Human 5-HT3A -- Arg 436

Species Original Mutated to Mutation
Human Arg 436 Asp

Glu

Gln

Phe

Cys

R436D

R436F

R436E

R436Q

R436C

Rat Equivalent Arg 435    
Mouse Equivalent Arg 441    

 

hArg436Asp

This mutation caused a 5-fold increase in the single-channel conductance. When it was combined with R432Q, the double mutation produced conductance that was greater than the sum of the individual mutations.The results were the same when it was combined with R440A. In a triple mutation of R432Q, R436D, and R440A, the single-channel conductance increased substantially.

Kelley SP, Dunlop JI, Kirkness EF, Lambert JJ, Peters JA (2003) A Cytoplasmic Region Determines Single-Channel Conductance in 5-HT3 Receptors. Nature 424(6946):321-324

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In this study the cytoplasmic membrane-associated (MA) helix arginine residues 432 (-4'), 436 (0'), and 440 (4') of a homomeric 5HT3AR were replaced with equivalent residues found in 5HT3B to determine if these mutations had any effect on single channel conductance (γ). The techniques of whole-cell and outside-out patch recording were used to measure γ indirectly (by variance analysis) and directly (from single channel amplitudes), respectively. The study concluded that the MA 0' position had the greatest effect on γ.

The R436D mutation caused an ~9-fold increase in γ. The R432Q, R436D two-point mutation caused an ~18-fold increase in γ. The R436D, R440A two-point mutation caused an ~26-fold increase in γ. The R432Q, R436D, R440A three-point mutation caused an ~38-fold increase in γ.

Hales TG, Dunlop JI, Deeb TZ, Carland JE, Kelley SP, Lambert JJ, Peters JA (2006) Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors. The Journal of Biological Chemistry 281(12):8062-71

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This paper analyzed the effects of point mutations in the MA-helix on the relative Ca2+ permeability of 5-HT3A receptors. While the R432Q and R440A mutations had no effect on relative Ca2+ permeability, the R436D mutation increased Ca2+ permeability by more than two-fold. Simultaneous mutation of all three residues did not further increase the relative Ca2+ permeability. In order to determine the mechanism by which the R436 exerts its effect, several residues (Ala, Phe, Asp, Arg) were susbstituted into the 0' position in the background of the R432Q and R440A mutations. The researchers found that the charge of the 0' residue was the main determinant of Ca2+ permeability. Further analysis demonstrated that while relative Ca2+ permeability was significantly increased, the conductivity was supressed with increasing concentrations of extracellular Ca2+. This paper demonstrates that the MA-helix of the 5-HT3A receptor has a significant role in determining the relative Ca2+ permeability.

Livesey MR, Cooper MA, Deeb TZ, Carland JE, Kozuska J, Hales TG, Lambert JJ, Peters JA. (2008) Structural Determinants of Ca2+ Permeability and Conduction in the Human 5-Hydroxytryptamine Type 3A Receptor. The Journal of Biological Chemistry 283(28):19301-13

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hArg436Glu

In this study the cytoplasmic membrane-associated (MA) helix arginine residues 432 (-4'), 436 (0'), and 440 (4') of a homomeric 5HT3AR were replaced with equivalent residues found in 5HT3B to determine if these mutations had any effect on single channel conductance (γ). The techniques of whole-cell and outside-out patch recording were used to measure γ indirectly (by variance analysis) and directly (from single channel amplitudes), respectively. The study concluded that the MA 0' position had the greatest effect on γ.

The R436E mutation caused an ~13-fold increase in γ. The R432Q, R436E, R440A three-point mutation caused an ~36-fold increase in γ.

Hales TG, Dunlop JI, Deeb TZ, Carland JE, Kelley SP, Lambert JJ, Peters JA (2006) Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors. The Journal of Biological Chemistry 281(12):8062-71

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hArg436Gln

In this study the cytoplasmic membrane-associated (MA) helix arginine residues 432 (-4'), 436 (0'), and 440 (4') of a homomeric 5HT3AR were replaced with equivalent residues found in 5HT3B to determine if these mutations had any effect on single channel conductance (γ). The techniques of whole-cell and outside-out patch recording were used to measure γ indirectly (by variance analysis) and directly (from single channel amplitudes), respectively. The study concluded that the MA 0' position had the greatest effect on γ.

The R436Q mutation caused an ~7-fold increase in γ. The R432Q, R436Q, R440A three-point mutation caused an ~24-fold increase in γ.

Hales TG, Dunlop JI, Deeb TZ, Carland JE, Kelley SP, Lambert JJ, Peters JA (2006) Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors. The Journal of Biological Chemistry 281(12):8062-71

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hArg436Phe

In this study the cytoplasmic membrane-associated (MA) helix arginine residues 432 (-4'), 436 (0'), and 440 (4') of a homomeric 5HT3AR were replaced with equivalent residues found in 5HT3B to determine if these mutations had any effect on single channel conductance (γ). The techniques of whole-cell and outside-out patch recording were used to measure γ indirectly (by variance analysis) and directly (from single channel amplitudes), respectively. The study concluded that the MA 0' position had the greatest effect on γ.

The R436F mutation had little effect on γ. The R432Q, R436F, R440A three-point mutation caused an ~2-fold increase in γ.

Hales TG, Dunlop JI, Deeb TZ, Carland JE, Kelley SP, Lambert JJ, Peters JA (2006) Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors. The Journal of Biological Chemistry 281(12):8062-71

 

 

hArg436Cys

This study elucidated the mechanism by which the 0' residue of the 5-HT3A MA-helix affected g. Here Arg436 was mutated to a Cys residue while Arg432 and Arg440 were mutated to Gln and Ala, respectively, yielding the 5-HT3A(QCA) receptor. The Cys436 residue was then dynamically modified using a series of the sulfhydryl reactive methanethiosulfonate (MTS) compounds, with each compound in the series having a similar volume but a different charge. Using the excised patch configuration to analyze unitary conductance, this study demonstrated that both the volume and charge of this residue affects g. Furthermore, the effects of charge overwhelmed the effects of volume, e.g. while an uncharged voluminous residue (Cys modified with propyl-MTS) impeded cation flow due to a steric hinderance in the ion pathway, a negatively charged residue of similar volume (Cys modified with ethylsulfate-MTS) increased conductance, despite the steric impediment, due to favorable electrostatic interactions between the permeant cation and the negatively charged residue. This study further supported the portal hypothesis: the cytoplasmic portals are narrow enough to affect ion flow through direct interactions with permeating ions (Unwin, 2005).

Deeb TZ, Carland JE, Cooper MA, Livesey MR, Lambert JJ, Peters JA, Hales TG (2007) Dynamic modification of a mutant cytoplasmic cysteine residue modulates the conductance of the human 5-HT3A receptor. The Journal of Biological Chemistry 282(9):6172-82

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