Characterization of Synthetic Tf2 as a NaV1.3 Selective Pharmacological Probe

Mathilde R Israel; Thomas S Dash; Stefanie N Bothe; Samuel D Robinson; Jennifer R Deuis; David J Craik; Angelika Lampert; Irina Vetter; Thomas Durek

doi:10.3390/biomedicines8060155

Characterization of Synthetic Tf2 as a Na_V1.3 Selective Pharmacological Probe

Biomedicines. 2020 Jun 11;8(6):155. doi: 10.3390/biomedicines8060155.

Authors

Mathilde R Israel¹, Thomas S Dash¹, Stefanie N Bothe^{2

3}, Samuel D Robinson¹, Jennifer R Deuis¹, David J Craik¹, Angelika Lampert^{2

3

4}, Irina Vetter^{1

5}, Thomas Durek¹

Affiliations

¹ Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.
² Institute of Physiology, Medical Faculty, RWTH Aachen University, 52074 Aachen, Germany.
³ Research Training Group 2416 MultiSenses-MultiScales, RWTH Aachen University, 52074 Aachen, Germany.
⁴ Research Training Group 2415 ME3T, RWTH Aachen University, 52074 Aachen, Germany.
⁵ School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia.

Abstract

Na_V1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate Na_V1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for Na_V1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of Na_V1.3 activation in the hyperpolarizing direction by -11.3 mV, with no activity at Na_V1.1, Na_V1.2, and Na_V1.4-Na_V1.8. Additional activity was found at Na_V1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by -6.3 mV. In an attempt to convert Tf2 into an Na_V1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at Na_V1.3, although it still caused a small -5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the Na_V1.1/1.3 inhibitor ICA-121431 nor in Na_V1.9^-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at Na_V1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.

Keywords: NaV1.3; NaV1.9; Tf2; scorpion; sodium channel; toxin.