The 4 ' lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors

1. The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop su perfamily. Uniquely 5-HT3 receptor subunits (5-HT3A and 5-HT3A) possess a p ositively charged lysine residue within the putative channel lining M2 doma in (4' position). Using whole cell recording techniques, we examined the ro le of this residue in receptor function using wildtype (WT) and mutant 5-HT 3A receptor subunits of murine origin transiently expressed in human embryo nic kidney (HEK 293) cells. 2. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10-90 % rise time, 103 ms; EC50, 2.34 mu M; Hill coefficient, n(H), 2 .87). The currents rectified inwardly, reversed in sign at a potential of - 9 mV and desensitized in the continuous presence of agonist (half-time of d esensitization, t(1/2), 2.13 s). 3. 5-HT3A receptor subunits in which the 4'lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coef ficients, kinetics of current activation, rectification, and reversal poten tials were unaltered. 4. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor , with the arginine and glycine mutations exhibiting the greatest effect (5 -fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. 5. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4'lysine mutant receptors. Thus it appears unlikely th at 4'lysine is exposed to the channel lumen. 6. Mutation of residues immediately adjacent to 4'lysine to glutamate or ly sine resulted in lack of receptor expression or function. We conclude that 4'lysine does not form part of the channel lining, but may play an importan t role in 5-HT3 receptor desensitization.