Synthesis, bioactivity, and cloning of the L-type calcium channel blocker omega-conotoxin TxVII.
omega-Conotoxin TxVII is the first conotoxin reported to block L-type currents. In contrast to other omega-conotoxins, its sequence is characterized by net negative charge and high hydrophobicity, although it retains the omega-conotoxin cysteine framework. In order to obtain structural information and to supply material for further characterization of its biological function, we synthesized TxVII and determined its disulfide bond pairings. Because a linear precursor with free SH groups showed a strong tendency to aggregate and to polymerize, we examined many different conditions for air oxidation and concluded that a mixture of cationic buffer and hydrophobic solvent was the most effective for the folding of TxVII. Synthetic TxVII was shown to suppress the slowly inactivating voltage-dependent calcium current in cultured Lymnaea RPeD1 neurons and furthermore to suppress synaptic transmission between these neurons and their follower cells. In contrast, TxVII did not block calcium flux through L-type channels in PC12 cells, suggesting a phyletic or subtype specificity in this channel family. Disulfide bond pairings of TxVII and its isomers were determined by enzymatic fragmentation in combination with chemical synthesis, thus revealing that TxVII has the same disulfide bond pattern as other omega-conotoxins. Furthermore, the CD spectrum of TxVII is similar to those of omega-conotoxins MVIIA and MVIIC. The precursor sequence of TxVII was determined by cDNA cloning and shown to be closest to that of delta-conotoxin TxVIA, a sodium channel inactivation inhibitor. Thus TxVII conserves the structural fold of other omega-conotoxins, and the TxVIA/TxVII branch of this family reveals the versatility of its structural scaffold, allowing evolution of structurally related peptides to target different channels.
Sasaki
T
T
Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan.
Feng
Z P
ZP
Scott
R
R
Grigoriev
N
N
Syed
N I
NI
Fainzilber
M
M
Sato
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002121:Calcium Channel Blockers / Q000138:chemical synthesis* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004220:Disulfides / Q000737:chemistry; D008195:Lymnaea; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000235:genetics / Q000494:pharmacology; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D018076:DNA, Complementary; D004220:Disulfides; D010455:Peptides; D020960:omega-Conotoxins; C100425:omega-conotoxin TxVII