Human 5-HT3A -- Ile 289

Species Original Mutated to Mutation
Human Ile 289  

 

Rat Equivalent Ile 294    
Mouse Equivalent Ile 294

Cys

Thr

Leu

I294C

I294T

I294L

 

mIle294Cys

After the application of 1mM MTSET with 10uM 5-HT, The EC50 current was irreversibly reduced. The reaction of MTSET with I294C was shown to be voltage-dependent. After the application of 1mM MTSES with 10uM 5-HT, the channels were locked in an open. When the locked channel was given 300uM diltiazem, there was no effect. The reaction of MTSES with I294C 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

 

  Wild Type I294C
EC50 1.67 +/- 0.09 0.94 +/- 0.06
Hill Coefficient 2.0 +/- 0.1 2.6 +/- 0.1
Mg Inhibition (before MTSET) -41 +/- 3 -71 +/- 2
Mg Inhibition (after MTSET) -39 +/- 3 -59 +/- 2
Deactivation T50 ratio 0.95 +/- 0.03 1.46 +/- 0.11

 

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

 

mIle294Thr

The I294T and I294L mutations in the mouse 5-HT3A subunit affected the 5-HT concentration-response relationships, current kinetics and modulation by alcohols.

Recombinant 5-HT3A receptors were expressed in Xenopus oocytes and in HEK 293 cells. 5-HT-activated currents were recorded under voltage-clamp. The effect of replacing isoleucine by threonine was an increase in the potency of 5-HT, demonstrated by a leftward shift of the 5-HT concentration-response curve.

The EC50 value for wild-type 5-HT3A receptors was 0.93 +/- 0.023 mM and for the mutant 5-HT3A(I294T) receptor was 0.65 +/- 0.015 mM determined from experiments using Xenopus oocytes. Values derived from experiments performed using HEK cells were: EC50 value for wild-type 5-HT3A receptors was 3.82 +/- 0.11 mM and for the mutant 5-HT3A(I294T) was 2.73 +/- 0.18 mM.

The mutant 5-HT3A(I294T) receptor exhibited a marked reduction in the extent of desensitization by 5-HT. Furthermore ethanol and
2,2,2-trichloroethanol (TCEt) enhanced 5-HT-mediated currents medi ated by wild-type receptors, but inhibited or had little stimulatory effect on currents mediated by 5-HT3A(I294T) receptors. In the presence of TCEt, the slope of the 5-HT-activated current mediated by 5-HT3A(I294T) receptors was unchanged while the slope of the 5-HT-activated current mediated by the WT 5-HT3A receptor was increased by TCEt.

mIle294Leu

The I294T and I294L mutations in the mouse 5-HT3A subunit affected the 5-HT concentration-response relationships, current kinetics and modulation by alcohols.

Recombinant 5-HT3A receptors were expressed in Xenopus oocytes and in HEK 293 cells. 5-HT-activated currents were recorded under voltage-clamp. The effect of replacing isoleucine by leucine was a decrease in the potency of 5-HT, demonstrated by a rightward shift of the 5-HT concentration-response curve.

The EC50 value for wild-type 5-HT3A receptors was 0.93 +/- 0.023 mM and for the mutant 5-HT3A(I294L) receptor was 1.15 +/- 0.14mM determined from experiments using Xenopus oocytes.

Ethanol and 2,2,2-trichloroethanol (TCEt) enhanced 5-HT-mediated currents mediated by wild-type and 5-HT3A(I294L) receptors, but inhibited or had little stimulatory effect on currents mediated by 5-HT3A(I294T) receptors.

 

Sessoms-Sikes JS, Hamilton ME, Liu LX, Lovinger DM, Machu TK (2003) A mutation in transmembrane domain II of the 5-hydroxytryptamine(3A)receptor stabilizes channel opening and alters alcohol modulatory actions. The Journal of pharmacology and experimental therapeutics 306(2):595-604

 

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