| Summary: | 博士 === 國立臺灣大學 === 生命科學系 === 103 === Sodium channelopathy caused by dysfunction of voltage-gated sodium channel (VGSC) has been implicated in neuropathic pain and metastasis of tumor cells. In the thesis, we investigated two different effects of sodium channelopathy in pain disorder and tumor cells. We characterized the biophysical properties of mutant Nav1.7 channel, which causes primary erythromelalgia (PE), and effect of lidocaine on NF1-derived malignant peripheral nerve sheath tumor (MPNST) cells with expression of sodium channels. The symptoms of PE are extreme burning pain and erythema in the extremities upon heat challenge or exercise. We examined the electrophysiological properties of three mutant Nav1.7 channels, I136V, I848T, and V1316A. Mutant channels displayed hyperpolarizing shift of voltage dependence activation, depolarizing shift in steady state fast inactivation, and faster recovery from inactivation as compared to wild type channel. Elevating temperature is critical for inducing pain attack in PE. We compared the temperatures at 25 and 35°C to evaluate the effect on wild type and mutant channels. Wild type, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift in activation at 35°C than that at 25°C, nevertheless, mutant channels still produce hyperpolarizing shift compared to wild type. Increasing temperature caused depolarizing shift in steady state fast inactivation among three mutant channels except for the wild type channel. These results suggest mutant channels may contribute to hyperexcitability of sensory neurons that explains part of the symptoms of PE, especially at high temperature. We tested whether selective sodium channel blockers such as lidocaine and mexiletine exert differential antagonistic effect on mutant sodium channels. The mutant channels exhibited higher IC50 values for lidocaine compared to wild type channel, which suggests that lidocaine would not be effective medication for the patients carrying these mutations. However, IC50 for mexiletine is lower for I848T mutant channel. The result consists with the clinical observations that mexiletine alleviates symptoms in the patient with I848T mutations. MPNST is a rare soft tissue sarcoma originated from Schwann cells. Twenty to fifty percent of the MPNSTs occur in patients with neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant disorder resulted from a mutation of human NF1 gene. In our NF1-derived MPNST cell lines (S462, ST8814 and T265), the expression of VGSC increased significantly as compared to normal human Schwann cells. Further analysis demonstrated that the mRNA level of Nav-1.3, -1.5, -1.6 and -1.7 were significantly increased in S462 MPNST cells. The overexpression of Nav channels in MPNST cells brings about the idea that blockade of sodium channel might implicate in the cell phenotypes of MPNST. The tetrodotoxin has been used to treat without a positive effect in inhibiting the cell proliferation and invasiveness. However, lidocaine has shown a cytotoxic effect in a dose dependent manner whereas the normal Schwann cells were much resistant than the MPNST. Concerning the decrease of cell viability after treatment of lidocaine, cell death pathways were investigated. The activity of caspase-3 was not changed in treatment cells; however, the increase of autophagy by high level of transformation from LC3-I to LC3-II was identified in MPNST cells treated with lidocaine. Further studies on the molecular mechanism involving the lidocaine induced cell death identified that the treatment resulted in reduction the level ribosomal protein S6 kinase as well as its substrates, S6 and eIF4B. The S6 and eIF4B play a role in protein translation machinery. These findings suggest that lidocaine, a sodium channel blocker can induce the autophagy process of MPNST associated with inhibition of ribosomal protein kinase S6 which can hinder the protein synthesis in MPNST. In this study, we demonstrated that two different mechanisms result in the sodium channelopathies. It suggests that voltage-gated sodium channels might play a role in progression of diseases.
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