Cloning and functional expression of B chains of beta-bungarotoxins from Bungarus multicinctus (Taiwan banded krait).

The cDNA species encoding the B chains (B1 and B2) of beta-bungarotoxins (beta-Bgt) were constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequences of the B chains were different from those determined previously by a protein sequencing technique. One additional Arg residue is inserted between Val-19 and Arg-20 of the B1 chain. Similarly the insertion of one additional Val residue between Val-19 and Arg-20 of the B2 chain is noted. Thus the B chains should comprise 61 amino acid residues. Moreover, the residues at positions 44-46 are Gly-Asn-His, in contrast with a previous result showing the sequence His-Gly-Asn. Instead of Asp, the residues at positions 41 and 43 are Asn. The B chain was subcloned into the expression vector pET-32a(+) and transformed into Escherichia coli strain BL21(DE3). The recombinant B chain was expressed as a fusion protein and purified on a His-Bind resin column. The yield of affinity-purified fusion protein was increased markedly by replacing Cys-55 of the B chain with Ser. However, the isolated B(C55S) chain became insoluble in aqueous solution after removal of the fused protein from the affinity-purified product, suggesting that protein-protein interactions might be crucial for stabilizing the structure of the B chain. The B(C55S) chain fusion protein showed activity in blocking the voltage-dependent K+ channel, but did not inhibit the binding of beta-Bgt to synaptosomal membranes. These results, together with the finding that modification of His-48 of the A chain of beta-Bgt caused a marked decrease in the ability to bind toxin to its acceptor proteins, suggest that the B chain is involved in the K+ channel blocking action observed with beta-Bgt, and that the binding of beta-Bgt to neuronal receptors is not heavily dependent on the B chain.

Ionic mechanisms of tetrandrine in cultured rat aortic smooth muscle cells.

The ionic mechanism of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Radix stephania tetrandrae, was investigated in A7r5 vascular smooth muscle cells. The nystatin-perforated whole-cell voltage-clamp technique was performed to examine the effects of tetrandrine on ionic currents. Tetrandrine (1-100 microM) reversibly caused an inhibition of L-type voltage-dependent Ca2+ current (I(Ca,L)) in a concentration-dependent manner. Tetrandrine did not cause any change in the overall shape of the current-voltage relationship of I(Ca,L). The IC50 value of tetrandrine-induced inhibition of I(Ca,L) was 5 microM. In the presence of Bay K 8644 (3 microM) or cyclopiazonic acid (30 microM), tetrandrine still produced a significant inhibition of I(Ca,L). The inhibitory effects of tetrandrine on I(Ca,L) exhibited tonic and use-dependent characteristics. Moreover. tetrandrine (3 microM) shifted the steady-state inactivation curve of I(Ca,L) to more negative membrane potentials by approximately -15 mV. These results indicate that tetrandrine directly inhibits the voltage-dependent L-type Ca2+ current in vascular smooth muscle cells, which may predominantly contribute to the vasodilatory actions of tetrandrine.