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Human ?3?4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.

ABSTRACT: 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.

SUBMITTER: Krashia P

PROVIDER: S-EPMC2964301 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.

Krashia Paraskevi P Moroni Mirko M Broadbent Steven S Hofmann Giovanna G Kracun Sebastian S Beato Marco M Groot-Kormelink Paul J PJ Sivilotti Lucia G LG

PloS one 20101026 10

<h4>Background</h4>The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expre ...[more]

PMID: 21049012

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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.

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Human ?3?4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.

Publications

Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.

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