Human 5-HT3A -- Phe 287

Species Original Mutated to Mutation
Human Phe 287  

 

Rat Equivalent Phe 292    
Mouse Equivalent Phe 292

Cys

Ala

 

F292C

F292A

 

 

mPhe292Ala

The F269A, L270A, L270F, L270S, L270V and L270Y mutations in the mouse 5-HT3A subunits enhanced alcohol and anesthetic function on 5-HT3 receptor. Recombinant 5-HT3A receptors were expressed in Xenopus oocytes and 5-HT-activated currents were recorded under voltage-clamp. The wild-type receptors were used to compare the potency of 5-HT on mutants. The potency of 5-HT increased in F269A, L270A, L270F, L270S, L270V and L270Y mutants demonstrated by a leftward shift of the 5-HT concentration-response curves.

The mutation was made in the mouse 5-HT3A subunit at Phe292. This residue was numbered Phe269 in the paper in which numbering began at the end of the signal sequence.

Receptor EC50 (uM) Hill Coefficient
Wild-Type 1.64 3.09
F292A 0.28 2.60

The resulting receptors displayed either anesthetic-induced inhibition or decreased enhancement compared to WT. It also exhibited inhibition of receptor functin by both 23uM decanol and 2uM propofol. 200mM ethanol, which enhances WT function, inhibited F292A mutant.

 

Lopreato GF, Banerjee P, Mihic SJ (2003) Amino Acids in Transmembrane Domain Two Influence Anesthetic Enhancement of Serotonin-3A Receptor Function. Molecular Brain Research 118:45-51

 

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mPhe292Cys

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

 

  Wild Type F292C
EC50 1.67 +/- 0.09 not tested
Hill Coefficient 2.0 +/- 0.1 not tested
Mg Inhibition (before MTSET) -41 +/- 3 -41 +/- 3
Mg Inhibition (after MTSET) -39 +/- 3 -39 +/- 2
Deactivation T50 ratio 0.95 +/- 0.03 2.09 +/- 0.16

 

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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