J Biol Chem, Vol. 274, Issue 7, 3934-3936, February 12, 1999

COMMUNICATION
The Nicotinic 4 Receptor Subunit Contributes to the Lining of the Ion Channel Pore When Expressed with the 5-HT₃ Receptor Subunit

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

To understand the wide variation of calcium permeability seen in native and recombinant 5-HT₃ receptor (5-HT₃R) channels, we reported previously the novel hypothesis that the serotonin 5-HT₃R subunit can co-assemble with the 4 subunit of the nicotinic acetylcholine receptor (van Hooft, J. A., Spier, A. D., Yakel, J. L., Lummis, S. C. R. & Vijverberg, H. P. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11456-11461). To test the hypothesis that the 4 subunit contributes to the lining of the pore of the resulting 5-HT₃R channel, a mutant nicotinic 4 subunit with a reactive cysteine residue engineered into the putative pore region was constructed by substituting the leucine at position 285 (4-L285C). The sulfhydryl-modifying reagent [2-(trimethylammonium) ethyl]methanethiosulfonate (MTSET) reduced the acetylcholine-induced current in oocytes expressing this mutant nicotinic 4-L285C subunit along with the nicotinic 2 subunit by ~60%. When the 4-L285C subunit was co-expressed with the 5-HT₃R subunit, both MTSET and silver nitrate (AgNO₃), another cysteine-modifying reagent, significantly reduced the serotonin-induced current. No reduction was seen when the 5-HT₃R was expressed alone or with the wild-type 4 subunit. These data provide direct molecular evidence that the nicotinic 4 subunit co-assembles with the 5-HT₃R subunit and forms an integral part of the ion channel pore.