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Received June 20, 2005
Revised July 26, 2005
Accepted after revision August 5, 2005
1 National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health
* To whom correspondence should be addressed. E-mail: lovindav{at}mail.nih.gov.
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 TM2-TM3 loop, in the mouse 5-hydroxytryptamine3A (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 DR 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 DA mutation. The DE 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+]o 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 DA and DR 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.
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