Human 5-HT3A -- Val 286 (V13')
|Rat Equivalent||Val 291
This mutation was combined with E-1'A (E272A) 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 activationwas much slower, as was the desnsitization in the continued presence of a maximal concentration agonist.
Shorter duration appliacations of agonist to cells produced whole-cell responses where the receptors appeared to reamin 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
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The EC50 value of this mutant is 70 fold less than the WT.
The current-voltage profile and voltage jump relaxation kinetics were similar to the WT, but there was a much slower densisitization kinetics.
Overexpression of mutant receptor with injection of large amounts of mRNA produced a standing voltage clamp current (1-2uA at -60mV).
It was 10-fold less sensitive to blockade by the open channel blocker TMB-8 than the WT.
When doubly mutated with M1 mPro257Ala, mPro257Leu, or mPro257Gly, substantial standing currents were produced. They are changed little by agonists or antagonists. The currents are blocked by the channel blocker TMB-8 at concentrations near those that block Val13'Ser.
Dang H, England PM, Farivar SS, Dougherty DA, Lester HA (2000) Probing the Role of a Conserved M1 Proline Residue in 5-Hydroxtryptamine3 Receptor Gating. Molecular Pharmacology 57:1114-1122
In this study the 5-HT3A(V291S) mutant receptor was expressed in mice by targeted exon replacement. The functional effect of introducing the V291S mutation (13’ Val in the TM2) into the mouse 5-HT3A receptor was studied in Xenopus oocytes using twin point voltage-clamp to record 5-HT activated currents. The mutation caused a 70-fold increase in the potency of 5-HT and induced spontaneous (constitutive) gating in the absence of agonist. Both these effects were seen upon expression of homomeric 5-HT3A and heteromeric 5-HT3A/B receptors. Homozygous mutant mice had decreased 5-HT3A mRNA levels and 5-HT-activated currents recorded from sympathetic ganglion cells were significantly smaller than those of wild type mice. Heterozygous and homozygous mice had extensive histopathology of the lower urinary tract and died earlier than did WT (gene-dose dependent) from an obstructive uropathy.
Bhattacharya A, Dang H, Zhu QM, Schnegelsberg B, Rozengurt N, Cain G, Prantil R, Vorp DA, Guy N, Julius D, Ford AP, Lester HA, Cockayne DA. (2004) Uropathic observations in mice expressing a constitutively active point mutation in the 5-HT3A receptor subunit. The Journal of Neuroscience, 24(24):5537-48
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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 V291C was shown to be voltage-dependent. After the application of 1mM MTSES with 10uM 5-HT, the channels were locked in an open state. When these locked channels were given 300uM diltiazem, the current was then blocked by 37%. The reaction of MTSES with V291C was shown to be voltage-dependent.
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||0.35 +/- 0.02|
|Hill Coefficient||2.0 +/- 0.1||3.0 +/- 0.2|
|Mg Inhibition (before MTSET)||-41 +/- 3||-53 +/- 5|
|Mg Inhibition (after MTSET)||-39 +/- 3||-61 +/- 4|
|Deactivation T50 ratio||0.95 +/- 0.03||1.00 +/- 0.07|
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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