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 Na_v (fNa_v1.4a), 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 rNa_v1.4 [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 K_d (dissociation constants) values of TTX for F385N, F385A, F385Q, G1718T, and F385N/G1718T mutants of rNa_v1.2a, transiently expressed in HEK-293 cells. F385N caused 3000-fold increase of the K_d, 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 K_d 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 K_d, the mutations at F385 caused only small changes in the k_off from that of WT, suggesting that the K_d for TTX receptors are mainly determined by the k_on.