Human 5-HT3A -- Ser 275
|Rat Equivalent||Ser 274|
|Mouse Equivalent||Ser 280|
After the application of 1mM MTSET with 10uM 5-HT, The EC50 current was irreversibly reduced. The channels were locked in the open state. The reaction of MTSET with S280C 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||2.21 +/- 0.03|
|Hill Coefficient||2.0 +/- 0.1||3.3 +/- 0.1|
|Mg Inhibition (before MTSET)||-41 +/- 3||-26 +/- 2|
|Mg Inhibition (after MTSET)||-39 +/- 3||-36 +/- 2|
|Deactivation T50 ratio||0.95 +/- 0.03||1.09 +/- 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
back to top
go back to 5-HT3A
The functional effects of introducing mutations S280A (2’ Ser in the TM2), T284F (6’ Thr in the TM2) and L285T (7’ Leu in the TM2) into the mouse 5-HT3A subunit and N280T, N280S (6’ Asn in the TM2) and V281T (7’ Val in the TM2) into the mouse 5-HT3B subunit were studied in HEK-293 cells using the whole-cell patch clamp technique. 5-HT3A subunit is a functional homomeric receptors and 5-HT3B subunit coexpressed with 5-HT3A subunit to form functional heteromeric receptors.
In addition, [3H]EBOB and TBPS were used to study the interaction of 5-HT3 receptors with these antagonists. Unlike picrotoxin, [3H]EBOB and TBPS, which potently inhibit GABAA receptors, did not interact with 5-HT3 receptors.
The S280A mutation did not alter the sensitivity of the 5-HT3A receptor to picrotoxin. The IC50 of picrotoxin at the 5-HT3A(S280A) receptor was 45.6 +/- 5.2 µM compared to 41.2 +/- 5.6 µM at the WT 5-HT3A receptor. The S280A mutant did not significantly alter the potency of 5-HT (EC50 = 1.0 +/- 0.03 µM ) when compared with wild-type (EC50 =1.2 +/- 0.07 µM ).
The T284F mutant decreased the sensitivity of the 5-HT3A receptor to picrotoxin by 50-fold (see table below). By contrast, mutations (N280T and N280S) at the equivalent (6') position in the 5-HT3B subunit increased the sensitivity of heteromeric 5-HT3A/B receptors to picrotoxin.
The L285T mutant increased the sensitivity of the receptor to picrotoxin by 10-fold and also altered the gating kinetics of the 5-HT3A receptor. However, the equivalent mutation at the 7' position in the 5-HT3B subunit (V281T) did not increase the sensitivity of 5-HT3A/B receptors to picrotoxin.
|Receptor||5-HT EC50 (mM)||PTX IC50 (mM)|
|5-HT3A Wild-Type||1.2 +/- 0.07||41.2 +/- 5.6|
|3A: S280A (S2'A)||1.0 +/- 0.03*||45.6 +/- 5.2|
|3A: T284F (T6'F)||0.9 +/- 0.09*||1900 +/- 300**|
|3A: L285T( L 7'T)||2.0 +/- 0.05**||4.0 +/- 0.5**|
|5-HT3A/B Wild-Type||1.0 +/- 0.1||1135 +/- 131|
|3B: N280T (N6'T)||0.5 +/- 0.03**||213 +/- 24**|
|3B: N280S(N6'S)||0.4 +/- 0.01**||173 +/- 28**|
|3B: V281T (V7'T)||0.6 +/- 0.09**||866 +/- 35|
|3B: N280T, V281T (N6'T,V7'T)||0.4 +/- 0.01**||343 +/- 45**|
*Significantly different from the wild-type receptor (p<0.05)
**Significantly different from equivalent wild-type receptor (p<0.01)
Das P, Dillion GH. (2005) Molecular determinants of picrotoxin inhibition of 5-hydroxytryptamine type 3 receptors. The Journal of pharmacology and experimental therapeutics 314(1):320-8
back to top
go back to 5-HT3A