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Synthesis, bioactivity, and cloning of the L-type calcium channel blocker omega-conotoxin TxVII
1Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan.
Abstract:
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.
Insights
Omega-conotoxin TxVII, a novel L-type calcium channel blocker, was synthesized and structurally characterized. This conotoxin exhibits unique properties and specific targeting, highlighting the versatility of conotoxin scaffolds.
Area of Science:
- * Neuroscience and Pharmacology
- * Peptide Chemistry and Structural Biology
Background:
- * Omega-conotoxins are known calcium channel blockers, but TxVII is the first reported to target L-type currents.
- * TxVII possesses a unique sequence with a net negative charge and high hydrophobicity, differing from other omega-conotoxins while retaining the conserved cysteine framework.
Purpose of the Study:
- * To synthesize omega-conotoxin TxVII and determine its disulfide bond pairings for structural insights.
- * To investigate the biological function of TxVII, including its effects on voltage-dependent calcium currents and synaptic transmission.
- * To explore the specificity of TxVII's interaction with L-type calcium channels.
Main Methods:
- * Chemical synthesis of TxVII and optimization of air oxidation conditions for proper folding.
- * Determination of disulfide bond pairings using enzymatic fragmentation and chemical synthesis.
- * Assessment of TxVII's biological activity on cultured Lymnaea RPeD1 neurons and PC12 cells.
Main Results:
- * Optimized folding conditions using a cationic buffer and hydrophobic solvent facilitated TxVII synthesis.
- * Synthetic TxVII suppressed slowly inactivating voltage-dependent calcium currents and synaptic transmission in Lymnaea neurons.
- * TxVII did not block L-type calcium channels in PC12 cells, indicating potential phyletic or subtype specificity.
- * Disulfide bond analysis revealed TxVII shares the same pattern as other omega-conotoxins, and its CD spectrum is similar to MVIIA and MVIIC.
Conclusions:
- * TxVII is a structurally conserved omega-conotoxin that specifically blocks L-type calcium currents and synaptic transmission.
- * The study highlights the evolutionary versatility of the conotoxin scaffold, enabling the targeting of different ion channels.
- * TxVII's unique properties and specificity offer potential for further research in neuropharmacology.