Functional Hypervariability and Gene Diversity of Cardioactive Neuropeptides

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions. Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions. IntroductionPhysiological processes such as neurotransmission, metabolism, and cardiac function are regulated by peptides. The structural diversity of these regulatory peptidic scaffolds varies from small linear peptides such as enkephalins to more elaborate disulfide-constrained peptide hormones such as insulin. Related to these peptides, endogenous-like toxins are utilized exogenously by venomous animals as evident by their frequent discovery in venom (1Pimenta A.M. De Lima M.E. J. Pept. Sci. 2005; 11: 670-676Crossref PubMed Scopus (66) Google Scholar). For example, sarafotoxins are potent mammalian vasoconstrictors found in snake venom (2Ducancel F. Toxicon. 2002; 40: 1541-1545Crossref PubMed Scopus (29) Google Scholar), and are members of the endogenous hormone endothelin family (∼60% sequence homology). Sequence differences between the endogenous and their exogenous counterparts can account for their change of activity. Post-translational modifications can transform endogenous peptides for exogenous use as in the cases of the epimerization of C-type natriuretic peptide in platypus venom (3Torres A.M. Menz I. Alewood P.F. Bansal P. Lahnstein J. Gallagher C.H. Kuchel P.W. FEBS Lett. 2002; 524: 172-176Crossref PubMed Scopus (73) Google Scholar), glycosylation of neurotensin-related peptides (4Craig A.G. Norberg T. Griffin D. Hoeger C. Akhtar M. Schmidt K. Low W. Dykert J. Richelson E. Navarro V. Mazella J. Watkins M. Hillyard D. Imperial J. Cruz L.J. Olivera B.M. J. Biol. Chem. 1999; 274: 13752-13759Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), and carboxylation of vasopressin/oxytocin-related peptides in cone snail venom (5Möller C. Marí F. Biochem. J. 2007; 404: 413-419Crossref PubMed Scopus (28) Google Scholar).Among venomous animals, cone snails are exquisitely adapted to immobilize their prey (fish, worms, and mollusks), because their venom is composed of a complex multitargeting concoction of highly modified peptides (conopeptides) (6Franco A. Pisarewicz K. Moller C. Mora D. Fields G.B. Marì F. Prog. Mol. Subcell. Biol. 2006; 43: 83-103Crossref PubMed Scopus (41) Google Scholar, 7Terlau H. Olivera B.M. Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (800) Google Scholar). Conopeptides are typically expressed by exogenes with characteristic signal sequences followed by a pre-pro region and a hypervariable toxin region (8Olivera B.M. J. Biol. Chem. 2006; 281: 31173-31177Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Additionally, cone snail venom includes several families of endogenous-like peptides, such as the conopressins (vasopressin/oxytocin) (5Möller C. Marí F. Biochem. J. 2007; 404: 413-419Crossref PubMed Scopus (28) Google Scholar, 9Dutertre S. Croker D. Daly N.L. Andersson A. Muttenthaler M. Lumsden N.G. Craik D.J. Alewood P.F. Guillon G. Lewis R.J. J. Biol. Chem. 2008; 283: 7100-7108Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) contulakins (neurotensin) (4Craig A.G. Norberg T. Griffin D. Hoeger C. Akhtar M. Schmidt K. Low W. Dykert J. Richelson E. Navarro V. Mazella J. Watkins M. Hillyard D. Imperial J. Cruz L.J. Olivera B.M. J. Biol. Chem. 1999; 274: 13752-13759Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), conophysin (neurophysin) (10Lirazan M. Jimenez E.C. Grey Craig A. Olivera B.M. Cruz L.J. Toxicon. 2002; 40: 901-908Crossref PubMed Scopus (15) Google Scholar), conorfamides (RFamides) (11Aguilar M.B. Luna-Ramírez K.S. Echeverría D. Falcón A. Olivera B.M. Heimer de la Cotera E.P. Maillo M. Peptides. 2008; 29: 186-195Crossref PubMed Scopus (30) Google Scholar, 12Maillo M. Aguilar M.B. Lopéz-Vera E. Craig A.G. Bulaj G. Olivera B.M. Heimer de la Cotera E.P. Toxicon. 2002; 40: 401-407Crossref PubMed Scopus (51) Google Scholar), conomaps (myoactive tetradecapeptide) (13Dutertre S. Lumsden N.G. Alewood P.F. Lewis R.J. FEBS Lett. 2006; 580: 3860-3866Crossref PubMed Scopus (32) Google Scholar), and conkunitzins (Kunitz-type proteins) (14Bayrhuber M. Vijayan V. Ferber M. Graf R. Korukottu J. Imperial J. Garrett J.E. Olivera B.M. Terlau H. Zweckstetter M. Becker S. J. Biol. Chem. 2005; 280: 23766-23770Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Here, we report the discovery and characterization of a new class of endogenous-like conopeptides in the venom duct of Conus villepinii that belongs to the superfamily of cardioactive peptides (CAPs). 2The abbreviations used are: CAPcardioactive peptideCCAPcrustacean cardioactive peptideRACErapid amplification of cDNA endsESTexpressed sequence tagIAMiodoacetamideSPPSsolid phase peptide synthesisDMSOdimethyl sulfoxideMABPmean arterial blood pressureHRheart rate[Ca2+]iintracellular calciumLTCCL-type calcium channelRyRryanodine receptorCICRcalcium-induced calcium releaseSRsarcoplasmic reticulumβ1-AR and β2-ARβ1- and β2-adrenergic receptorHCN1 and HCN2potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1 and 2Kv1.4voltage-gated potassium channel 1.4Nav1.5voltage-gated sodium channel 1.5hERGhuman ether-à-go-go-related geneV1aV1b, V2, vasopressin receptor type 1a, 1b, and 2GPCRG protein-coupled receptorsPAMPAparallel artificial membrane permeability assayTICtotal ion current detection.The first CAP was isolated from the shore crab Carcinus maenas (15Stangier J. Hilbich C. Beyreuther K. Keller R. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 575-579Crossref PubMed Scopus (198) Google Scholar) and described as crustacean cardioactive peptide (CCAP). Subsequently, this peptide was also isolated from several other arthropod species (16Furuya K. Liao S. Reynolds S.E. Ota R.B. Hackett M. Schooley D.A. Biol. Chem. Hoppe-Seyler. 1993; 374: 1065-1074Crossref PubMed Scopus (35) Google Scholar, 17Lehman H.K. Murgiuc C.M. Miller T.A. Lee T.D. Hildebrand J.G. Peptides. 1993; 14: 735-741Crossref PubMed Scopus (75) Google Scholar, 18Stangier J. Keller R. Gen. Comp. Endocrinol. 1989; 74: 271-272Google Scholar). In mollusks, two related CCAP-like peptides, M-CCAP1 and M-CCAP2, were isolated from the pulmonate snail Helix pomatia (19Minakata H. Ikeda T. Fujita T. Kiss T. Hiripi L. Muneoka Y. Nomoto K. Yanaihara N. Peptide Chemistry. ESCOM, Leiden1992Google Scholar, 20Muneoka Y. Takahashi T. Kobayashi M. Ikeda T. Minataka H. Nomoto K. Davey K.G. Peter R.E. Tobe S.S. Perspectives in Comparative Endocrinology. National Research Council of Canada, Ottawa, CA1994Google Scholar). Sequences encoding CCAP-like peptides have also been found in the genome of the sea snail Lottia gigantea and in ESTs from the California sea hare Aplysia californica and the Pacific oyster Crassostrea gigas (21Veenstra J.A. Gen. Comp. Endocrinol. 2010; 167: 86-103Crossref PubMed Scopus (169) Google Scholar) (Table 1). CCAP was originally described as a cardioaccelerator in arthropods (22Tublitz N.J. Evans P.D. J. Neurosci. 1986; 6: 2451-2456Crossref PubMed Google Scholar, 23Dulcis D. Davis N.T. Hildebrand J.G. J. Comp. Phys. A. 2001; 187: 837-849Crossref PubMed Scopus (40) Google Scholar, 24Dulcis D. Levine R.B. Ewer J. J. Neurobiol. 2005; 64: 259-274Crossref PubMed Scopus (77) Google Scholar, 25Fort T.J. García-Crescioni K. Agricola H.J. Brezina V. Miller M.W. J. Neurophysiol. 2007; 97: PubMed Scopus (41) Google Scholar, R. S. E. E. Peptides. 1999; PubMed Scopus Google Scholar, Physiol. 1993; Google Scholar). studies found that CCAP to be multifunctional that is in several physiological processes such as and A. C. Peptides. 2002; PubMed Scopus Google Scholar, A. Peptides. 2002; PubMed Scopus Google Scholar), as as the of hormone from the D. P. J. J. L. De A. Endocrinology. PubMed Scopus Google Scholar), the cardiac E. J. Biol. 2007; PubMed Scopus Google Scholar), and and activity T. H. H. M. Endocrinology. 2004; PubMed Scopus Google Scholar, T. H. M. Peptides. 2006; PubMed Scopus Google Scholar). CCAP is also in and processes Ewer J. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. D. J. Comp. 2002; PubMed Scopus Google Scholar, J. Neurosci. PubMed Google Scholar, H. J. Biol. PubMed Google Scholar, 2004; PubMed Scopus Google of and predicted conoCAPs and CCAP-like Conus (conoCAP-a, have to sequence with the heart which is the opposite to the of We found cardiac of CCAP in we that conoCAP-a decreases the heart and blood effects with the systolic calcium and in single ventricular myocytes from rat precursor of the conoCAPs encodes for conoCAPs in an analogous to the CCAP-like precursor predicted from the genome of L. gigantea (21Veenstra J.A. Gen. Comp. Endocrinol. 2010; 167: 86-103Crossref PubMed Scopus (169) Google Scholar). In to a new class of conopeptides with discovery of conoCAPs suggests the of an of peptides the venom duct of an peptides to the CCAP superfamily in venom is a new class of conopeptides in the of cone The conoCAPs sequences are analogous to other molluscan CCAP-like peptides (Table in and as as the that the with conoCAPs are is to be for activity of hormones and in R.E. Endocrinology. PubMed Scopus Google the of CCAP-like peptides in peptides can be in to their amino (Table 1). The can the in peptide A. D. A. 1986; PubMed Scopus Google and CCAP-like peptides, which CCAP, are predicted to be the is as by the of activity for the of conoCAP-a The CCAP-like was not found in the venom duct of C. villepinii in the of H. pomatia (19Minakata H. Ikeda T. Fujita T. Kiss T. Hiripi L. Muneoka Y. Nomoto K. Yanaihara N. Peptide Chemistry. ESCOM, Leiden1992Google Scholar, 20Muneoka Y. Takahashi T. Kobayashi M. Ikeda T. Minataka H. Nomoto K. Davey K.G. Peter R.E. Tobe S.S. Perspectives in Comparative Endocrinology. National Research Council of Canada, Ottawa, CA1994Google Scholar). The CCAP-like is the was not found in the venom duct of C. villepinii was found in the of H. pomatia (19Minakata H. Ikeda T. Fujita T. Kiss T. Hiripi L. Muneoka Y. Nomoto K. Yanaihara N. Peptide Chemistry. ESCOM, Leiden1992Google Scholar). The of such peptides in venom might be to of with conoCAP-a their in other of the snail for the precursor of conoCAP-a that sequences encoding two other CCAP-like peptides: conoCAP-b and conoCAP-c. the conoCAPs precursor organization is analogous to the predicted CCAP-like of L. A. and C. gigas (21Veenstra J.A. Gen. Comp. Endocrinol. 2010; 167: 86-103Crossref PubMed Scopus (169) Google Scholar) discovery of conoCAP-a and its precursor the diversification of the gene superfamily for peptides. In Lottia and Aplysia the precursor encodes such peptides two in linear peptides can be predicted from the of conoCAPs and the CCAP-like peptides from Aplysia molluscan CCAP-like peptides were isolated from the of H. pomatia (Table 1). their precursor not been we the first of a CCAP-like precursor as as its peptide in the venom of a cone snail an of the functional diversity of these of conoCAPs the cDNA of C. molluscan CCAP-like peptides the genome of L. ESTs of A. californica and C. (21Veenstra J.A. Gen. Comp. Endocrinol. 2010; 167: 86-103Crossref PubMed Scopus (169) Google Scholar), the cDNA of C. N. Y. H. J. Biol. 2006; PubMed Scopus Google Scholar) as CCAP the cDNA of P. T. H. M. Peptides. 2006; PubMed Scopus Google described a structure of signal and peptide (8Olivera B.M. J. Biol. Chem. 2006; 281: 31173-31177Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). the precursor is for peptides expressed in the cone snail venom this precursor the molluscan CCAP-like in an organization of gene encoding a of This within the gene to a mechanism for biological diversification without a for gene In the of other peptides family are expressed in single venom to B.M. Hillyard M. D. Full Text PDF PubMed Scopus Google Scholar). The of the conoCAPs encoding sequences in a single precursor might be a mechanism to such in while a single peptide CCAP-like in are also of CCAP-like peptides, sequences differ between that encoding for CCAP-like peptides an to their This gene organization is also in enkephalins and of the in several peptides sequences within a in to adaptive and mutations and related peptides Biol. J. Scopus Google a of functional in and R. S. E. E. Peptides. 1999; PubMed Scopus Google Scholar), we found that CCAP the in Drosophila we found that conoCAPs elicit a the in by these effects were also in mammalian cardiac as conoCAP-a decreased the and of rats by and (Table In the injection of CCAP did not elicit any cardiac in CCAP-like peptides are found in to this their effects mammalian not been was that small variations in the sequence of CCAP, such as the found in such physiological in of conoCAP-a rat cardiac myocytes decreased systolic and activity. The systolic in cardiac is by via the channel which from the calcium via by a calcium 2002; PubMed Scopus Google Scholar). decreased systolic calcium from effects of conoCAP-a we this to that conoCAP-a as a of the conoCAP-a did not affect other membrane and membrane in the and did a of and decreased the systolic calcium in a This with the of effects the membrane that conoCAP-a to an intracellular that is for intracellular calcium a be the This in by the that conoCAP-a membrane CCAP not the effects also is a of a in Drosophila G. F. S. M. Biochem. PubMed Scopus (51) Google Scholar). This receptor not with other Drosophila of CCAP to this is predicted to the of the receptor K. M. Pept. Sci. 2005; Scholar). the sequence of conoCAPs with CCAP, the by conoCAPs suggests that their receptor is from the In this is that the cardiac of and might and that this affect the cardiac by these peptides Comp. Biochem. Physiol. A. Mol. Physiol. 2001; PubMed Scopus Google cardiac effects of conoCAP-a and CCAP that molluscan CCAP-like peptides might also have functions that differ from those of their arthropod counterparts. the of several peptide sequences in the precursor is of functional in the sequences differences can have functional This in a for functional hypervariability of these peptides cardiac activity is by single amino cases have been for other related peptides such as and vasopressin in which in amino acids and their to the and vasopressin and type and their physiological effects S. Croker D. Daly N.L. Andersson A. Muttenthaler M. Lumsden N.G. Craik D.J. Alewood P.F. Guillon G. Lewis R.J. J. Biol. Chem. 2008; 283: 7100-7108Abstract Full Text Full Text PDF PubMed Scopus (71) Google functional studies the effects of conoCAPs in cardiac the of these peptides in venom as toxins be the within the snail from which their precursor was In this is also that conoCAP-a was in C. venom in to its and peptide sequence C. villepinii is a cone snail species that might use conoCAPs for a receptor in their prey and their discovery a new class of conopeptides related to the superfamily and for the first the of such peptides in the venom of an The diversity of of this peptide superfamily emphasizes the significance of their plasticity to mutations as an adaptive in terms of structure, cellular site of and physiological functions. This suggests that their functions not by the peptide sequence also by the in which are IntroductionPhysiological processes such as neurotransmission, metabolism, and cardiac function are regulated by peptides. The structural diversity of these regulatory peptidic scaffolds varies from small linear peptides such as enkephalins to more elaborate disulfide-constrained peptide hormones such as insulin. Related to these peptides, endogenous-like toxins are utilized exogenously by venomous animals as evident by their frequent discovery in venom (1Pimenta A.M. De Lima M.E. J. Pept. Sci. 2005; 11: 670-676Crossref PubMed Scopus (66) Google Scholar). For example, sarafotoxins are potent mammalian vasoconstrictors found in snake venom (2Ducancel F. Toxicon. 2002; 40: 1541-1545Crossref PubMed Scopus (29) Google Scholar), and are members of the endogenous hormone endothelin family (∼60% sequence homology). Sequence differences between the endogenous and their exogenous counterparts can account for their change of activity. Post-translational modifications can transform endogenous peptides for exogenous use as in the cases of the epimerization of C-type natriuretic peptide in platypus venom (3Torres A.M. Menz I. Alewood P.F. Bansal P. Lahnstein J. Gallagher C.H. Kuchel P.W. FEBS Lett. 2002; 524: 172-176Crossref PubMed Scopus (73) Google Scholar), glycosylation of neurotensin-related peptides (4Craig A.G. Norberg T. Griffin D. Hoeger C. Akhtar M. Schmidt K. Low W. Dykert J. Richelson E. Navarro V. Mazella J. Watkins M. Hillyard D. Imperial J. Cruz L.J. Olivera B.M. J. Biol. Chem. 1999; 274: 13752-13759Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), and carboxylation of vasopressin/oxytocin-related peptides in cone snail venom (5Möller C. Marí F. Biochem. J. 2007; 404: 413-419Crossref PubMed Scopus (28) Google Scholar).Among venomous animals, cone snails are exquisitely adapted to immobilize their prey (fish, worms, and mollusks), because their venom is composed of a complex multitargeting concoction of highly modified peptides (conopeptides) (6Franco A. Pisarewicz K. Moller C. Mora D. Fields G.B. Marì F. Prog. Mol. Subcell. Biol. 2006; 43: 83-103Crossref PubMed Scopus (41) Google Scholar, 7Terlau H. Olivera B.M. Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (800) Google Scholar). Conopeptides are typically expressed by exogenes with characteristic signal sequences followed by a pre-pro region and a hypervariable toxin region (8Olivera B.M. J. Biol. Chem. 2006; 281: 31173-31177Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Additionally, cone snail venom includes several families of endogenous-like peptides, such as the conopressins (vasopressin/oxytocin) (5Möller C. Marí F. Biochem. J. 2007; 404: 413-419Crossref PubMed Scopus (28) Google Scholar, 9Dutertre S. Croker D. Daly N.L. Andersson A. Muttenthaler M. Lumsden N.G. Craik D.J. Alewood P.F. Guillon G. Lewis R.J. J. Biol. Chem. 2008; 283: 7100-7108Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) contulakins (neurotensin) (4Craig A.G. Norberg T. Griffin D. Hoeger C. Akhtar M. Schmidt K. Low W. Dykert J. Richelson E. Navarro V. Mazella J. Watkins M. Hillyard D. Imperial J. Cruz L.J. Olivera B.M. J. Biol. Chem. 1999; 274: 13752-13759Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), conophysin (neurophysin) (10Lirazan M. Jimenez E.C. Grey Craig A. Olivera B.M. Cruz L.J. Toxicon. 2002; 40: 901-908Crossref PubMed Scopus (15) Google Scholar), conorfamides (RFamides) (11Aguilar M.B. Luna-Ramírez K.S. Echeverría D. Falcón A. Olivera B.M. Heimer de la Cotera E.P. Maillo M. Peptides. 2008; 29: 186-195Crossref PubMed Scopus (30) Google Scholar, 12Maillo M. Aguilar M.B. Lopéz-Vera E. Craig A.G. Bulaj G. Olivera B.M. Heimer de la Cotera E.P. Toxicon. 2002; 40: 401-407Crossref PubMed Scopus (51) Google Scholar), conomaps (myoactive tetradecapeptide) (13Dutertre S. Lumsden N.G. Alewood P.F. Lewis R.J. FEBS Lett. 2006; 580: 3860-3866Crossref PubMed Scopus (32) Google Scholar), and conkunitzins (Kunitz-type proteins) (14Bayrhuber M. Vijayan V. Ferber M. Graf R. Korukottu J. Imperial J. Garrett J.E. Olivera B.M. Terlau H. Zweckstetter M. Becker S. J. Biol. Chem. 2005; 280: 23766-23770Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Here, we report the discovery and characterization of a new class of endogenous-like conopeptides in the venom duct of Conus villepinii that belongs to the superfamily of cardioactive peptides (CAPs). 2The abbreviations used are: CAPcardioactive peptideCCAPcrustacean cardioactive peptideRACErapid amplification of cDNA endsESTexpressed sequence tagIAMiodoacetamideSPPSsolid phase peptide synthesisDMSOdimethyl sulfoxideMABPmean arterial blood pressureHRheart rate[Ca2+]iintracellular calciumLTCCL-type calcium channelRyRryanodine receptorCICRcalcium-induced calcium releaseSRsarcoplasmic reticulumβ1-AR and β2-ARβ1- and β2-adrenergic receptorHCN1 and HCN2potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1 and 2Kv1.4voltage-gated potassium channel 1.4Nav1.5voltage-gated sodium channel 1.5hERGhuman ether-à-go-go-related geneV1aV1b, V2, vasopressin receptor type 1a, 1b, and 2GPCRG protein-coupled receptorsPAMPAparallel artificial membrane permeability assayTICtotal ion current detection.The first CAP was isolated from the shore crab Carcinus maenas (15Stangier J. Hilbich C. Beyreuther K. Keller R. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 575-579Crossref PubMed Scopus (198) Google Scholar) and described as crustacean cardioactive peptide (CCAP). Subsequently, this peptide was also isolated from several other arthropod species (16Furuya K. Liao S. Reynolds S.E. Ota R.B. Hackett M. Schooley D.A. Biol. Chem. Hoppe-Seyler. 1993; 374: 1065-1074Crossref PubMed Scopus (35) Google Scholar, 17Lehman H.K. Murgiuc C.M. Miller T.A. Lee T.D. Hildebrand J.G. Peptides. 1993; 14: 735-741Crossref PubMed Scopus (75) Google Scholar, 18Stangier J. Keller R. Gen. Comp. Endocrinol. 1989; 74: 271-272Google Scholar). In mollusks, two related CCAP-like peptides, M-CCAP1 and M-CCAP2, were isolated from the pulmonate snail Helix pomatia (19Minakata H. Ikeda T. Fujita T. Kiss T. Hiripi L. Muneoka Y. Nomoto K. Yanaihara N. Peptide Chemistry. ESCOM, Leiden1992Google Scholar, 20Muneoka Y. Takahashi T. Kobayashi M. Ikeda T. Minataka H. Nomoto K. Davey K.G. Peter R.E. Tobe S.S. Perspectives in Comparative Endocrinology. National Research Council of Canada, Ottawa, CA1994Google Scholar). Sequences encoding CCAP-like peptides have also been found in the genome of the sea snail Lottia gigantea and in ESTs from the California sea hare Aplysia californica and the Pacific oyster Crassostrea gigas (21Veenstra J.A. Gen. Comp. Endocrinol. 2010; 167: 86-103Crossref PubMed Scopus (169) Google Scholar) (Table 1). CCAP was originally described as a cardioaccelerator in arthropods (22Tublitz N.J. Evans P.D. J. Neurosci. 1986; 6: 2451-2456Crossref PubMed Google Scholar, 23Dulcis D. Davis N.T. Hildebrand J.G. J. Comp. Phys. A. 2001; 187: 837-849Crossref PubMed Scopus (40) Google Scholar, 24Dulcis D. Levine R.B. Ewer J. J. Neurobiol. 2005; 64: 259-274Crossref PubMed Scopus (77) Google Scholar, 25Fort T.J. García-Crescioni K. Agricola H.J. Brezina V. Miller M.W. J. Neurophysiol. 2007; 97: PubMed Scopus (41) Google Scholar, R. S. E. E. Peptides. 1999; PubMed Scopus Google Scholar, Physiol. 1993; Google Scholar). studies found that CCAP to be multifunctional that is in several physiological processes such as and A. C. Peptides. 2002; PubMed Scopus Google Scholar, A. Peptides. 2002; PubMed Scopus Google Scholar), as as the of hormone from the D. P. J. J. L. De A. Endocrinology. PubMed Scopus Google Scholar), the cardiac E. J. Biol. 2007; PubMed Scopus Google Scholar), and and activity T. H. H. M. Endocrinology. 2004; PubMed Scopus Google Scholar, T. H. M. Peptides. 2006; PubMed Scopus Google Scholar). CCAP is also in and processes Ewer J. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. D. J. Comp. 2002; PubMed Scopus Google Scholar, J. Neurosci. PubMed Google Scholar, H. J. Biol. PubMed Google Scholar, 2004; PubMed Scopus Google of and predicted conoCAPs and CCAP-like Conus (conoCAP-a, have to sequence with the heart which is the opposite to the of We found cardiac of CCAP in we that conoCAP-a decreases the heart and blood effects with the systolic calcium and in single ventricular myocytes from rat precursor of the conoCAPs encodes for conoCAPs in an analogous to the CCAP-like precursor predicted from the genome of L. gigantea (21Veenstra J.A. Gen. Comp. Endocrinol. 2010; 167: 86-103Crossref PubMed Scopus (169) Google Scholar). In to a new class of conopeptides with discovery of conoCAPs suggests the of an of peptides the venom duct of an

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