JVATiTD - Synthesis and electrophysiological characterization of TxO6, an O1 gene superfamily

Abstract

Background: Conotoxins of the O1 gene superfamily are valuable ion channel research tools and potential drug candidates because they selectively and efficiently target sodium, potassium, or calcium channels. Among them, the calcium channel-targeting ω-conotoxin MVIIA (ziconotide) was approved by the FDA in 2004 to treat intractable pain, whereas several Nav1.8-targeting μO-conotoxins have undergone preclinical investigation as potential treatments for neuropathic pain. In this study, TxO6, an O1 gene superfamily conotoxin with sequence similarity to μO-conotoxins, was synthesized, and its physiological targets were investigated. Methods: TxO6, containing three disulfide bonds, was synthesized using solid-phase peptide synthesis, purified by HPLC, and characterized by mass spectrometry. The effects of synthetic TxO6 on sodium, potassium and calcium currents were evaluated using patch-clamp recordings in acutely isolated rat dorsal root ganglion (DRG) neurons. The effects of TxO6 on four human sodium and potassium channel subtypes expressed in HEK293 or ND7/23 cells were also evaluated. Results: TxO6 (1 μM) had no apparent effect on the current-voltage (I-V) relationships, activation, inactivation, or recovery of sodium currents. TxO6 (1 μM) also had no apparent effect on the I-V relationships of calcium currents. In contrast, TxO6 inhibited rat DRG potassium currents in a concentration-dependent manner, with an IC₅₀ of 0.89 ± 0.24 μM. Regarding specific subtypes, TxO6 (1 μM) had no apparent effect on hNav1.7 currents but appeared to slightly delay the inactivation of hNav1.8 currents. Furthermore, TxO6 (1 μM) inhibited hKv1.1 and hKv7.1 currents by 4.75% ± 0.65% and 11.20% ± 1.87%, respectively. Conclusion: These results indicate that TxO6 preferentially inhibits native potassium currents in rat DRG neurons and may be a candidate for further investigation as a pharmacological probe for potassium channels.

Keywords: Conotoxin; Conopeptide; TxO6; Potassium currents; Patch clamp.