Human 5-HT3A -- Glu 272
|Rat Equivalent||Glu 271|
|Mouse Equivalent||Glu 277||Cys||E277C|
This mutation was combined with V13'T (V286T) and a proline insertion between -1' and -2' (G271 and E272) positions on the M2 region. The resulting currents were completely blocked when combined with granisetron. The EC50 value decreased 10-fold. The Hill coefficient also decreased. The activation was much slower, as was the desensitization in the continued presence of a maximal concentration agonist.
Shorter duration applications of agonist to cells produced whole-cell responses where the receptors appeared to remain in a conducting state for more than 20 seconds after agonist removal.
Antagonists, (granisetron or D-tubocurarine), appeared to have no effect and they did not reduce leak currents or cause activation of the receptors.
When exposed to various ion concentrations, the receptors appeared to have converted from cation- selective to anion-selective.
Gunthorpe MJ, Lummis SCR (2001) Conversion of the Ion Selectivity of the 5-HT3A receptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily. The Journal of Biological Chemistry 276 (14):10977-10983
This mutation- E-1'A (EA) generated functional 5-HT-gated receptors. When it was doubly mutated with S292R (S19'R or SR), functional receptors were also generated. The EC50 values for 5-HT and retification properties were similar to those of WT receptors. The activation kinetics were also similar, but the desensitization rates did vary in that the time constant for EA was less than the WT. The double mutation EASR caused a greater decrease in the time constant. (WT>EA>SR>EASR)
However, when it was combined with A297K (either EA alone or EASR), no functional receptors were detected.
The EA receptors showed almost no ion selectivity. But the EASR receptors were anionic, while the SR receptors were cationic.
Thompson AJ, Lummis SCR (2003) A Single Ring of Charged Amino Acids at One End of the Pore Can Control Ion Selectivity in the 5-HT3 Receptor. British Journal of Pharmaoclogy 140(s) 359-365
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After the application of 1mM MTSET with 10uM 5-HT, the EC50 current was irreversibly reduced. The reaction of MTSET with E277C was shown to be voltage- dependent. After the application of 1mM MTSES with 10uM 5-HT, there was no significant change.
Reeves DC, Goren EN, Akabas MH, Lummis SCR (2001) Structural and Electrostatic Properties of the 5-HT3 Receptor Pore Revealed by Substituted Cysteine Accessibility Mutagenesis. The Journal of Biological Chemistry 276(45):42035-42042
|EC50||1.67 +/- 0.09||1.36 +/- 0.07|
|Hill Coefficient||2.0 +/- 0.1||2.0 +/- 0.2|
|Mg Inhibition (before MTSET)||-41 +/- 3||-13 +/- 1|
|Mg Inhibition (after MTSET)||-39 +/- 3||-7 +/- 1|
|Deactivation T50 ratio||0.95 +/- 0.03||1.01 +/- 0.04|
Panicker S, Cruz H, Arrabit C, Slesinger PA (2002) Evidence for a Centrally Located Gate in the Pore of a Serotonin-Gated Ion Channel. The Journal of Neuroscience 22(5): 1629-1639
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