The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of Na1.7 subtype of voltage-gated sodium (Na) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and Na1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of Na1.6, compared to Na1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNa1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the Na1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity.

Background and Purpose: Genetic deletion and pharmacological studies suggest a role for lysophosphatidic acid (LPA 1) receptor in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA 1 receptor antagonist using dermal fibroblasts from patients and an animal model of skin fibrosis. Experimental Approach: Dermal fibroblast and skin biopsies from systemic sclerosis patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Pharmacolgical effect of SAR100842 was assessed in the tight skin 1 (Tsk1) mouse model. Key Results: SAR100842 is equipotent against various LPA isoforms. Dermal fibroblasts and skin biopsies from patients with systemic sclerosis expressed high levels of LPA 1 receptor. The LPA functional response (Ca 2+) in systemic sclerosis dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in systemic sclerosis dermal and idiopathic pulmonary fibrosis lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from systemic sclerosis dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content.

Background and Purpose Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Nav1.7 voltage‐dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Nav channel targets. Experimental Approach We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high‐throughput automated patch‐clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Nav‐expressing cells in pull‐down and cell distribution studies. Key Results Two of the produced ProTx II analogues, Biot‐ProTx II and ATTO488‐ProTx II, best emulate the pharmacological properties of unlabelled ProTx II, whereas other analogues remain high affinity blockers of Nav1.7. The biotinylated version of ProTx II efficiently works for the pull‐down of several Nav isoforms tested in a concentration‐dependent manner, whereas the fluorescent ATTO488‐ProTx II specifically labels the Nav1.7 channel over other Nav isoforms tested in various experimental conditions. Conclusions and Implications The properties of these ProTx II analogues as tools for Nav channel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also be useful in the design of new drug screening strategies.

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