Biophys J, September 2000, p. 1379-1387, Vol. 79, No. 3

Residues in Na⁺ Channel D3-S6 Segment Modulate both Batrachotoxin and Local Anesthetic Affinities

Sho-Ya Wang,^* Carla Nau, and Ging Kuo Wang

 ^*Department of Biology, State University of New York, Albany, New York 12222, and  Department of Anesthesia, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115 USA

Batrachotoxin (BTX) alters the gating of voltage-gated Na⁺ channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na⁺ conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na⁺ channel -subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle µ1 Na⁺ channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, µ1-S1276K and µ1-L1280K, when expressed in mammalian cells, become completely resistant to 5 µM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display ~10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues µ1-S1276 and µ1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na⁺ channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.

Biophys J, September 2000, p. 1379-1387, Vol. 79, No. 3
© 2000 by the Biophysical Society   0006-3495/00/09/1379/09  $2.00

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