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J. Biol. Chem., Vol. 278, Issue 47, 46583-46589, November 21, 2003

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Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT_3A Receptors Links Agonist Binding to Channel Gating^*

Xiang-Qun Hu, Li Zhang, Randall R. Stewart, and Forrest F. Weight

From the Laboratory of Molecular and Cellular Neurobiology, NIAAA, National Institutes of Health, Bethesda, Maryland 20892-8115

Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT_3A receptors and 7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT_3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT₃ receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT_3A receptors.

Received for publication, August 13, 2003 , and in revised form, September 8, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Molecular and Cellular Neurobiology, NIAAA, National Institutes of Health, Park Bldg., Rm. 150, Bethesda, MD 20892-8115. Tel.: 301-443-8163; Fax: 301-480-6882; E-mail: xhu{at}mail.nih.gov.

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