which were similar to the two potency components in inhibition of the steady-state current by SR95531. Bicuculline caused similar facilitation of desensitization recovery.ConclusionSR95531 facilitates ?1?2?2 GABAA receptor recovery from desensitization, possibly through two mechanisms: binding to the desensitized receptor and converting it to the non-desensitized state, and binding to the resting state receptor and preventing re-desensitization.
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Project description: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.
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Project description:Boric acid is a vital micronutrient that is toxic at high concentrations in animals. However, the mechanisms underlying boric acid transport in animal cells remain unclear. To identify the plasma membrane boric acid channels in animals, we analyzed the function of human aquaporins (AQPs), which are homologous to the nodulin-like intrinsic protein family of plant boric acid channels. When human AQPs were expressed in Xenopus laevis oocytes, the results of the swelling assay showed that boric acid permeability significantly increased in oocytes expressing AQP3, 7, 8, 9, and 10, but not in those expressing AQP1, 2, 4, and 5. The boric acid influxes of these oocytes were also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these AQPs transported boric acid (B(OH)3 ) but not borate ions (B(OH)4- ). These results indicate that AQP3, 7, 8, 9, and 10 act as boric acid transport systems, likely as channels in humans.
| S-EPMC8749175 | biostudies-literature
Project description:Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co-expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8β1, α7, α2α8β1 and α2α7α8β1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR-1) and potential chaperone proteins (e.g., SOL-1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR-1 and α1α8β1 when co-expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N-terminal sequence. We recorded small ACh-gated currents in few oocytes when the α7 subunit was co-expressed with Caenorhabditis elegans RIC-3, but none of the chaperone proteins allowed efficient expression of α2α8β1 or α2α7α8β1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species.
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Project description:The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the ?3 and ?4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the ?5 and/or ?2 subunits. We compared the properties of human ?3?4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and ?:? subunit ratio.Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the ? subunit, as reported for ?4?2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike ?4?2 channels, for ?3?4 receptors the putative two-? form is the predominant one in oocytes (at 1:1 ?:? cRNA ratio). This two-? form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-? form is the predominant one in HEK cells transfected with a 1:1 ?:? DNA ratio or in oocytes at 9:1 ?:? RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-? form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-? form gave rise to short bursts (14 ms) of high conductance (39 pS).Like other neuronal nicotinic receptors, the ?3?4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.
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Project description:Functional modifications of neuronal P/Q-type voltage-dependent Ca2+ channels expressed in Xenopus oocytes by oxidation were examined electrophysiologically. Oxidation by external H2O2 enhanced the whole-oocyte currents through the Ca2+ channels composed of the alpha1A, alpha2/delta, and beta3 subunits at negative voltages (<0 mV) without markedly affecting the currents at more positive voltages. Single-channel analysis showed that oxidation accelerates the overall channel opening process. The effect of H2O2 to enhance the Ca2+ channel activity did not require heterologous expression of the alpha2/delta subunit, and it was not mimicked by a cysteine-specific oxidizing agent. The results suggest that oxidative stress may regulate the activity of neuronal Ca2+ channels and that regulation by oxidation may be important in some clinical situations, such as in reperfusion injury after ischemic episodes.
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Project description:Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin-like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.
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Project description:We studied the properties of GABAA receptors microtransplanted from the human temporal lobe epilepsy (TLE)-associated brain regions to Xenopus oocytes. Cell membranes, isolated from surgically resected brain specimens of drug-resistant TLE patients, were injected into frog oocytes, which rapidly incorporated human GABAA receptors, and any associated proteins, into their surface membrane. The receptors originating from different epileptic brain regions had a similar run-down but an affinity for GABA that was approximately 60% lower for the subiculum receptors than for receptors issuing from the hippocampus proper or the temporal lobe neocortex. Moreover, GABA currents recorded in oocytes injected with membranes from the subiculum had a more depolarized reversal potential compared with the hippocampus proper or neocortex of the same patients. Quantitative RT-PCR analysis was performed of the GABAA receptor alpha1- to alpha5-, beta1- to beta3-, gamma2- to gamma3-, and delta-subunit mRNAs. The levels of expression of the alpha3-, alpha5-, and beta1- to beta3- subunit mRNAs are significantly higher, with the exception of gamma2-subunit whose expression is lower, in subiculum compared with neocortex specimens. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE subiculum leads to the expression of GABAA receptors with a relatively low affinity. This abnormal behavior of the subiculum GABAA receptors may contribute to epileptogenesis.
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