The nicotinic α4 receptor subunit contributes to the lining of the ion channel pore when expressed with the 5-HT3 receptor subunit

Abstract

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 α4 subunit of the nicotinic acetylcholine receptor (van Hooft, J. A., Spier, A. D., Yakel, J. L., Lummis, S. C. R. and Vijverberg, H. P. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11456-11461). To test the hypothesis that the 34 subunit contributes to the lining of the pore of the resulting 5- HT3R channel, a mutant nicotinic 34 subunit with a reactive cysteine residue engineered into the putative pore region was constructed by substituting the leucine at position 285 (α4-L285C). The sulfhydryl-modifying reagent [2- (trimethylammonium)ethyl]methanethiosulfonate (MTSET) reduced the acetylcholine-induced current in oocytes expressing this mutant nicotinic α4-L285C subunit along with the nicotinic β2 subunit by ~60%. When the α4-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 α4 subunit. These data provide direct molecular evidence that the nicotinic α4 subunit co-assembles with the 5-HT3R subunit and forms an integral part of the ion channel pore.