Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+

The TM2-TM3 extracellular loop is critical for activation of the Cys-loop family of ligand-gated ion channels. The contribution of aspartate 298 (D298), an amino acid that links the transmembrane domain 2 (TM2) to the TM2-TM3 loop, in mouse 5-hydroxytryptamine(3A) (5-HT3A) receptor function was probed with site-directed mutagenesis in the present study. This negatively charged residue was replaced with an alanine to neutralize the charge, with a glutamate to conserve the charge, or with an arginine to reverse the charge. Human embryonic kidney 293 (HEK 293) cells transfected with the wild-type and mutant receptors were studied by combining whole-cell patch-clamp recording with fast agonist application. The D -> A or D -> R mutations resulted in a receptor with reduced 5-HT potency, and accelerated kinetics of desensitization and deactivation. In addition, the efficacy of partial agonists was reduced by the D -> A mutation. The D -> E mutation produced a receptor with properties similar to those of the wild-type receptor. In addition, the potential role of this residue in modulation of the receptor by extracellular calcium ([Ca2+](o)) was investigated. Increasing [Ca2+]. inhibited 5-HT-activated currents and altered receptor kinetics in a similar manner in the wild-type and D298E receptors, and this alteration was eliminated by the D -> A and D -> R mutations. Our data suggest that the charge at D298 participates in transitions between functional states of the 5-HT3A receptor, and provide evidence that the charge of the side-chain at residue D298 contributes to channel gating kinetics and is crucial for Ca2+ modulation.