Patch Clamp Study of Serotonin-Gated Currents via 5-HT Type 3 Receptors by Using a Novel Approach SHAM for Receptor Channel Scanning.
We studied 5-hydroxy tryptamine type 3 (5-HT(3)) receptors transfected in tsA-201 cell line to examine serotonin-induced whole cell currents. Using the site-directed mutagenesis technique, we individually mutated each residue in the membrane-spanning M2 segment to histidine. A high proportion of tsA-201 cells cotransfected with the cDNAs of 5-HT(3)R and CD8 produced large amplitude responses (0.57.0 nA) to serotonin. The dose-response curve of wild-type (WT) receptor ranging from 0.5 to 500 $\mu$ mole increases its K(d) values, and I_\mathrm{max} of 5-HT(3)R falls at low external pH as if protonation of an acid group is enough to block the channel. Lysine at position 281, a basic residue, is more susceptible to acidification-induced blockade of the 5-HT(3)R channel. Dose-response curves of K281S (replacing lysine at the 281 position with serine) at different pH are not significantly modulated, and histidine substitutions at the three consecutive positions 293, 294, and 296 eliminate the pH block of the channel.
Kaneez
Fatima-Shad
FS
White
M.
M