Electrophysiological characterization of Tst2 peptide isolated from the scorpion Tityus stigmurus in the voltage-gated sodium channels Nav 1.3 and Nav 1.7

Introduction
Voltage-gated sodium channels (Nav) regulate Na+ flow in cells, crucial for depolarization and initiating action potentials. Scorpion venom, responsible for millions of accidents annually, contains peptides that target ion channels, including Nav channels. These peptides are classified as non-disulfide-bridged peptides (NDBPs) and disulfide-bridged peptides (DBPs), with α-toxins and β-toxins modulating Nav activity. Tst2, a peptide from Tityus stigmurus, comprises 62 amino acids and 4 disulfide bonds. 

Objectives
To purify and characterize the Tst2 peptide in Nav1.3 and Nav1.7 subtypes at 1 µM concentration.

Methodology
Venom was extracted in collaboration with Prof. Matheus de Freitas Fernandes-Pedrosa (UFRN). Purification employed three RP-HPLC methods, with mass confirmed via MALDI-TOF mass spectrometry. Multiple sequence alignment compared Tst2 to peptides with high sequence identity. Quantification used a BCA Protein Assay Kit. HEK 293 and CHO cells expressing Nav1.3 and Nav1.7 were cultured. Electrophysiological assays were conducted using the EPC 10 HEKA amplifier and PatchMaster software in Whole-Cell mode. Sodium currents were analyzed with Fitmaster software.

Results
Purification
Tst2 eluted at 26.42% ACN in method 3, with an average molecular mass of [M+H]+= 6988.23 Da and a monoisotopic mass of [M+H]+ 6984.74 Da. Sequence alignment revealed high identity between Tst2 and toxins such as Tf2, Tf1a (Tityus fasciolatus), Tb1, Tb2 (Tityus bahiensis), Tst1 (Tityus stigmurus), To12 (Tityus obscurus), and Ts2 (Tityus serrulatus), with Ts2 showing 100% identity.

Electrophysiological Assays
Tst2, tested at 1 µM, exhibited β-NaScTx activity. It shifted activation voltage to more hyperpolarized potentials in Nav1.3 and Nav1.7 channels with and without pre-pulse, with small effects on inactivation.

Conclusion
Tst2 and Ts2, sharing 100% identity, were tested at 1 µM. Ts2 inhibited fast inactivation in Nav1.3, Nav1.6, and Nav1.7 channels (α-NaScTx). In this study, Tst2 showed β-NaScTx activity, with a stronger effect on Nav1.3, significantly shifting its activation probability. Tst2 affected Nav1.3 and Nav1.7 activation and inactivation under various conditions, contributing to the understanding of its function.