Two critical residues in p-loop regions of puffer fish Na+ channels on TTX sensitivity

We previously showed that Asn-383 and Thr-1569 residues of p-loop regions in domains I and IV, respectively, of the puffer fish, Fugu pardialis, skeletal muscle Nav (fNav1.4a), are anomalous to those of other species of TTX-sensitive Na+ channels, where the aromatic residues of Phe or Tyr, and Gly are the counterparts [Yotsu-Yamashita, M., Nishimori, K., Nitanai, Y., Isemura, M., Sugimoto, A., Yasumoto, T., 2000. Binding properties of 3H-PbTx-3 and 3H-saxitoxin to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardalis and the primary structure of a voltage-gated Na+ channel α-subunit (fMNa1) from skeletal muscle of F. pardalis. Biochem. Biophys. Res. Commun. 267, 403–412]. The former was suggested to confer TTX resistance by using Y401N mutant of rNav1.4 [Venkatesh, V., Lu, S.Q., Dandona, N., See, S.L., Benner, S., Soong, T.W., 2005. Genetic basis of tetrodotoxin resistance in pufferfishes. Curr. Biol. 15, 2069–2072]. The latter function remained to be elucidated. Thus, we further explored the function of these two residues, electrophysiologically, by evaluating the Kd (dissociation constants) values of TTX for F385N, F385A, F385Q, G1718T, and F385N/G1718T mutants of rNav1.2a, transiently expressed in HEK-293 cells. F385N caused 3000-fold increase of the Kd, while G1718T and F385N/G1718T caused 2- and 3-fold increases compared with those of WT and F385N, respectively, suggesting that G1718T further enhanced TTX resistivity caused by F385N. The Kd for F385A and F385Q were 2- and 11-fold larger than that of F385N, respectively, suggesting that the longer side chain in the non-aromatic amino acid residue causes the larger decrease of TTX sensitivity. Despite drastic changes in the Kd, the mutations at F385 caused only small changes in the koff from that of WT, suggesting that the Kd for TTX receptors are mainly determined by the kon.