title: Flanking proline residues identify the L-type Ca2+ channel binding site of calciseptine and FS2.
abstract: Calciseptine and FS2 are 60-amino acid polypeptides, isolated from venom of the black mamba (Dendroaspis polylepis polylepis), that block voltage-dependent L-type Ca2+ channels. We predicted that these polypeptides contain an identical functional site between residues 43 and 46 by searching for proline residues that mark the flanks of protein-protein interaction sites [Kini, R. M., and Evans, H. J. (1966) FEBS Lett. 385, 81-86]. The predicted Ca2+ channel binding site also occurs in closely related toxins, C10S2C2 and S4C8. Therefore, it is likely that these toxins also will block L-type Ca2+ channels. To test the proposed binding site on calciseptine and FS2, an eight-residue peptide, named L-calchin (L-type calcium channel inhibitor), was synthesized and examined for biological activity. As expected for an L-type Ca2+ channel blocker, L-calchin reduced peak systolic and developed pressure in isolated rat heart Langendorff preparations without affecting diastolic pressure or heart rate. Furthermore, L-calchin caused a voltage-independent block of L-type Ca2+ channel currents in whole-cell patch-clamped rabbit ventricular myocytes. Thus the synthetic peptide exhibits the L-type Ca2+ channel blocking properties of the parent molecules, calciseptine and FS2, but with a lower potency. These results strongly support the identification of a site in calciseptine and FS2 that is important for binding to L-type Ca2+ channels and reinforce the importance of proline brackets flanking protein-protein interaction sites.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Kini
authors.forename: R A
authors.initials: RA
authors.lastname: Caldwell
authors.forename: Q Y
authors.initials: QY
authors.lastname: Wu
authors.forename: C M
authors.initials: CM
authors.lastname: Baumgarten
authors.forename: J J
authors.initials: JJ
authors.lastname: Feher
authors.forename: H J
authors.initials: HJ
authors.lastname: Evans
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000138:chemical synthesis / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D004546:Elapid Venoms / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D012910:Snake Venoms; D016277:Ventricular Function, Left / Q000187:drug effects
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004546:Elapid Venoms; D010455:Peptides; D012910:Snake Venoms; C093284:toxin FS2; C067971:calciseptine; D011392:Proline