Role of the cationic residues arginine 14 and lysine 48 in the function of the cardiotonic polypeptide anthopleurin B.
Polypeptide neurotoxins from sea anemones have been useful biological probes for sodium channel function. Cationic residues, specifically Arg-14, which is conserved in essentially all known sea anemone toxins, have been generally thought to be important determinants of their binding affinity and/or efficacy. In the present study, we have constructed site-directed mutants of the Anthopleura xanthogrammica toxin anthopleurin B (ApB) at Arg-14 and Lys-48 to characterize the roles played by these cationic residues in the biological activity of the toxin. Using a bacterial expression system developed in this laboratory, we have produced recombinant proteins having three substitutions at each of these positions. The proteins produced have been purified to homogeneity and have structures and conformational stabilities identical to wild-type ApB. We have assayed the mutants by determining their ability to enhance the veratridine-dependent uptake of sodium by both N1E-115 neuroblastoma cells and RT4-B cells, which express the cardiac/denervated skeletal muscle sodium channel. All mutants showed activities only slightly reduced from that of wild-type ApB, with the greatest reductions (4- and 6-fold) being observed for the mutants R14A and K48A, respectively. We conclude that contrary to results from chemical modification studies, Arg-14 is not essential for the biological activity of the toxin. Our data also indicate that Lys-48 plays a small but discernible role in the toxin-receptor interaction.
Khera
P K
PK
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Blumenthal
K M
KM
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D002316:Cardiotonic Agents / Q000737:chemistry; D003001:Cloning, Molecular; D036341:Intercellular Signaling Peptides and Proteins; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins; D012615:Sea Anemones; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship; D014701:Veratridine / Q000494:pharmacology
D002316:Cardiotonic Agents; D036341:Intercellular Signaling Peptides and Proteins; D009498:Neurotoxins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D014701:Veratridine; C026132:anthopleurin B; D001120:Arginine; D012964:Sodium; D008239:Lysine