Abstract
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-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A 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-HT3A receptors.
Highlights
5-HT3A receptor subunits [3]. 5-HT3A and 5-HT3B receptor subunits have different distribution patterns in the nervous system
Functional Characterization of the Wild-type and Mutant (R222A) 5-HT3A Receptors—The wild-type and R222A 5-HT3A receptors were transiently expressed in human embryonic kidney (HEK) 293 cells, and their responses to the full agonist, 5-HT, or the partial agonist, 2-Me-5-HT, were recorded in the whole-cell configuration with fast solution exchange
In the wild-type receptor the current activated by 100 M 2-Me-5-HT was much smaller in amplitude than the current activated by 30 M 5-HT (Fig. 2A), whereas in the R222A mutant receptor the current activated by 100 M 2-Me-5-HT was similar in amplitude to that activated by 30 M 5-HT (Fig. 2B)
Summary
5-HT3, serotonin type 3; nACh, acetylcholine; TM, transmembrane domain; 2-Me-5-HT, 2-methyl-5-hydroxytryptamine; HEK cells, human embryonic kidney cells; WT, wild type. 5-HT3 receptors belong to a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh) receptors, glycine receptors, and ␥-aminobutyric acid type A receptors [5] The subunits in this superfamily are thought to assemble as pentamers with each subunit containing a large extracellular N-terminal domain, four transmembrane domains (TM1-TM4), a large intracellular loop between TM3 and TM4, and an extracellular C-terminal domain (Fig. 1A) [6]. The results suggest that Arg-222 is involved in transducing the signal that couples agonist binding to channel opening in 5-HT3A receptors. The time constants for activation, deactivation, and desensitization were determined with Levenberg-Marquardt algorithms
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