Two Tarantula Peptides Inhibit Activation of Multiple Sodium Channels

Abstract

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

“…This library could be engineered from the terrific variety of potent, VGIC subtype-selective VSTs that are increasingly being discovered. Examples of VSTs from spiders include ω-Agatoxin-IVA, which binds P-type Ca 2+ channels (58) and the ProTx-I and -II peptides, which bind Na + channel subtypes (59)(60)(61)(62), and heteropodatoxins, which bind Kv4 channels (63). The library of known VSTs is growing rapidly as venom peptides and mRNAs of more predatory animals are sequenced (64).…”

Tarantula Huwentoxin-IV Inhibits Neuronal Sodium Channels by Binding to Receptor Site 4 and Trapping the Domain II Voltage Sensor in the Closed Configuration

“…Although these toxins may trap the IV-S4 in the closed configuration, they impair inactivation and are excitatory. ProTx-II, from the venom of the tarantula Thrixopelma pruriens, has been shown to inhibit sodium currents, but in contrast to HWTX-IV, ProTx-II obviously modifies the voltage dependence of VGSC activation in the physiological voltage range (22). It also had been proposed that ProTx-II might interact with neurotoxin receptor site 4 (52).…”

“…Many toxins from spiders, such as ␦-atracotoxins; Magi5; CcoTx1, CcoTx2, and CcoTx3; PaurTx3; and ProTx-II, are able to shift the threshold of sodium channel activation to more negative or positive potentials (21)(22)(23)(24)(25)(26). Therefore we investigated the effects of subsaturating toxin concentrations on the voltage dependence of current activation.…”

Structures of μO-conotoxins from Conus marmoreus

“…The only sequence conservation between these peptides and MrVIA/MrVIB is the high content of cysteine residues, with the ProTx peptides containing six cysteine residues and kurtoxin containing eight. The disulfide connectivity has been elucidated for the ProTx peptides and shown to be Cys I -Cys IV , Cys II -Cys V , and Cys III -Cys VI (48). It has been suggested that they contain an inhibitor cystine knot motif based on this connectivity.…”