Human 5-HT3A -- Leu 288

Species Original Mutated to Mutation
Human Leu 288  

 

Rat Equivalent Leu 293    
Mouse Equivalent Leu 293

Ala

Cys

Gly

Phe

Ile

Ser

Val

Tyr

L293A

L293C

L293G

L293F

L293I

L293S

L293V

L293Y

 

mLeu293Ala

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 Leu293. This residue was numbered Leu270 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
L293A 0.18 1.69

Sensitivity to the enhancing effects of enflurane were retained in this mutant. Considerable inhibition was observed in the presence of chloroform.

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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mLeu293Cys

After the application of 1mM MTSET with 10uM 5-HT, The EC50 current was irreversibly reduced. The reaction of MTSET with L293C was shown to be voltage-dependent. After the application of 1mM MTSES with 10uM 5-HT, the channels were locked in an open state, and when the locked channel was given 300uM diltiazem there was no effect. The reaction of MTSES with L293C 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

The mouse 5-HT3A receptor was expressed in HEK293 cells, and was also studied in N1E-115 neuroblastoma cells expressing the native 5-HT3A receptor. Currents activated by 5-HT in the L293C mutant were slightly inhibited by co-application with 5-HI. 5-HT activated with an initial rising phase faster than that reported for the wild type. A rebound 5-HT-evoked current was observed in the mutant receptors when the co-application of 5-HT and 5-HI was terminated, and a faster apparent desensitization of 5-HT activated current was reported than in the wild type receptor.

Hu XQ, Lovinger DM (2008) The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole. Neuropharmacology 54(8):1153-65

  Wild Type L293C
EC50 1.67 +/- 0.09 1.37 +/- 0.12
Hill Coefficient 2.0 +/- 0.1 1.6 +/- 0.1
Mg Inhibition (before MTSET) -41 +/- 3 -42 +/- 3
Mg Inhibition (after MTSET) -39 +/- 3 -38 +/- 3
Deactivation T50 ratio 0.95 +/- 0.03 0.74 +/- 0.02

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

Mutation of the TM2 15' residue in GABAA and glycine receptors affects sensitivity to alcohol and some general anesthetics. The equivalent mouse 5-HT3A L293 (TM2 15') residue was mutated to determine if this residue is active in gating changes that reduce or eliminate n-chain alcohol and TCEt-induced enhancement of receptor function.

Serotonin concentration-response curves generated using oocytes and HEK293 cells expressing the mutant receptor showed increased 5-HT potency. Resting currents in the absence of agonists were not increased relative to the wild-type receptor, nor did MDL72222 application produce a change in holding current, indicating that the L293C mutation does not produce spontaneous opening of a significant fraction of the available channel. The current-voltage relationships suggest that the L293C variant does not affect relative ion permeability of the channel pore. Activation with 1 mM 5-HT caused a biexponential deactivation , composed of a fast component (~65% of total) faster than the wild type, followed by a slow component.

Dopamine efficacy was increased in the mutant receptor when compared to the wild-type receptor, suggesting an increase in probability of opening.

Ethanol enhances the wild type receptor's function; however, ethanol inhibited the mutant receptor's function. This inhibition was rapidly reversible. Low levels of hexanol inhibited L293C receptor function; high levels of hexanol enhanced L293C receptor function, although not to the level observed in the wild type receptor. TCEt potency and efficacy were reduced in the L293C variant.

Hu XQ , Hayrapetyan V , Gadhiya JJ , Rhubottom HE , Lovinger DM , Machu TK . (2006) Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions. Br J Pharmacol. 148(1):88-101.

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mLeu293Phe

 

Receptor EC50 (uM) Hill Coefficient
Wild-Type 1.64 3.09
L293F 0.10 3.37

Sensitivity to the enhancing effects of enflurane were retained in this mutant. However, L293F was almost completely insensitive to enhancing effects of isoflurane. Chloroform failed to enhance the receptor function.

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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mLeu293Gly

Mutation of the TM2 15' residue in GABAA and glycine receptors affects sensitivity to alcohol and some general anesthetics. The equivalent mouse 5-HT3A L293 (TM2 15') residue was mutated to determine if this residue is active in gating changes that reduce or eliminate n-chain alcohol and TCEt-induced enhancement of receptor function.

Serotonin concentration-response curves generated using oocytes and HEK293 cells expressing the mutant receptor showed increased 5-HT potency. Resting currents in the absence of agonists were not increased relative to the wild-type receptor, nor did MDL72222 application produce a change in holding current, indicating that the L293G mutation does not produce spontaneous opening of a significant fraction of the available channel. The current-voltage relationships suggest that the L293G variant does not affect relative ion permeability of the channel pore. Activation with 1 mM 5-HT caused a biexponential deactivation , composed of a fast component (~23% of total) faster than the wild type, followed by a slow component.

Dopamine efficacy was increased in the mutant receptor when compared to the wild-type receptor, suggesting an increase in probability of opening. L293G had a significantly greater deactivation time constant than the wild type after removal of 3mM dopamine.

Ethanol enhances the wild type receptor's function; however , ethanol inhibited the mutant receptor's function. This inhibition was rapidly reversible. Hexanol enhanced the function of the L293G receptor to an extent similar to the wild type receptor, except at 5 and 10 mM, where enhancement was reduced. TCEt potency and efficacy were reduced in the L293G variant. TCEt had no effect on L293G receptor function. Ethanol and TCEt increase activation rates and peak current amplitudes in the wild type receptor. However, neither ethanol nor TCEt affected L293G activation rates, nor did they alter peak current amplitudes.

Hu XQ , Hayrapetyan V , Gadhiya JJ , Rhubottom HE , Lovinger DM , Machu TK . (2006) Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions. Br J Pharmacol. 148(1):88-101.

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mLeu293Ile

Mutation of the TM2 15' residue in GABAA and glycine receptors affects sensitivity to alcohol and some general anesthetics. The equivalent mouse 5-HT3A L293 (TM2 15') residue was mutated to determine if this residue is active in gating changes that reduce or eliminate n-chain alcohol and TCEt-induced enhancement of receptor function.

Serotonin concentration-response curves generated using oocytes and HEK293 cells expressing the mutant receptor showed increased 5-HT potency. Resting currents in the absence of agonists were not increased relative to the wild-type receptor, nor did MDL72222 application produce a change in holding current, indicating that the L293I mutation does not produce spontaneous opening of a significant fraction of the available channel. The current-voltage relationships suggest that the L293I variant does not affect relative ion permeability of the channel pore. Activation with 1 mM 5-HT caused a biexponential deactivation , composed of a fast component (~65% of total) faster than the wild type, followed by a slow component.

Dopamine efficacy was increased in the mutant receptor when compared to the wild-type receptor, suggesting an increase in probability of opening.

Ethanol enhanced the mutant receptor's function, similar to the wild-type receptor. This stimulation was rapidly reversible. Hexanol enhanced the function of the L293I receptor to an extent similar to the wild type receptor, except at 5 and 10 M, where enhancement was reduced. Similar to the wild type receptor, TCEt enhanced 5-HT mediated currents in the L293I variant.

Hu XQ , Hayrapetyan V , Gadhiya JJ , Rhubottom HE , Lovinger DM , Machu TK . (2006) Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions. Br J Pharmacol. 148(1):88-101.

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mLeu293Ser

Receptor EC50 (uM) Hill Coefficient
Wild-Type 1.64 3.09
L293S 0.67 1.82

Sensitivity to the enhancing effects of enflurane were retained in this mutant. L293S also showed similar effects to isoflurane. Chloroform failed to enhance the receptor function. The greatest degree of inhibition was observed using the lowest 5-HT concentration.

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

The mouse 5-HT3A receptor was expressed in HEK293 cells, and was also studied in N1E-115 neuroblastoma cells expressing the native 5-HT3A receptor. 5-HI acts as a partial agonist in the 5-HT3A (L293S) construct that lacks 5-HI induced potentiation. The direct activation of the mutant by 5-HI occurs at drug concentrations higher than those needed to elicit potentiation in the wild type receptor. Currents activated by 5-HT in the L293S mutants were slightly inhibited by co-application on 5 mM 5-HI. 5-HT (1 ?M) activated an initial rising phase faster than that reported for the wild type. A rebound current was observed in the mutant receptor when the co-application of 5-HT and 5-HI was terminated, and a faster apparent desensitization of 5-HT activated current was reported than in the wild type receptor. The concentration response curve for the partial agonist dopamine was shifted to the left (3.5 fold increase in dopamine potency) and maximal dopamine efficacy was increased in the 5-HT3A(L293S) receptor compared to the wild type, suggesting that the L293 residue is important for the coupling of agonist binding to channel gating in the 5-HT3A receptor. A decreased Hill slope was also reported for the mutant. Neither TMB-8 nor MDL72222 altered the holding current in cells expressing the mutant receptor, suggesting that the mutation does not produce spontaneous channel activity.

Hu XQ, Lovinger DM (2008) The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole. Neuropharmacology 54(8):1153-65

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Mutation of the TM2 15' residue in GABAA and glycine receptors affects sensitivity to alcohol and some general anesthetics. The equivalent mouse 5-HT3A L293 (TM2 15') residue was mutated to determine if this residue is active in gating changes that reduce or eliminate n-chain alcohol and TCEt-induced enhancement of receptor function.

Serotonin concentration-response curves generated using oocytes and HEK293 cells expressing the mutant receptor showed increased 5-HT potency. Resting currents in the absence of agonists were not increased relative to the wild-type receptor, nor did MDL72222 application produce a change in holding current, indicating that the L293S mutation does not produce spontaneous opening of a significant fraction of the available channel. The current-voltage relationships suggest that the L293S variant does not affect relative ion permeability of the channel pore. Activation with 1 mM 5-HT caused a biexponential deactivation , composed of a fast component (~65% of total) faster than the wild type, followed by a slow component.

Dopamine efficacy was increased in the mutant receptor when compared to the wild-type receptor, suggesting an increase in probability of opening.

Ethanol enhances the wild type receptor's function; however, ethanol inhibited the mutant receptor's function. This inhibition was rapidly reversible. Hexanol enhanced the function of the L293S receptor, but at concentrations of 2 mM and lower, receptor potentiation was less than that observed in the wild type receptor. TCEt potency and efficacy were reduced in the L293S variant. TCEt inhibited L293S receptor function. Ethanol and TCEt increase activation rates and peak current amplitudes in the wild type receptor. However, neither ethanol nor TCEt affected L293S activation rates, nor did they alter peak current amplitudes. TCEt enhanced the fast phase of desensitization and enhanced current decay in the L293S mutant.

Hu XQ , Hayrapetyan V , Gadhiya JJ , Rhubottom HE , Lovinger DM , Machu TK . (2006) Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions. Br J Pharmacol. 148(1):88-101.

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mLeu293Val

Receptor EC50(uM) Hill Coefficient
Wild-Type 1.64 3.09
L293V 0.26 1.47

This mutant showed inhibition by enflurane. Considerable inhibition was observed under the presence of chloroform.

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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mLeu293Tyr

Receptor EC50 (uM) Hill Coefficient
Wild-Type 1.64 3.09
L293Y 0.24 2.45

Sensitivity to the enhancing effects of enflurane were retained in this mutant. However, L293Y was almost completely insenitive to the effects of isoflurane. Considerable inhibition was observed under the presence of chloroform. The Wild-type showed enflurane, isoflurane, chloroform, and halothane all enhancing wild-type receptor function in a concentration-dependent manner. However, L293Y only showed enflurane producing any such enhancement of receptor function.

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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