title: Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type Cav channels.
abstract: Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address: j.klint@imb.uq.edu.au.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Klint
authors.affiliation: Health Innovations Research Institute, RMIT University, Bundoora, VIC 3083, Australia. Electronic address: geza.berecki@rmit.edu.au.
authors.forename: Géza
authors.initials: G
authors.lastname: Berecki
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address: t.durek@imb.uq.edu.au.
authors.forename: Thomas
authors.initials: T
authors.lastname: Durek
authors.affiliation: Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address: m.mobli@uq.edu.au.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mobli
authors.affiliation: Health Innovations Research Institute, RMIT University, Bundoora, VIC 3083, Australia. Electronic address: knapplowitsch@yahoo.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Knapp
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address: g.king2@uq.edu.au.
authors.forename: Glenn F
authors.initials: GF
authors.lastname: King
authors.affiliation: Health Innovations Research Institute, RMIT University, Bundoora, VIC 3083, Australia. Electronic address: david.adams@rmit.edu.au.
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address: P.Alewood@imb.uq.edu.au.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia. Electronic address: l.rash@uq.edu.au.
authors.forename: Lachlan D
authors.initials: LD
authors.lastname: Rash
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002121:Calcium Channel Blockers / Q000008:administration & dosage / Q000138:chemical synthesis / Q000302:isolation & purification; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D013111:Spider Venoms / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000302:isolation & purification*; D013112:Spiders; D014982:Xenopus laevis
chemical_list: D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D010446:Peptide Fragments; D013111:Spider Venoms
keywords: Citharischius crawshayi; Peptide isomer; Tarantula; Venom-peptide; Voltage-gated calcium channel; Voltage-gated sodium channel