Human 5-HT3A -- Ser 275

Species Original Mutated to Mutation
Human Ser 275  

 

Rat Equivalent Ser 274    
Mouse Equivalent Ser 280

Cys

Ala

S280C

S280A

 

mSer280Cys

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

 

  Wild Type S280C
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

 

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mSer280Ala

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 M at the WT 5-HT3A receptor. The S280A mutant did not significantly alter the potency of 5-HT (EC50 = 1.0 +/- 0.03 M ) when compared with wild-type (EC50 =1.2 +/- 0.07 M ).

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

 

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