Abstract
NaV1.3 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 NaV1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at NaV1.1, NaV1.2, and NaV1.4-NaV1.8. Additional activity was found at NaV1.9, 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 NaV1.3 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 NaV1.3, 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 NaV1.1/1.3 inhibitor ICA-121431 nor in NaV1.9-/- 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 NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.
Highlights
Voltage-gated sodium channels (VGSCs) are large pore-forming transmembrane-spanning proteins with four homologous domains (I-IV) that regulate the influx of Na+ ions across neuronal membranes in response to local changes in voltage [1]
NaV 1.3 is expressed at very low levels in adult rodent dorsal root ganglion (DRG) neurons but is upregulated in DRGs following nerve injury or streptozotocin-induced diabetes, suggesting the channel may contribute to the development and maintenance of neuropathic pain [4,5,6]
ShRNA-mediated knockdown of NaV 1.3 in DRG neurons attenuates the development of mechanical allodynia in rodent models of spared nerve injury (SNI) and diabetic neuropathy [7,8], while global NaV 1.3 knockout has confounding results, with minimal effect on the development of mechanical allodynia following chronic constriction injury (CCI), spinal nerve transection (SNT), and spinal nerve ligation (SNL) [9,10]
Summary
Voltage-gated sodium channels (VGSCs) are large pore-forming transmembrane-spanning proteins with four homologous domains (I-IV) that regulate the influx of Na+ ions across neuronal membranes in response to local changes in voltage [1]. This Na+ influx is essential for action potential initiation and propagation in electrically excitable cells and crucial for the physiological function of neurons. NaV 1.3 is expressed at very low levels in adult rodent dorsal root ganglion (DRG) neurons but is upregulated in DRGs following nerve injury or streptozotocin-induced diabetes, suggesting the channel may contribute to the development and maintenance of neuropathic pain [4,5,6]. Selective pharmacological modulators of NaV 1.3 are required to further elucidate the contribution of the channel to neuronal excitability in physiological and pathological pain states
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