Interaction of a Toxin from the Scorpion Tityus serrulatus with a Cloned K⁺ Channel from Squid (sqKv1A)

Abstract

A toxin from the scorpion Tityus serrulatus (TsTX-Kalpha) blocks native squid K(+) channels and their cloned counterpart, sqKv1A, at pH 8 ((native)K(d) approximately 20 nM; (sqKv1A)K(d) approximately 10 nM). In both cases, decreasing the pH below 7.0 significantly diminishes the TsTX-Kalpha effect (pK = 6.6). In the cloned squid channel, the pH dependence of the block is abolished by a single point mutation (H351G), and no change in toxin affinity was observed at higher pH values (pH > or =8.0). To further investigate the TsTX-Kalpha-sqKv1A interaction, the three-dimensional structure of TsTX-Kalpha was determined in solution by NMR spectroscopy, and a model of the TsTX-Kalpha-sqKv1A complex was generated. As found for other alpha-K toxins such as charybdotoxin (CTX), site-directed mutagenesis at toxin residue K27 (K27A, K27R, and K27E) significantly reduced the toxin's affinity for sqKv1A channels. This is consistent with the TsTX-Kalpha-sqKv1A model reported here, which has K27 of the toxin inserted into the ion conduction pathway of the K(+) channel. This toxin-channel model also illustrates a possible mechanism for the pH-dependent block whereby lysine residues from TsTX-Kalpha (K6 and K23) are repelled by protonated H351 on sqKv1A at low pH.

Use of Venom Peptides to Probe Ion Channel Structure and Function

“…However, KTxs have evolved to use different faces of their structure to interact with different K ϩ channels (9). For instance, charybdotoxin (KTx1.1) and agitoxin 2 (KTx3.2) interact with the Shaker (or K v 1) channel using residues protruding from the ␤-strand motif (10,11), whereas BmPO5 (KTx5.3) binds to the K Ca 2.2 channel using residues in the ␣-helical motif (12).…”

Characterization of a Novel Gastropod Toxin (6-Bromo-2-mercaptotryptamine) That Inhibits Shaker K Channel Activity

“…In every case where the mechanism of inhibition has been investigated, these peptide toxins have been found to block K conduction by directly occluding the entrance to the pore (31). However, some peptides (perhaps most) lead to an appearance of slowed I K activation due to voltage-dependent unbinding at positive voltages (27,32,33 and unpublished data on TsTX-K␣, a scorpion toxin (34)). This important mechanism (35) predicts that, at a given voltage, the rate of I K rise will be faster at higher toxin concentrations because of faster equilibrium binding kinetics (33).…”

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus

“…Experiments were also carried out on K + currents, but only small and inconsistent effects of C. californicus venom were observed. At least 90% of the K + channels under the currents analyzed are of one type that is blocked by TsTX-K, a peptide toxin isolated from the scorpion, Tityus serrulatus (Mathes et al, 1997;Ellis et al, 2001), and no consistent effect on these squid K + channels was thus observed. Active toxin was purified by conventional HPLC from duct and milked venom (Fig.2C).…”

“…Giant-fiber-lobe (GFL) neurons were prepared from excised stellate ganglia following established procedures and maintained in vitro for up to 7days on glass coverslips at 14°C (Gilly et al, 1990). These cells were used for recording voltage-gated Na + (Gilly et al, 1990), Ca + (McFarlane and Gilly, 1996) and K + currents (Ellis et al, 2001) using whole-cell patch-clamp methods at 12-13°C. Electrodes had a resistance of <0.7M when filled with internal recording solution, and series resistance compensation was employed to the greatest extent possible.…”

“…amino acids 9-18, 23-28 and 37-45) and the modular diversity in other regions (amino acids 19-23 and 34-37), it is possible that all isoforms block voltage-gated Na + channels. This idea is based on a similar modular structure of scorpion toxins that block voltagegated potassium (K v 1) channels, with selectivity for specific channel isoforms being dictated by amino-acid side chains in variable regions that are flanked by highly conserved sequences (Ellis et al, 2001;Garcia et al, 2001;Gross and MacKinnon, 1996;Miller, 1995). In general, the variable regions form the active surface that defines target specificity.…”