Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

Abstract

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2·10−9M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0·10−7M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl− current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation. A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2·10−9M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0·10−7M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl− current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation. INTRODUCTIONG-protein 1The abbreviations used are: G-proteinguanine nucleotide-binding regulatory proteinHEK293human embryonic kidney 293 cellsTMtransmembrane regionPCRpolymerase chain reactionIVcurrent voltagePLCphospholipase C.-coupled receptors form a large superfamily of membrane receptors that can be found in all species ranging from unicellular eukaryotes to mammals and that can interact with a large variety of signals (e.g. light, odorants, Ca2+, biogenic amines, glycoprotein hormones, etc.) (1Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar).Bioamines like dopamine, epinephrine, norepinephrine, and octopamine all interact with specific G-protein-coupled receptors. Dopamine is present in high amounts in both vertebrate and invertebrate species. Epinephrine and norepinephrine, on the other hand, are present predominantly in vertebrates, whereas octopamine is considered to act as a major neurotransmitter in invertebrate species only. Octopamine is often referred to as the invertebrate counterpart of norepinephrine because of the structural similarity between these neurotransmitters (they differ only in the presence of a single catecholic hydroxyl group) as well as their functional similarity (both serve an important role in stress adaptation) (2David J.-C. Coulon J.-F. Prog. Neurobiol.(Oxf.). 1985; 24: 141-185Crossref PubMed Scopus (177) Google Scholar). Consequently, the adrenergic and octopaminergic receptors share pharmacological as well as structural properties (3Evans P.D. Robb S. Neurochem. Res. 1993; 18: 869-874Crossref PubMed Scopus (93) Google Scholar, 4Hiripi L. Juhos S. Downer R.G.H. Brain Res. 1994; 633: 119-126Crossref PubMed Scopus (58) Google Scholar).The role of octopamine as a neurotransmitter has been studied particularly well in insects, where it has been shown to interact with at least four different octopamine receptor subtypes (3Evans P.D. Robb S. Neurochem. Res. 1993; 18: 869-874Crossref PubMed Scopus (93) Google Scholar). The central nervous systems of a number of snails have also been used to study octopaminergic neurotransmission. The presence of octopamine has been demonstrated in the sea slug Aplysia (5Saavedra J.M. Brownstein M.J. Carpenter D.O. Axelrod J. Science. 1974; 185: 364-365Crossref PubMed Scopus (74) Google Scholar, 6Brownstein M.J. Saavedra J.M. Axelrod J. Zeman G.H. Carpenter D.O. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4662-4665Crossref PubMed Scopus (131) Google Scholar, 7McCaman M.W. Mosnaim A.D. Wolff M.A. Noncatecholic Phenylethylamines: Octopamine and Phenylethanolamine in the Central Nervous System of Aplysia. Marcel Dekker Inc., New York1980: 193-200Google Scholar), the land snail Helix (8Walker R.J. Ralph K.L. Woodruff G.N. Experientia (Basel). 1972; 28: 1173-1174Crossref PubMed Scopus (37) Google Scholar, 9Guthrie P.B. Neuhoff V. Osborne N.N. Comp. Biochem. Physiol. 1975; 52: 109-111Crossref Scopus (7) Google Scholar), and the pond snail Lymnaea (10Elekes K. Eckert M. Rapus J. Brain Res. 1993; 16: 191-197Crossref Scopus (28) Google Scholar). In these species, the interactions of octopamine with its receptors have been described mainly at the electrophysiological level (7McCaman M.W. Mosnaim A.D. Wolff M.A. Noncatecholic Phenylethylamines: Octopamine and Phenylethanolamine in the Central Nervous System of Aplysia. Marcel Dekker Inc., New York1980: 193-200Google Scholar, 8Walker R.J. Ralph K.L. Woodruff G.N. Experientia (Basel). 1972; 28: 1173-1174Crossref PubMed Scopus (37) Google Scholar, 11Nathanson J.A. Greengard P. Science. 1973; 180: 308-310Crossref PubMed Scopus (163) Google Scholar, 12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar, 13Carpenter D.O. Gaubatz G.L. Nature. 1974; 252: 483-485Crossref PubMed Scopus (49) Google Scholar, 14Bahls F.H. Neurosci. Lett. 1990; 120: 131-133Crossref PubMed Scopus (13) Google Scholar, 15Pellmar T.C. Brain Res. 1981; 223: 448-454Crossref PubMed Scopus (14) Google Scholar). In general, application of octopamine induces a hyperpolarization of susceptible snail neurons, a process that is thought to be mediated by an increase in cAMP (2David J.-C. Coulon J.-F. Prog. Neurobiol.(Oxf.). 1985; 24: 141-185Crossref PubMed Scopus (177) Google Scholar, 11Nathanson J.A. Greengard P. Science. 1973; 180: 308-310Crossref PubMed Scopus (163) Google Scholar, 12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar), leading to an increased potassium (12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar, 13Carpenter D.O. Gaubatz G.L. Nature. 1974; 252: 483-485Crossref PubMed Scopus (49) Google Scholar, 14Bahls F.H. Neurosci. Lett. 1990; 120: 131-133Crossref PubMed Scopus (13) Google Scholar) or calcium (15Pellmar T.C. Brain Res. 1981; 223: 448-454Crossref PubMed Scopus (14) Google Scholar) conductance. In addition, a pharmacological characterization of octopamine receptors has been described for Lymnaea 2S. Juhos, L. Hiripi, M. Eckert, J. Rapus, and K. Elekes, submitted for publication.We have recently cloned and characterized a cDNA encoding an octopamine receptor expressed in the brain of Lymnaea stagnalis (16Gerhardt C.C. Bakker R.A. Piek G.J. Planta R.J. Vreugdenhil E. Leysen J.E. van Heerikhuizen H. Mol. Pharmacol. 1997; 51: 293-300Crossref PubMed Scopus (92) Google Scholar). This receptor (Lym oa1) shows moderate homology to the Drosophila tyramine/octopamine receptor (17Arakawa S. Gocayne J.D. McCombie W.R. Urquhart D.A. Hall L.M. Fraser C.M. Venter J.C. Neuron. 1990; 2: 343-354Abstract Full Text PDF Scopus (166) Google Scholar, 18Saudou F. Amlaiky N. Plassat J.-L. Borelli E. Hen R. EMBO J. 1990; 9: 3611-3617Crossref PubMed Scopus (208) Google Scholar), to the Locusta tyramine receptor (19van den Broeck J. Vulsteke V. Huybrechts R. De Loof A. J. Neurochem. 1995; 64: 2387-2395Crossref PubMed Scopus (123) Google Scholar) and to the vertebrate α-adrenergic receptors. Activation of this receptor, when expressed in human embryonic kidney (HEK293) cells, leads to elevated concentrations of both intracellular inositol phosphates and cAMP. The pharmacological profile of Lym oa1 suggests that this receptor represents a previously unknown octopamine receptor subtype.This paper describes the structure of a second octopamine receptor cDNA (Lym oa2) cloned from Lymnaea The predicted amino acid sequence of this receptor only shows limited similarity to Lym oa1, as well as to the insect tyramine receptors and the α-adrenergic receptors. Its pharmacological profile clearly differs from that of previously described receptors. When the novel receptor is stably expressed in HEK293 cells, application of octopamine does not lead to changes in the intracellular concentration of cAMP or inositol phosphates. Activation of Lym oa2 does, however, induce a long lasting opening of Cl− channels. G-protein-mediated activation of a similar Cl− current was recently described by Postma et al. (20Postma F.R. Jalink K. Hengeveld T. Bot A.G. Alblas J. de Jonge H.R. Moolenaar W.H. EMBO J. 1996; 15: 63-72Crossref PubMed Scopus (47) Google Scholar) in rat fibroblasts. Their study also indicated that none of the known second messenger system was involved in the Cl− channel activation. Nevertheless, it was suggested that the increase in chloride conductance was closely associated with the activation of phosphoinositide hydrolysis. In contrast, our results clearly indicate that this novel signaling pathway functions independently from the activation of phospholipase C (PLC). We further show that although protein kinase A and protein kinase C are most probably not involved in the signaling pathway, protein phosphorylation is important for the opening of the Cl− channel.DISCUSSIONWe have used a degenerate PCR strategy to isolate cDNAs encoding G-protein-coupled bioamine receptors that are expressed in the central nervous system of the pond snail L. stagnalis Recently, we reported the cloning and expression of a serotonin receptor (5-HT2Lym; Ref. 23Gerhardt C.C. Leysen J.E. Planta R.J. Vreugdenhil E. van Heerikhuizen H. Eur. J. Pharmacol. 1996; 311: 249-258Crossref PubMed Scopus (51) Google Scholar) and an octopamine receptor (Lym oa1; Ref. 16Gerhardt C.C. Bakker R.A. Piek G.J. Planta R.J. Vreugdenhil E. Leysen J.E. van Heerikhuizen H. Mol. Pharmacol. 1997; 51: 293-300Crossref PubMed Scopus (92) Google Scholar) using the same strategy. This paper describes the isolation of a cDNA encoding a second octopamine receptor, designated Lym oa2. The predicted amino acid sequence of Lym oa2 exhibits the highest similarity to the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and Lym oa1, and to the vertebrate α-adrenergic receptors. Although both Lym oa2 and Lym oa1 encode Lymnaea octopamine receptors, their amino acid identity is only moderate, i.e. 45% in the TM regions. An interesting difference in the sequence of both octopamine receptors can be found in TM5. Mutagenesis studies on catecholaminergic receptors have indicated that two conserved serine residues in this domain (Ser204 and Ser207 in the β-adrenergic receptor) play a crucial role in ligand binding. Supposedly, the two Ser hydroxyl groups can hydrogen bond to the catechol hydroxyl groups of the ligand (see for instance Refs. 26Strader C.D. Candelore M.R. Hill W.S. Sigal I.S. Dixon R.A.F. J. Biol. Chem. 1989; 264: 13572-13578Abstract Full Text PDF PubMed Google Scholar, 27Wang C.D. Buck M.A. Fraser C.M. Mol. Pharmacol. 1991; 40: 168-179PubMed Google Scholar, 28Cox B.A. Henningsen R.A. Spanoyannis A. Neve R.L. Neve K.A. J. Neurochem. 1992; 59: 627-635Crossref PubMed Scopus (121) Google Scholar). In octopamine receptors there is no obvious need for conservation of both serine residues in TM5 because the aromatic ring of octopamine is monohydroxylated. Indeed, in Lym oa1 only a single serine is found at the relevant position in TM5. In Lym oa2, however, both serines are present. Interestingly, the catecholamines (epinephrine, norepinephrine, dopamine) show a considerably higher affinity for Lym oa2 than for Lym oa1 (see Table I). This suggests that the serine residues in TM5 play a role in agonist binding in the Lymnaea octopamine receptors similar to their role in the vertebrate catecholamine receptors.In general, agonists exhibit higher affinities for Lym oa2 than for Lym oa1, while antagonists have higher affinities for Lym oa1 than for Lym oa2. Another interesting difference between the binding properties of both octopamine receptors is the opposite order of affinities for the isoschizomers rauwolscine and yohimbine. Lym oa1 has a higher affinity for yohimbine than for rauwolscine (pKi = 8.9 versus 7.5), while Lym oa2 has a higher affinity for rauwolscine than for yohimbine (pKi = 8.0 versus 6.2). Both the pharmacological profiles of Lym oa1 and Lym oa2 indicate a closer relationship to the α-adrenergic receptors than to the β-adrenergic receptors. In that respect it is noteworthy to mention the much higher affinity of the α1 antagonist prazosine for Lym oa1 (pKi = 7.0) than for Lym oa2 (pKi = 4.7). Whereas the pharmacological profile of Lym oa1 still shows a moderate similarity to that of the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and the α2-adrenergic receptors, the pharmacological profile of Lym oa2 clearly differs from that of Lym oa1, the tyramine receptors, the adrenergic receptors, and the insect octopamine receptor subtypes as described in tissue preparations.Stimulation of Lym oa2 did not lead to the activation of the classical signal transduction pathways mediated by adenylyl cyclase and PLC. The receptor is, however, clearly able to transduce signals since we found that HEK293 cells expressing Lym oa2 showed octopamine-induced changes in their membrane conductance. More specifically, we observed a slow but large increase in the holding current of voltage-clamped HEK293 cells upon application of octopamine. At potentials below the Cl− equilibrium potential, this response was observed as an increase in inward current, most likely caused by an efflux of Cl− ions. The process underlying the opening of the Cl− channels may involve protein phosphorylation, since the (nonselective) protein kinase inhibitor HA1004 inhibited the inward the Cl− channels involved may need to be in a to be able to in response to the octopamine the relationship of the current response to be and because no changes in inositol phosphates observed upon octopamine the Cl− channels involved are suggested to be both voltage-independent and of octopamine on also observed but not in stimulation of HEK293 cells expressing the other Lymnaea octopamine receptor, Lym oa1, in a similar Cl− In to Lym oa2, Lym oa1 has previously been shown to activate both and adenylyl the on the Cl− channel be to the on adenylyl and or represent an signaling we the of activation of two other receptors, expressed in HEK293 of these receptors, i.e. a Lymnaea serotonin receptor or an β-adrenergic receptor, to activation of and adenylyl The in of or cAMP activate protein kinase C or protein kinase of these receptors however, not show on the inward current, that protein kinase C or protein kinase A is not involved in the phosphorylation process underlying Cl− channel to be stimulation of Lym oa2 in Lymnaea also the Cl− conductance. Activation of octopamine receptors on of other snails has been described to lead to potassium (12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar, 13Carpenter D.O. Gaubatz G.L. Nature. 1974; 252: 483-485Crossref PubMed Scopus (49) Google Scholar, 14Bahls F.H. Neurosci. Lett. 1990; 120: 131-133Crossref PubMed Scopus (13) Google Scholar) and inward calcium (15Pellmar T.C. Brain Res. 1981; 223: 448-454Crossref PubMed Scopus (14) Google Scholar). studies on Aplysia octopamine receptors Proc. Natl. Acad. Sci. U. S. A. 1974; 71: PubMed Scopus Google Scholar) have shown that stimulation of with octopamine induces the specific phosphorylation of a In this however, it was suggested that the process involve protein kinase since the intracellular concentration of cAMP a similar it was shown that activation of the G-protein-coupled acid receptor present on leads to a long lasting of these cells to an efflux of chloride (20Postma F.R. Jalink K. Hengeveld T. Bot A.G. Alblas J. de Jonge H.R. Moolenaar W.H. EMBO J. 1996; 15: 63-72Crossref PubMed Scopus (47) Google Scholar). was found in our the signal transduction pathway for opening the chloride channel was suggested to be of known second The opening of this chloride channel also be by activation of the receptor, the receptor, and the A these receptors well as the acid receptor) all to it was suggested that the signaling pathway leading to the increased Cl− conductance is closely associated with phosphoinositide (20Postma F.R. Jalink K. Hengeveld T. Bot A.G. Alblas J. de Jonge H.R. Moolenaar W.H. EMBO J. 1996; 15: 63-72Crossref PubMed Scopus (47) Google that the chloride channel in HEK293 cells that we have found to be is similar to the channel described by Postma et al. (20Postma F.R. Jalink K. Hengeveld T. Bot A.G. Alblas J. de Jonge H.R. Moolenaar W.H. EMBO J. 1996; 15: 63-72Crossref PubMed Scopus (47) Google Scholar). however, the that phosphoinositide is important in the signaling pathway leading from the receptor to the opening of the chloride since stimulation of Lym oa2 does not in in intracellular concentrations of inositol phosphates. activation of a serotonin receptor that has been shown to be to the activation of does not the Cl− The G-protein-mediated activation of the chloride channel described by Postma et al. in (20Postma F.R. Jalink K. Hengeveld T. Bot A.G. Alblas J. de Jonge H.R. Moolenaar W.H. EMBO J. 1996; 15: 63-72Crossref PubMed Scopus (47) Google Scholar) and by in HEK293 cells an as unknown signaling we show that protein phosphorylation is important in this pathway but that protein kinase A and protein kinase C are not The of the relevant kinase and the further of the signal transduction pathway be the of INTRODUCTIONG-protein 1The abbreviations used are: G-proteinguanine nucleotide-binding regulatory proteinHEK293human embryonic kidney 293 cellsTMtransmembrane regionPCRpolymerase chain reactionIVcurrent voltagePLCphospholipase C.-coupled receptors form a large superfamily of membrane receptors that can be found in all species ranging from unicellular eukaryotes to mammals and that can interact with a large variety of signals (e.g. light, odorants, Ca2+, biogenic amines, glycoprotein hormones, etc.) (1Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar).Bioamines like dopamine, epinephrine, norepinephrine, and octopamine all interact with specific G-protein-coupled receptors. Dopamine is present in high amounts in both vertebrate and invertebrate species. Epinephrine and norepinephrine, on the other hand, are present predominantly in vertebrates, whereas octopamine is considered to act as a major neurotransmitter in invertebrate species only. Octopamine is often referred to as the invertebrate counterpart of norepinephrine because of the structural similarity between these neurotransmitters (they differ only in the presence of a single catecholic hydroxyl group) as well as their functional similarity (both serve an important role in stress adaptation) (2David J.-C. Coulon J.-F. Prog. Neurobiol.(Oxf.). 1985; 24: 141-185Crossref PubMed Scopus (177) Google Scholar). Consequently, the adrenergic and octopaminergic receptors share pharmacological as well as structural properties (3Evans P.D. Robb S. Neurochem. Res. 1993; 18: 869-874Crossref PubMed Scopus (93) Google Scholar, 4Hiripi L. Juhos S. Downer R.G.H. Brain Res. 1994; 633: 119-126Crossref PubMed Scopus (58) Google Scholar).The role of octopamine as a neurotransmitter has been studied particularly well in insects, where it has been shown to interact with at least four different octopamine receptor subtypes (3Evans P.D. Robb S. Neurochem. Res. 1993; 18: 869-874Crossref PubMed Scopus (93) Google Scholar). The central nervous systems of a number of snails have also been used to study octopaminergic neurotransmission. The presence of octopamine has been demonstrated in the sea slug Aplysia (5Saavedra J.M. Brownstein M.J. Carpenter D.O. Axelrod J. Science. 1974; 185: 364-365Crossref PubMed Scopus (74) Google Scholar, 6Brownstein M.J. Saavedra J.M. Axelrod J. Zeman G.H. Carpenter D.O. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4662-4665Crossref PubMed Scopus (131) Google Scholar, 7McCaman M.W. Mosnaim A.D. Wolff M.A. Noncatecholic Phenylethylamines: Octopamine and Phenylethanolamine in the Central Nervous System of Aplysia. Marcel Dekker Inc., New York1980: 193-200Google Scholar), the land snail Helix (8Walker R.J. Ralph K.L. Woodruff G.N. Experientia (Basel). 1972; 28: 1173-1174Crossref PubMed Scopus (37) Google Scholar, 9Guthrie P.B. Neuhoff V. Osborne N.N. Comp. Biochem. Physiol. 1975; 52: 109-111Crossref Scopus (7) Google Scholar), and the pond snail Lymnaea (10Elekes K. Eckert M. Rapus J. Brain Res. 1993; 16: 191-197Crossref Scopus (28) Google Scholar). In these species, the interactions of octopamine with its receptors have been described mainly at the electrophysiological level (7McCaman M.W. Mosnaim A.D. Wolff M.A. Noncatecholic Phenylethylamines: Octopamine and Phenylethanolamine in the Central Nervous System of Aplysia. Marcel Dekker Inc., New York1980: 193-200Google Scholar, 8Walker R.J. Ralph K.L. Woodruff G.N. Experientia (Basel). 1972; 28: 1173-1174Crossref PubMed Scopus (37) Google Scholar, 11Nathanson J.A. Greengard P. Science. 1973; 180: 308-310Crossref PubMed Scopus (163) Google Scholar, 12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar, 13Carpenter D.O. Gaubatz G.L. Nature. 1974; 252: 483-485Crossref PubMed Scopus (49) Google Scholar, 14Bahls F.H. Neurosci. Lett. 1990; 120: 131-133Crossref PubMed Scopus (13) Google Scholar, 15Pellmar T.C. Brain Res. 1981; 223: 448-454Crossref PubMed Scopus (14) Google Scholar). In general, application of octopamine induces a hyperpolarization of susceptible snail neurons, a process that is thought to be mediated by an increase in cAMP (2David J.-C. Coulon J.-F. Prog. Neurobiol.(Oxf.). 1985; 24: 141-185Crossref PubMed Scopus (177) Google Scholar, 11Nathanson J.A. Greengard P. Science. 1973; 180: 308-310Crossref PubMed Scopus (163) Google Scholar, 12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar), leading to an increased potassium (12Cox R.T.L. Walker R.J. Comp. Biochem. Physiol. 1988; 91: 541-547Google Scholar, 13Carpenter D.O. Gaubatz G.L. Nature. 1974; 252: 483-485Crossref PubMed Scopus (49) Google Scholar, 14Bahls F.H. Neurosci. Lett. 1990; 120: 131-133Crossref PubMed Scopus (13) Google Scholar) or calcium (15Pellmar T.C. Brain Res. 1981; 223: 448-454Crossref PubMed Scopus (14) Google Scholar) conductance. In addition, a pharmacological characterization of octopamine receptors has been described for Lymnaea 2S. Juhos, L. Hiripi, M. Eckert, J. Rapus, and K. Elekes, submitted for publication.We have recently cloned and characterized a cDNA encoding an octopamine receptor expressed in the brain of Lymnaea stagnalis (16Gerhardt C.C. Bakker R.A. Piek G.J. Planta R.J. Vreugdenhil E. Leysen J.E. van Heerikhuizen H. Mol. Pharmacol. 1997; 51: 293-300Crossref PubMed Scopus (92) Google Scholar). This receptor (Lym oa1) shows moderate homology to the Drosophila tyramine/octopamine receptor (17Arakawa S. Gocayne J.D. McCombie W.R. Urquhart D.A. Hall L.M. Fraser C.M. Venter J.C. Neuron. 1990; 2: 343-354Abstract Full Text PDF Scopus (166) Google Scholar, 18Saudou F. Amlaiky N. Plassat J.-L. Borelli E. Hen R. EMBO J. 1990; 9: 3611-3617Crossref PubMed Scopus (208) Google Scholar), to the Locusta tyramine receptor (19van den Broeck J. Vulsteke V. Huybrechts R. De Loof A. J. Neurochem. 1995; 64: 2387-2395Crossref PubMed Scopus (123) Google Scholar) and to the vertebrate α-adrenergic receptors. Activation of this receptor, when expressed in human embryonic kidney (HEK293) cells, leads to elevated concentrations of both intracellular inositol phosphates and cAMP. The pharmacological profile of Lym oa1 suggests that this receptor represents a previously unknown octopamine receptor subtype.This paper describes the structure of a second octopamine receptor cDNA (Lym oa2) cloned from Lymnaea The predicted amino acid sequence of this receptor only shows limited similarity to Lym oa1, as well as to the insect tyramine receptors and the α-adrenergic receptors. Its pharmacological profile clearly differs from that of previously described receptors. When the novel receptor is stably expressed in HEK293 cells, application of octopamine does not lead to changes in the intracellular concentration of cAMP or inositol phosphates. Activation of Lym oa2 does, however, induce a long lasting opening of Cl− channels. G-protein-mediated activation of a similar Cl− current was recently described by Postma et al. (20Postma F.R. Jalink K. Hengeveld T. Bot A.G. Alblas J. de Jonge H.R. Moolenaar W.H. EMBO J. 1996; 15: 63-72Crossref PubMed Scopus (47) Google Scholar) in rat fibroblasts. Their study also indicated that none of the known second messenger system was involved in the Cl− channel activation. Nevertheless, it was suggested that the increase in chloride conductance was closely associated with the activation of phosphoinositide hydrolysis. In contrast, our results clearly indicate that this novel signaling pathway functions independently from the activation of phospholipase C (PLC). We further show that although protein kinase A and protein kinase C are most probably not involved in the signaling pathway, protein phosphorylation is important for the opening of the Cl−