Abstract
The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily.
Highlights
5-HT31 receptors are ligand-gated ion channels (LGICs) whose activation results in membrane depolarization due to the gating of an integral cation-selective channel [3, 4]. 5-HT3 receptors can exist as homomeric assemblies of 5-HT3A recep
Shorter duration applications of agonist (30 M 5-HT, 5 s) to cells expressing the 5-HT3A-STM receptor produced whole-cell responses with a different time course of recovery; receptors appeared to remain in a conducting state for Ͼ20 s after agonist removal. This combination of slower activation, reduced desensitization, and increased affinity exhibited by the 5-HT3A-STM suggests that this receptor may attain a desensitized but conducting state (D*) in the presence of agonist, similar to that identified in some mutant acetylcholine receptors [1, 24, 25]
Gating and Desensitization of the 5-HT3A-STM—Ligandgated ion channels, exemplified by the nicotinic acetylcholine (nACh) receptor, open rapidly following agonist binding and, in the continued presence of agonist, may enter an agonist-bound but nonconducting “desensitized” state [23]; this desensitized state of a receptor possesses a higher affinity for agonist than the closed resting or open states of the channel
Summary
5-HT31 receptors are ligand-gated ion channels (LGICs) whose activation results in membrane depolarization due to the gating of an integral cation-selective channel [3, 4]. 5-HT3 receptors can exist as homomeric assemblies of 5-HT3A recep-. Once the channels have opened, one crucial difference becomes clear; whereas the integral ion channels of the 5-HT3 and nACh receptors are cation-selective and are primarily concerned with excitatory neurotransmission, glycine and ␥-aminobutyric acid receptors are anion-selective and are involved in inhibitory neurotransmission. The occurrence of both anionic and cationic-selective receptors in such a closely related family provides a basis for the identification of the molecular determinants of ion selectivity.
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