Discovery of a novel allosteric modulator of 5-HT3 receptor: Inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site

TY - JOUR

T1 - Discovery of a novel allosteric modulator of 5-HT3 receptor

T2 - Inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site

AU - Trattnig, Sarah M

AU - Harpsøe, Kasper

AU - Thygesen, Sarah B

AU - Rahr, Louise M

AU - Ahring, Philip K

AU - Balle, Thomas

AU - Jensen, Anders A

PY - 2012/7/20

Y1 - 2012/7/20

N2 - The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT3 receptors (5-HT 3Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC50 values of -1μM at 5-HT 3Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT3A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit andTM1andTM2in the (-)-subunit. The Ser248, Leu 288, Ile290, Thr294, and Gly306 residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser292 and Val310, and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser248, Thr294, and Gly306 convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT 3R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.

AB - The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT3 receptors (5-HT 3Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC50 values of -1μM at 5-HT 3Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT3A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit andTM1andTM2in the (-)-subunit. The Ser248, Leu 288, Ile290, Thr294, and Gly306 residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser292 and Val310, and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser248, Thr294, and Gly306 convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT 3R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.

U2 - 10.1074/jbc.M112.360370

DO - 10.1074/jbc.M112.360370

M3 - Journal article

C2 - 22589534

SN - 0021-9258

VL - 287

SP - 25241

EP - 25254

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 30

ER -