Human 5-HT3A -- Asp 293

Species Original Mutated to Mutation
Human Asp 293  

 

Rat Equivalent Asp 292    
Mouse Equivalent Asp 298

Cys

Ala

Arg

Glu

D298C

D298A

D298R

D298E

 

mAsp298Cys

No current was detected after the application of 5-HT up to 1mM or when it was perfused with 10mM dithiothreitol for 3 minutes. After the application of 1mM MTSET with 10uM 5-HT, no current was detected. After the application of 1mM MTSES with 10uM 5-HT, no current was detected.

 

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

 

back to top
go back to 5-HT3A

 

mAsp298Ala

The functional effects of introducing mutations D298A, D298E and D298R into the mouse 5-HT3A subunit were studied in HEK-293 cells using the whole-cell patch clamp technique. This experiment suggested that charge at residue D298 plays an important role in gating kinetics and modulation of extracellular Ca2+.

In order to neutralize the charge of residue 298, Asp was substituted by Ala. The D298A mutant decreased the sensitivity to 5-HT (EC50 = 3.05 +/- 0.14 ÁM) when compared with wild-type (EC50 = 1.74 +/- 0.09 ÁM ). The mutant also significantly altered the gating kinetics of the 5-HT3A receptor by increasing the rate of desensitization and deactivation of the 5-HT3A receptor when compared with wild-type.

The mutant decreased the relative efficacy of partial agonists, 2-Me-5-HT and dopamine when compared with wild type.

The mutant increased the gating kinetics of 5-HT3A receptor which activated by partial agonist, 2-Me-5-HT and decreased the gating kinetics of 5-HT3A receptor which activated by partial agonist, dopamine.

When [Ca2+]o was increased from 1.8mM to 10mM, the rate of activation, desensitization and deactivation did not change in the mutant D298A. In contrast, when [Ca2+]o was decreased from 1.8mM to 0.1mM, the rate of kinetics still did not change in that mutant.

  Wild Type D298A D298E D298R
EC50 (mM ) 1.74 +/- 0.09 3.05 +/- 0.14 1.84 +/- 0.10 7.20 +/- 0.20
Desensitization of 5-HT (ms) 3365 +/- 162 565 +/- 41** 4258 +/- 537 200 +/- 15**
Deactivation of 5-HT(ms) 3232 +/- 218 337 +/- 20** 4032 +/- 324 122 +/- 8**
Activation of 2-Me-5-HT (ms) 259 +/- 14 54 +/- 6** 298 +/- 12 NA
Desensitization of 2-Me-5-HT(ms) 5653 +/- 383 3462 +/- 223** 9730 +/- 1352** NA
Deactivation of 2-Me-5-HT(ms) 633 +/- 38 101 +/- 5** 879 +/- 53** NA
Activation of Dopamine (ms) 543 +/- 18 48 +/- 6** 832 +/- 70** NA
Desensitization of Dopamine(ms) 17967 +/- 2575 5686 +/- 761** 23794 +/- 2644 NA
Deactivation of Dopamine (ms) 184 +/- 15 69 +/- 4** 273 +/- 17** NA

Hu XQ, Lovinger DM (2005) Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+. The Journal of Physiology 568(Pt 2):381-96

back to top
go back to 5-HT3A

 

 

mAsp298Arg

The functional effects of introducing mutations D298A, D298E and D298R into the mouse 5-HT3A subunit were studied in HEK-293 cells using the whole-cell patch clamp technique. This experiment suggested that charge at residue D298 plays an important role in gating kinetics and modulation of extracellular Ca2+.

In order to reverse the charge of residue 298, Asp was substituted by Arg. The D298R mutant reduced the sensitivity to 5-HT (EC50 = 7.20 +/- 0.20 uM) when compared with wild-type (EC50 = 1.74 +/- 0.09 ÁM ).

The mutant significantly altered the gating kinetics by increasing the rate of desensitization (200 +/- 15** ms) when compared with wild-type (3365 +/- 162 ms) and deactivation (122 +/- 8** ms) when compared with wild-type (3232 +/- 218 ms) .

When [Ca2+]o was increased from 1.8mM to 10mM, the rate of activation, desensitization and deactivation did not change in the mutant D298E.

Wild Type D298A D298E D298R
EC50 (mM ) 1.74 +/- 0.09 3.05 +/- 0.14 1.84 +/- 0.10 7.20 +/- 0.20
Desensitization of 5-HT (ms) 3365 +/- 162 565 +/- 41** 4258 +/- 537 200 +/- 15**
Deactivation of 5-HT(ms) 3232 +/- 218 337 +/- 20** 4032 +/- 324 122 +/- 8**
Activation of 2-Me-5-HT (ms) 259 +/- 14 54 +/- 6** 298 +/- 12 NA
Desensitization of 2-Me-5-HT(ms) 5653 +/- 383 3462 +/- 223** 9730 +/- 1352** NA
Deactivation of 2-Me-5-HT(ms) 633 +/- 38 101 +/- 5** 879 +/- 53** NA
Activation of Dopamine (ms) 543 +/- 18 48 +/- 6** 832 +/- 70** NA
Desensitization of Dopamine(ms) 17967 +/- 2575 5686 +/- 761** 23794 +/- 2644 NA
Deactivation of Dopamine (ms) 184 +/- 15 69 +/- 4** 273 +/- 17** NA

Hu XQ, Lovinger DM (2005) Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+. The Journal of Physiology 568(Pt 2):381-96

back to top
go back to 5-HT3A

mAsp298Glu

The functional effects of introducing mutations D298A, D298E and D298R into the mouse 5-HT3A subunit were studied in HEK-293 cells using the whole-cell patch clamp technique. This experiment suggested that charge at residue D298 plays an important role in gating kinetics and modulation of extracellular Ca2+.

In order to conserve the charge of residue 298, Asp was substituted by Glu. The D298E mutant did not significantly alter the potency of 5-HT (EC50 = 1.84 +/- 0.10 uM) when compared with wild-type (EC50 = 1.74 +/- 0.09 ÁM ). The mutant also significantly altered the gating kinetics by decreasing the rate of desensitization (4258 +/- 537 ms) when compared with wild-type ( 3365 +/- 162 ms) and deactivation (4032 +/- 324 ms) when compared with wild-type (3232 +/- 218 ms) .

The mutant increased the relative efficacy of partial agonist, 2-Me-5-HT and did not significantly alter the relative efficacy of dopamine when compared with wild type.

When [Ca2+]o was increased from 1.8mM to 10mM, the rate of activation, desensitization and deactivation increased in the mutant D298E. In contrast, when [Ca2+]o was decreased from 1.8mM to 0.1mM, the rate of kinetics decreased in the mutant.

Wild Type D298A D298E D298R
EC50 (mM ) 1.74 +/- 0.09 3.05 +/- 0.14 1.84 +/- 0.10 7.20 +/- 0.20
Desensitization of 5-HT (ms) 3365 +/- 162 565 +/- 41** 4258 +/- 537 200 +/- 15**
Deactivation of 5-HT(ms) 3232 +/- 218 337 +/- 20** 4032 +/- 324 122 +/- 8**
Activation of 2-Me-5-HT (ms) 259 +/- 14 54 +/- 6** 298 +/- 12 NA
Desensitization of 2-Me-5-HT(ms) 5653 +/- 383 3462 +/- 223** 9730 +/- 1352** NA
Deactivation of 2-Me-5-HT(ms) 633 +/- 38 101 +/- 5** 879 +/- 53** NA
Activation of Dopamine (ms) 543 +/- 18 48 +/- 6** 832 +/- 70** NA
Desensitization of Dopamine(ms) 17967 +/- 2575 5686 +/- 761** 23794 +/- 2644 NA
Deactivation of Dopamine (ms) 184 +/- 15 69 +/- 4** 273 +/- 17** NA

 

Hu XQ, Lovinger DM (2005) Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+. The Journal of Physiology 568(Pt 2):381-96

back to top
go back to 5-HT3A

Previous Next