{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T07:34:04Z","timestamp":1791272044722,"version":"4.1.0"},"reference-count":30,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2012,8,27]],"date-time":"2012-08-27T00:00:00Z","timestamp":1346025600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Peptide Science"],"published-print":{"date-parts":[[2012,10]]},"abstract":"<jats:p>Hainantoxin\u2010IV (HNTX\u2010IV), isolated from the venom of the spider <jats:italic>Ornithoctonus hainana<\/jats:italic>, is a specific antagonist of tetrodotoxin\u2010sensitive (TTX\u2010S) voltage\u2010gated sodium channels in rat dorsal root ganglion (DRG) cells. It adopts an inhibitor cystine knot motif, and structural analysis revealed a positively charged patch consisting of Arg26, Lys27, His28, Arg29 and Lys32 distributed on its molecular surface. Our previous study demonstrated that Lys27 and Arg29 but not Arg26 were critical residues for HNTX\u2010IV binding to TTX\u2010S sodium channels. In the present study, we examined the roles of His28 and Lys32 in the interaction of HNTX\u2010IV with its target. Two mutants, HNTX\u2010IV\u2010H28D and HNTX\u2010IV\u2010K32A, were generated by solid\u2010phase chemical synthesis and purified by reverse\u2010phase HPLC after refolding and oxidation, yielding two compounds of high purity with monoisotopic masses of 3962.66 and 3927.70\u2009Da, respectively, as determined by MALDI\u2010TOF mass spectrometry. This indicated the presence of six cysteine residues forming three disulfide bonds. Moreover, circular dichroism spectroscopy analysis demonstrated that the substitution of His28 or Lys32 did not affect the overall structure of HNTX\u2010IV. The inhibitory activity of HNTX\u2010IV\u2010H28D and HNTX\u2010IV\u2010K32A against TTX\u2010S sodium channels in rat DRG cells was analyzed by whole\u2010cell patch\u2010clamp technique. The IC<jats:sub>50<\/jats:sub> values for the mutants were 0.57 and 5.80\u2009\u03bcM (17\u2010fold and 170\u2010fold lower than the activity of the native toxin), indicating that His28 and Lys32 may be important for the inhibitory activity of HNTX\u2010IV. Taken together, our results suggest that the positively charged patch might be the binding site for the interaction of HNTX\u2010IV with TTX\u2010S sodium channels. These findings might contribute to the elucidation of the structure and function relationship of HNTX\u2010IV. 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