Histamine Action on Vertebrate GABAA Receptors
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m709993200
ISSN1083-351X
AutoresArunesh Saras, Günter Gisselmann, Angela K. Vogt-Eisele, Katja S. Erlkamp, Olaf Kletke, H. Pusch, Hanns Hatt,
Tópico(s)Receptor Mechanisms and Signaling
ResumoHistamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified β subunits of GABAA receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric β channels, histamine evoked currents with an EC50 of 212 μm (β2) and 174 μm (β3), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for β channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of α1β2 or α1β2γ2 subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a γ2 subunit-controlled manner. Despite the vast number of synthetic modulators of GABAA receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABAA receptors and may thus represent an endogenous ligand for an allosteric site. Histamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified β subunits of GABAA receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric β channels, histamine evoked currents with an EC50 of 212 μm (β2) and 174 μm (β3), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for β channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of α1β2 or α1β2γ2 subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a γ2 subunit-controlled manner. Despite the vast number of synthetic modulators of GABAA receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABAA receptors and may thus represent an endogenous ligand for an allosteric site. Ligand-gated ion channels mediate the fast responses of cells to neurotransmitters (1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar). A universal feature of ligand-gated ion channels subunits is a common topology, comprising four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconserved cysteine loop motif. In vertebrates, this "Cys loop" family of phylogenetically related genes codes for anion and cation channels activated by acetylcholine and serotonin (cation channels) or GABA 3The abbreviations used are: GABA, γ-aminobutyric acid; GABAA, GABA, type A. and glycine (anion channels) (1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar, 2Ortells M.O. Lunt G.G. Trends Neurosci. 1995; 18 (121–127): 3Google Scholar). Despite the many years of intensive research on such ion channels, recent reports revealed unexpected new findings about this channel family. In vertebrates, a gene for zinc-gated ion channels was recently discovered (3Davies P.A. Wang W. Hales T.G. Kirkness E.F. J. Biol. Chem. 2003; 278: 712-717Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In insects, new classes of ligand-gated chloride channels gated by histamine or pH and cation channels gated by GABA were reported (4Zheng Y. Hirschberg B. Yuan J. Wang A.P. Hunt D.C. Ludmerer S.W. Schmatz D.M. Cully D.F. J. Biol. Chem. 2002; 277: 2000-2005Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 5Gisselmann G. Pusch H. Hovemann B.T. Hatt H. Nat. Neurosci. 2002; 5: 11-12Crossref PubMed Scopus (118) Google Scholar, 6Gisselmann G. Plonka J. Pusch H. Hatt H. Br. J Pharmacol. 2004; 142: 409-413Crossref PubMed Scopus (78) Google Scholar, 7Schnizler K. Saeger B. Pfeffer C. Gerbaulet A. Ebbinghaus-Kintscher U. Methfessel C. Franken E.M. Raming K. Wetzel C.H. Saras A. Pusch H. Hatt H. Gisselmann G. J. Biol. Chem. 2005; 280: 16254-16262Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Histamine is a neurotransmitter and neuromodulator acting on the metabotropic H1-H4 receptors (8Brown R.E. Stevens D.R. Haas H.L. Prog. Neurobiol. (Oxf.). 2001; 63: 637-672Crossref PubMed Scopus (881) Google Scholar). In addition, a direct activation of mammalian anion channels has been postulated (9Hatton G.I. Yang Q.Z. J. Neurosci. 2001; 21: 2974-2982Crossref PubMed Google Scholar). Histamine-containing neurons are found exclusively in the tuberomamillary nucleus of the hypothalamus (10Haas H. Panula P. Nat. Rev. Neurosci. 2003; 4: 121-130Crossref PubMed Scopus (768) Google Scholar). The GABA-synthesizing enzyme, glutamate acid decarboxylase, and GABA are also seen in most tuberomamillary neurons, and many neurons in the tuberomamillary nucleus contain both GABA and histamine (10Haas H. Panula P. Nat. Rev. Neurosci. 2003; 4: 121-130Crossref PubMed Scopus (768) Google Scholar). The molecular structure of a potential mammalian histamine-gated channel is still elusive, but in invertebrates, two genes for histamine-gated channels have been identified (4Zheng Y. Hirschberg B. Yuan J. Wang A.P. Hunt D.C. Ludmerer S.W. Schmatz D.M. Cully D.F. J. Biol. Chem. 2002; 277: 2000-2005Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 5Gisselmann G. Pusch H. Hovemann B.T. Hatt H. Nat. Neurosci. 2002; 5: 11-12Crossref PubMed Scopus (118) Google Scholar, 11Witte I. Kreienkamp H.J. Gewecke M. Roeder T. J. Neurochem. 2002; 83: 504-514Crossref PubMed Scopus (51) Google Scholar, 12Gengs C. Leung H.T. Skingsley D.R. Iovchev M.I. Yin Z. Semenov E.P. Burg M.G. Hardie R.C. Pak W.L. J. Biol. Chem. 2002; 277: 42113-42120Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In addition, GABA and histamine can activate the same ion channel (13Gisselmann G. Plonka J. Pusch H. Hatt H. Neurosci. Lett. 2004; 372: 151-156Crossref PubMed Scopus (25) Google Scholar). Therefore, we hypothesized that a subtype of mammalian GABA receptors may be a good candidate mediating the effect of histamine in vertebrates. GABA is the major inhibitory neurotransmitter in the central nervous system. Most of the rapid inhibitory neurotransmission in the central nervous system is mediated by the GABA type A receptors (14Whiting P.J. Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Thompson S.A. Wafford K.A. Ann. N. Y. Acad. Sci. 1999; 868: 645-653Crossref PubMed Scopus (299) Google Scholar). GABAA receptors are heteropentameric proteins constructed of various subunits (α, β, γ, δ, θ, ϵ, and π). The most prominent native receptors are heteromultimers of α, β, and γ subunits (14Whiting P.J. Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Thompson S.A. Wafford K.A. Ann. N. Y. Acad. Sci. 1999; 868: 645-653Crossref PubMed Scopus (299) Google Scholar), but at least in recombinant systems, functional homomultimeric receptors composed of β or γ subunits alone exist (15Wooltorton J.R. Moss S.J. Smart T.G. Eur. J. Neurosci. 1997; 9: 2225-2235Crossref PubMed Scopus (117) Google Scholar, 16Cestari I.N. Uchida I. Li L. Burt D. Yang J. Neuroreport. 1996; 7: 943-947Crossref PubMed Scopus (58) Google Scholar, 17Miko A. Werby E. Sun H. Healey J. Zhang L. J. Biol. Chem. 2004; 279: 22833-22840Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 18Sanna E. Garau F. Harris R.A. Mol. Pharmacol. 1995; 47: 213-217PubMed Google Scholar, 19Martinez-Torres A. Miledi R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3220-3223Crossref PubMed Scopus (8) Google Scholar). Such homomultimeric ion channels differ in many aspects from the conventional, heteromultimeric GABAA receptors and were therefore candidates for receptors with unexpected new properties. GABAA cDNAs and RNAs—Rat α1 and β1 cDNAs were cloned by PCR-based methods using standard molecular biology procedures. Rat β2 cDNA was kindly provided by R. Rupprecht (Munich, Germany). Mouse γ2L and human β3 cDNA was obtained from ImaGenes (Berlin, Germany). All cDNAs were subcloned into pSGEM (courtesy of M. Hollmann, Bochum, Germany) or pCDNA3 (Invitrogen, Karlsruhe, Germany) for HEK293 cell expression. Expression of Receptor cRNA in Xenopus Oocytes—cRNAs were synthesized using the AmpliCap T7 high yield message maker kit (Epicenter, Madison, WI), according to the manufacturer's protocol, with PacI-linearized pSGEM plasmids as templates. Xenopus laevis oocytes were prepared by standard methods. After 24 h, stage V–VI oocytes were injected with cRNA (typically 5–25 ng/oocyte), incubated at 16 °C in Barth's solution, and tested for functional expression of GABAA receptors by two-electrode voltage clamp recording after 3–7 days. Agonists and antagonists were diluted to the concentrations indicated with Frog-Ringer's solution (115 mm NaCl, 2.5 mm KCl, 1.8 mm CaCl2, 10 mm HEPES, pH 7.2). Recording was done with a two-electrode voltage clamp amplifier (TURBO TEC-03, npi, Tamm, Germany) and pCLAMP software (Axon Instruments, Union City, CA) with typical membrane potential of –40 to –60 mV. The pH of all Ringer's solutions containing histamine was adjusted to pH 7.2 if necessary. The quality of the water used for solution preparation can be critical for the amount of contamination by traces of Zn2+ or Cu2+. Previously it was shown that in native cerebral Purkinje neurons, co-application of histidine enhances GABA-evoked currents by complexing trace amounts of copper ions (20Sharonova I.N. Vorobjev V.S. Haas H.L. Eur. J. Neurosci. 1998; 10: 522-528Crossref PubMed Scopus (52) Google Scholar). To exclude that the observed histamine effects depend on the complexation of such divalent cation contaminations, potentiation and dose-response curves experiments were performed in Ringer's solution prepared with ultrapure water (AMPUWA water, Fresenius, Bad Homburg, Germany) as suggested (20Sharonova I.N. Vorobjev V.S. Haas H.L. Eur. J. Neurosci. 1998; 10: 522-528Crossref PubMed Scopus (52) Google Scholar). In experiments targeting the effect of histamine on heteromultimeric receptors, it was vital to ensure the absence of any contaminating population of homomultimeric channels composed of β subunits when expressing α1β2γ2 receptors as such could mimic a histamine effect. In preliminary studies with oocytes injected with a 1:1:1 ratio of α1β2γ2 subunits, in some experiments, small currents were directly evocable by 1 mm histamine, obviously caused by a small, contaminating population of homomultimeric β2 channels. Therefore, we injected an excess of α1 and γ2 over β2 subunit RNA (ratio α1:10, β2:1, γ2:2). In these oocytes, 1 mm histamine itself evoked no detectable currents (≪1% of the maximum GABA evoked current), proving the absence of a contaminating population of homomultimeric β2-subunits. The same is valid for receptors composed of α1β2 subunits, where the α subunit was also injected in a 10-fold excess over β. To ensure the incorporation of the γ2L subunit, oocytes were screened with 10 μm Zn2+ in the presence of GABA. Although αβ subunit combinations are highly sensitive for an inhibition by Zn2+, the αβγ isoforms are insensitive. Statistics—For electrophysiological measurements, statistical analysis and curve fitting was done by the Hill equation using SigmaPlot V8.0 (Systat Software, San Jose, CA). All mean values are ± S.E. Patch Clamp Experiments—HEK293 cells were maintained under standard conditions in a minimum essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and streptomycin, and 2 mm l-glutamine. Semiconfluent cells were transfected in 35-mm dishes (BD Biosciences) by using standard calcium phosphate precipitation, 48–72 h after transfection. Recordings were performed using the whole-cell mode of the patch clamp technique. Cells were maintained in an extracellular recording solution containing 140 mm NaCl, 5 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, 10 mm glucose, pH 7.4. Patch electrodes were pulled from borosilicate glass and fire-polished to 4–6-megaohm tip resistance using a horizontal pipette puller (Zeitz Instruments, Munich, Germany). The pipette solution contained 140 mm KCl, 1 mm MgCl2, 0.1 mm CaCl2, 5 mm EGTA, 10 mm HEPES, pH 7.4 for recordings. Recordings were carried out using a HEKA EPC7 amplifier. Membrane potential was held at –40 mV. Homomultimeric β Channels—We investigated the action of histamine on homomultimeric GABAA receptors. Complementary RNAs (cRNA) from β2–3 subunits were expressed in X. laevis oocytes. 3–7 days after injection, histamine- and GABA-elicited currents were recorded in the voltage clamp configuration. We used the known β channel agonist pentobarbital (15Wooltorton J.R. Moss S.J. Smart T.G. Eur. J. Neurosci. 1997; 9: 2225-2235Crossref PubMed Scopus (117) Google Scholar, 16Cestari I.N. Uchida I. Li L. Burt D. Yang J. Neuroreport. 1996; 7: 943-947Crossref PubMed Scopus (58) Google Scholar, 18Sanna E. Garau F. Harris R.A. Mol. Pharmacol. 1995; 47: 213-217PubMed Google Scholar) as a positive control and found that 3 mm histamine could also directly open homomultimeric β3 channels with an average current amplitude of 536 nA (±95, n = 15). When compared with saturating concentrations of pentobarbital, histamine evoked currents were smaller, reaching 67% (±6%, n = 8, p = 0.008) of the pentobarbital response (Fig. 1A). 3 mm histamine also opened homomultimeric β2 channels, although with lower average amplitudes (132 nA (±21, n = 7), data not shown). Dose-response curves show that homomultimeric receptors composed of β2 or β3 were similarly sensitive to histamine, with an EC50 of 212 ± 29 μm (n = 4) and an EC50 of 174 ± 14 μm (n = 11), respectively (Fig. 1, B and C). It has been reported that homomultimeric β channels are activated by GABA (15Wooltorton J.R. Moss S.J. Smart T.G. Eur. J. Neurosci. 1997; 9: 2225-2235Crossref PubMed Scopus (117) Google Scholar, 16Cestari I.N. Uchida I. Li L. Burt D. Yang J. Neuroreport. 1996; 7: 943-947Crossref PubMed Scopus (58) Google Scholar, 17Miko A. Werby E. Sun H. Healey J. Zhang L. J. Biol. Chem. 2004; 279: 22833-22840Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 18Sanna E. Garau F. Harris R.A. Mol. Pharmacol. 1995; 47: 213-217PubMed Google Scholar). Our experiments expressing β2 or β3 confirmed the effect of GABA, but even at high, saturating GABA concentrations (3 mm), the amplitudes were only 10% of the histamine-activated currents (β3: IGABA (3 mM)/IHistamine (10 mM) = 0.11 ± 0.012 (n = 10)). GABA is a weak partial agonist when compared with histamine (Fig. 1A). Homomultimeric channels composed of GABAA β subunits are histamine-gated channels rather than GABA-gated channels. Histamine evoked a current reversing typically at –23 mV under standard ion conditions in oocytes expressing β3 (Fig. 1D), which is consistent with the previously reported chloride selectivity of β channels (15Wooltorton J.R. Moss S.J. Smart T.G. Eur. J. Neurosci. 1997; 9: 2225-2235Crossref PubMed Scopus (117) Google Scholar). The histamine effect is independent on the oocyte expression system. In whole-cell patch clamp experiments on human embryonic kidney 293 (HEK293) cells expressing β3 channels (Fig. 1E), 1 mm histamine evoked currents with an average amplitude of 203 pA (±31, n = 8), which was typically 60% of the pentobarbital response (21Taylor P.M. Thomas P. Gorrie G.H. Connolly C.N. Smart T.G. Moss S.J. J. Neurosci. 1999; 19: 6360-6371Crossref PubMed Google Scholar). The EC50 of histamine with 386 μm (±24, n = 6) was in the same range as in Xenopus oocytes (Fig. 1F). Pharmacology of the Histamine Response—The currents evoked by 1 mm histamine expressed in X. laevis oocytes expressing β3 were completely blocked by 10 μm picrotoxinin, but 100 μm of the GABA-antagonists bicuculline or gabazine were ineffective (data not shown). Histamine analogs such as histidine and tele-methylhistamine were also found to be agonists for β3 channels (Fig. 2A and supplemental Figs. 1 and 2), although with lower potencies (EC50 1.14 mm ± 0.15 μm (n = 5) and EC50 1.1 mm ± 0.13 μm (n = 3), respectively), demonstrating that metabolic precursors and metabolites could also be active agonists. For the histamine-binding sites of metabotropic H1–H4 receptors, specific agonists and antagonists are known (22van der Goot H. Timmerman H. Eur. J. Med. Chem. 2000; 35: 5-20Crossref PubMed Scopus (106) Google Scholar). We investigated whether the histamine-binding site of GABAA β subunits match one of those pharmacological profiles. In oocytes expressing β3 channels, current evoked by 300 μm histamine was effectively blocked by the H3/4 antagonist thioperamide. Higher concentrations of thioperamide additionally blocked a fraction of spontaneously open channels as indicated by the apparent "outward" current; it thus behaves like an "inverse agonist" (Fig. 2B). The IC50 was 7.2 ± 0.7 μm (n = 5) in the absence of histamine (Fig. 2D). In the presence of 300 μm histamine, the IC50 for thioperamide was 32 ± 3.8 μm (n = 5) determined by a four-parameter Hill fit of the complete blocking curve, but it would be in the range of 10 μm if one would relate it only to the histamine evoked current. Therefore, the found value is only a rough estimate as the population of open channels interferes with the analysis. Next, we tested whether thioperamide and histamine might compete for the same binding site and found that at 10 mm histamine, thioperamide was a far less effective blocker, a clear indication for a competitive mechanism (Fig. 2C). The H2 antagonist famotidine (IC50 = 154 ± 21 μm (n = 5)) and the H1/2 agonist histamine trifluoromethyl toluidide (IC50 = 162 ± 19 μm (n = 5)) were also found to be blockers of the histamine-evoked current as well as the population of open channels (supplemental Fig. 3). Other ligands for metabotropic histamine receptors, such as cimetidine (H2 antagonist), pyrilamine (H1 antagonist), and dimaprit (H2 agonist), were ineffective in concentrations up to 500 μm (data not shown). Our data reveal that the histamine-binding site of GABAA β subunits has a unique pharmacology not matching any of the metabotropic receptors. Histamine analog H2 blockers act on heteromultimeric GABAA receptors as reported previously (23Cannon K.E. Fleck M.W. Hough L.B. Life Sci. 2004; 75: 2551-2558Crossref PubMed Scopus (19) Google Scholar, 24Koutsoviti-Papadopoulou M. Nikolaidis E. Kounenis G. Pharmacol. Res. 2003; 48: 279-284Crossref PubMed Scopus (4) Google Scholar). Our findings may explain the molecular basis of this observed action as we found that such molecules directly act on β subunits. However, our findings imply that GABAA receptors are not specifically blocked by H2 antagonists alone but agonists or antagonists of other metabotropic histamine receptors also act. The Action of Histamine on α1β2γ2 Receptors—The action of histamine on the β subunits resembles that of allosteric GABAA modulators such as propofol and barbiturates, which also activate currents at homomultimeric β receptors directly (15Wooltorton J.R. Moss S.J. Smart T.G. Eur. J. Neurosci. 1997; 9: 2225-2235Crossref PubMed Scopus (117) Google Scholar, 16Cestari I.N. Uchida I. Li L. Burt D. Yang J. Neuroreport. 1996; 7: 943-947Crossref PubMed Scopus (58) Google Scholar, 18Sanna E. Garau F. Harris R.A. Mol. Pharmacol. 1995; 47: 213-217PubMed Google Scholar). At heteromultimeric GABAA receptors, such modulators potentiate the action of GABA (25Korpi E.R. Grunder G. Luddens H. Prog. Neurobiol. (Oxf.). 2002; 67: 113-159Crossref PubMed Scopus (417) Google Scholar). To investigate the action of histamine on heteromultimeric receptors, we investigated recombinant α1β2γ2 receptors, which are the most abundant GABAA synaptic receptor type in the central nervous system (14Whiting P.J. Bonnert T.P. McKernan R.M. Farrar S. le Bourdelles B. Heavens R.P. Smith D.W. Hewson L. Rigby M.R. Sirinathsinghji D.J. Thompson S.A. Wafford K.A. Ann. N. Y. Acad. Sci. 1999; 868: 645-653Crossref PubMed Scopus (299) Google Scholar). In oocytes expressing α1β2γ2 receptors, 1 mm histamine potentiated the current evoked by 10 μm GABA (about EC30) (Fig. 3A). The potentiation at 10 μm GABA was 1.5-fold on average (I(GABA + histamine)/I(GABA) = 1.52 ± 0.43, n = 12) but had a considerable variability, reaching from 0 in few oocytes up to 2.4-fold. In the same set of oocytes, 1 mm histamine was virtually ineffective on the current evoked by saturating concentrations (300 μm) of GABA (1.01 ± 0.06), pointing out that potentiation by histamine was significantly much more effective at submaximal GABA concentrations (p = 0.0012, n = 12). This fit to the observation that 1 mm histamine significantly lowered the EC50 for GABA from 15.8 ± 2.1 μm to 11.1 ± 1.7 μm (p = 0.0044, n = 5) (Fig. 3, A and B). 1 mm histamine itself did not evoke detectable currents (≪1% of the maximum GABA evoked current, Fig. 3A). The histamine potentiation of recombinant GABAA receptors was independent of the expression system and could also be observed at α1β2γ2 receptors expressed in HEK293 cells. Histamine showed potentiating effects similar to those in oocytes, demonstrating that the effect was not restricted to the oocyte expression system. At cells stimulated with 3 μm GABA, 1 mm histamine evoked an up to 2.0-fold potentiation of the GABA current (1.4-fold potentiation on average; n = 12) (Fig. 3C), but 1 mm histamine alone never evoked any detectable currents. The Action of Histamine on α1β2 Receptors—It is known that the presence of a γ2 subunit modulates the potentiation by allosteric modulators such as benzodiazepines, which are only effective in γ subunit-containing receptors (26Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar). In the case of potentiators acting on β subunits such as propofol, it was reported that the γ2 subunit alters the mode of potentiation (27Olsen R.W. Toxicol. Lett. 1998; 100-101: 193-201Crossref PubMed Scopus (44) Google Scholar). To address the question of whether the presence of a γ2 subunit alters the potentiation of histamine, we compared the effect of histamine on heteromultimeric α1β2 and α1β2γ2 receptors. In oocytes expressing α1β2, receptors, 1 mm histamine potentiated currents evoked by GABA. Histamine potentiation was strongly dependent on the GABA concentration but in a different manner as for α1β2γ2 receptors. At α1β2 GABA receptors, 1 mm histamine potentiated best at saturating GABA concentrations (300 μm) but on average did not potentiate at submaximal concentrations of 3 μm GABA (Fig. 3, D and E). At 300 μm GABA, average potentiation was 1.26-fold (±0.24, n = 14) and significantly greater (p = 0.013, n = 14) than at 3 μm GABA in the same set of oocytes (1.04 ± 0.07, n = 14). Also in these experiments, some oocytes had GABA currents not potentiated by histamine at all. 1 mm histamine itself did not evoke detectable currents (≪1% of the maximum GABA evoked current). The average EC50 for GABA was not significantly affected by 1 mm histamine (p = 0.22, n = 4). These experiments demonstrate that the γ2 subunit has a vital, modulatory role in histamine potentiation of GABAA receptors. Histamine potentiates GABA receptors in a dose-dependent manner. At α1β2 receptors, the EC50 of potentiation is 965 μm (±306, n = 4) (Fig. 3F). The potentiation effect requires higher histamine concentrations as the direct action on homomultimeric β2 channels. Our study shows that histamine directly opens homomultimeric GABAA receptors that thus can function as histamine-gated channels. The existence of such histamine-gated chloride channels in mammals has been suggested for a long time (4Zheng Y. Hirschberg B. Yuan J. Wang A.P. Hunt D.C. Ludmerer S.W. Schmatz D.M. Cully D.F. J. Biol. Chem. 2002; 277: 2000-2005Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 9Hatton G.I. Yang Q.Z. J. Neurosci. 2001; 21: 2974-2982Crossref PubMed Google Scholar). The putative histamine receptor described by Hatton and Yang (9Hatton G.I. Yang Q.Z. J. Neurosci. 2001; 21: 2974-2982Crossref PubMed Google Scholar) in the nucleus supraopticus shares some pharmacological similarities with homomultimeric β channels (e.g. the PTX sensitivity). However, there are also some pronounced differences (e.g. the affinity for histamine and the sensitivity to cimetidine), suggesting that the native receptor may need additional components. When compared with the high affinity of metabotropic histamine receptors, the EC50 of β channels with ∼200–400 μm is quite low, and they are not expected to be activated by typical extracellular histamine concentrations in the brain. However, at synaptic transmission, high enough concentrations could be reached in the synaptic cleft as histamine concentration in synaptic vesicles be as high as 670 mm (28Borycz J.A. Borycz J. Kubow A. Kostyleva R. Meinertzhagen I.A. J. Neurophysiol. 2005; 93: 1611-1619Crossref PubMed Scopus (31) Google Scholar). Nevertheless, our findings support the idea that GABAA subunits may be vital parts of potential native mammalian histamine-gated channels and could provide the histamine-binding site. Further, we demonstrated that histamine potentiates GABA responses in heteromultimeric receptors and thereby identified a new type of allosteric potentiator for GABAA receptors. The mode of potentiation resembles anesthetics such as propofol; like propofol, it targets β subunits and opens homomultimeric β channels directly but modulates the GABA response at heteromultimeric channels (15Wooltorton J.R. Moss S.J. Smart T.G. Eur. J. Neurosci. 1997; 9: 2225-2235Crossref PubMed Scopus (117) Google Scholar, 16Cestari I.N. Uchida I. Li L. Burt D. Yang J. Neuroreport. 1996; 7: 943-947Crossref PubMed Scopus (58) Google Scholar, 18Sanna E. Garau F. Harris R.A. Mol. Pharmacol. 1995; 47: 213-217PubMed Google Scholar, 27Olsen R.W. Toxicol. Lett. 1998; 100-101: 193-201Crossref PubMed Scopus (44) Google Scholar, 29Krasowski M.D. Koltchine V.V. Rick C.E. Ye Q. Finn S.E. Harrison N.L. Mol. Pharmacol. 1998; 53: 530-538Crossref PubMed Scopus (246) Google Scholar, 30Lam D.W. Reynolds J.N. Brain Res. 1998; 784: 179-187Crossref PubMed Scopus (53) Google Scholar). Also the influence of the γ2 subunit is similar. In receptors composed of α1β2 subunits, both histamine and propofol potentiate effectively at saturating GABA concentrations, increasing the maximally evoked currents. In contrast, at α1β2γ2 receptors, histamine and propofol were noneffective at high concentrations but are shifting the GABA dose-response curve leftwards (30Lam D.W. Reynolds J.N. Brain Res. 1998; 784: 179-187Crossref PubMed Scopus (53) Google Scholar). In our oocyte expression system, we found that at recombinant heteromultimeric receptors, the strength of histamine potentiation was quite variable, an indication that potentiation might be regulated for example posttranscriptionally by a yet unknown mechanism. Such variability of receptor properties in recombinant expression systems is often observed and can have several reasons: for example, differences in expression level (31Taleb O. Betz H. EMBO J. 1994; 13: 1318-1324Crossref PubMed Scopus (68) Google Scholar), receptor clustering (32Chen Z.W. Olsen R.W. J. Neurochem. 2007; 100: 279-294Crossref PubMed Scopus (149) Google Scholar), and different amounts of γ subunits relative to α or β in αβγ GABAA receptors (33Boileau A.J. Baur R. Sharkey L.M. Sigel E. Czajkowski C. Neuropharmacology. 2002; 43: 695-700Crossref PubMed Scopus (103) Google Scholar), as well as different receptor phosphorylation, just to mention a few. Interestingly, phosphorylation regulates GABAA receptor potentiation by neurosteroids (34Lambert J.J. Belelli D. Peden D.R. Vardy A.W. Peters J.A. Prog. Neurobiol. (Oxf.). 2003; 71: 67-80Crossref PubMed Scopus (436) Google Scholar). In contrast to the variability of the histamine effect on heteromultimeric channels, exclusively all measured homomultimeric β channels responded to histamine, and none were found that only respond to GABA or pentobarbital. For GABAA receptors, about 10 different sites for allosteric modulators are known including neurosteroids, benzodiazepines, general anesthetics, and ethanol (25Korpi E.R. Grunder G. Luddens H. Prog. Neurobiol. (Oxf.). 2002; 67: 113-159Crossref PubMed Scopus (417) Google Scholar, 35Olsen R.W. Chang C.S. Li G. Hanchar H.J. Wallner M. Biochem. Pharmacol. 2004; 68: 1675-1684Crossref PubMed Scopus (59) Google Scholar). With the exception of neurosteroids, no endogenous modulators have been identified so far. Our findings that histamine potentiates GABA action on GABAA receptors suggests that it is an endogenous ligand for an allosteric site located on the β subunits. Therefore, our results suggest an additional function for histamine in vivo, apart from the action on metabotropic histamine and N-methyl-d -aspartic acid receptors (36Williams K. Mol. Pharmacol. 1994; 46: 531-541PubMed Google Scholar). All histaminergic neurons in the mammalian brain are found in the tuberomamillary nucleus and send axons to almost all parts of the central nervous system (10Haas H. Panula P. Nat. Rev. Neurosci. 2003; 4: 121-130Crossref PubMed Scopus (768) Google Scholar). Some of these neurons contain both GABA and histamine. In addition, histamine could diffuse out of a histaminergic synapse by a "spillout" effect as described for GABA-ergic synapses (37Rossi D.J. Hamann M. Neuron. 1998; 20: 783-795Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) and thus may act on neighboring synaptic or extrasynaptic GABAA receptors. Further, mast cells in the brain are a source for histamine. Mast cells occur in the central nervous system of many species, and up to 50% of the brain histamine is attributable to the presence of these cells. By direct gating of channels or by affecting GABAA receptor currents, histamine should modulate processes in which rapid GABA-evoked currents participate (8Brown R.E. Stevens D.R. Haas H.L. Prog. Neurobiol. (Oxf.). 2001; 63: 637-672Crossref PubMed Scopus (881) Google Scholar). We thank R. Rupprecht and D. Benke for the gift of expression plasmids, acknowledge the excellent technical assistance of H. Bartel, W. Grabowsky, and K. Kallweit for HEK293 cell measurements, thank A. Kragler for substantial scientific support, and thank O. A. Sergeeva for discussion. Download .pdf (.03 MB) Help with pdf files
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