Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

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-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 wildtype 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-HT 3 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.In the nervous system, serotonin type 3 (5-HT 3 ) 1 receptors can mediate fast excitatory synaptic transmission and modulate neurotransmitter release (1). To date two 5-HT 3 receptor subunits have been identified: 5-HT 3A and 5-HT 3B (2, 3). The 5-HT 3A receptor subunits can form functional channels homomerically (2), whereas the 5-HT 3B receptor subunits are nonfunctional when expressed alone (3). However, the 5-HT 3B receptor subunits can form heteromeric channels with the 5-HT 3A receptor subunits, which results in modified biophysical characteristics compared with homomerically expressed 5-HT 3A receptor subunits (3). 5-HT 3A and 5-HT 3B receptor subunits also have different distribution patterns in the nervous system. The 5-HT 3A receptor subunits are expressed in both central and peripheral neurons, whereas the 5-HT 3B receptor subunits are restricted to peripheral neurons (4). This suggests that homomeric 5-HT 3A receptors play a dominant role in 5-HT 3 receptor-mediated responses in the central nervous system. 5-HT 3 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 be...

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

“…The L293S mutation could release the molecular constraints and lower these energy barriers such that 5-HI binding to the mutant receptor is able to produce a sufficient conformational change to open the channel. A similar finding was made with respect to the R222A mutation which converts the antagonist apomorphine into a potent and efficacious agonist by facilitating the conversion between different states of the 5-HT 3A receptor (Hu et al, 2003).…”

Pore conformations and gating mechanism of a Cys-loop receptor

“…Several studies have suggested that motions in the ligandbinding domain affect the pore conformation through residues that precede M1 (23,24) and by means of interactions between loops of the neurotransmitter-binding domain and the M2-M3 loop (25)(26)(27)(28)(29). Hence, the aforementioned rigid tilting motions can be reasonably achieved by simultaneous movements of the N-terminal tip of M1 and the C-terminal tip of M2 toward or away from the axis of ion conduction.…”

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

“…Characterization of Mutations in the Pre-TM1 Region of the GABA A -R ␤ 2 Subunit-A previous report has suggested that a cluster of conserved basic residues in the pre-TM1 region of the 5-HT 3 receptor are involved in receptor activation (13). To determine whether these residues have a similar role in the GABA A -R ␤ 2 subunit, we created the following ␤ 2 subunit mutations: K215D, R216D, and N217D.…”

“…Interactions between charged residues in the flexible loops 2 and 7 in the extracellular domain and those in the short linker between the second and third transmembrane domains (TM2-3L) of the GABA A -R ␣ 1 subunit have been implicated in the process of receptor activation (12). In addition, a recent report suggests that the pre-TM1 region is critical for receptor activation in the closely related serotonin (5-HT 3 ) receptor (13). In this study, we used site-directed mutagenesis and a number of GABA A -R agonists of varying efficacies to examine the contribution of the corresponding flexible loops in the GABA A -R ␤ 2 subunit to receptor activation.…”

“…In this study, we used site-directed mutagenesis and a number of GABA A -R agonists of varying efficacies to examine the contribution of the corresponding flexible loops in the GABA A -R ␤ 2 subunit to receptor activation. Based on previous studies (12)(13)(14) and the presence of highly conserved charged residues (see Fig. 1), we hypothesized that interactions between charged residues in these domains are crucial for coupling agonist binding to channel gating.…”