Abstract
Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca2+ entry into neurones. We examined the influence on Ca2+ permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT3B subunit (yielding 5-HT3A(QDA)) increased PCa/PCs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca2+, the single channel conductance of the 5-HT3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca2+. A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca2+ on macroscopic currents mediated by 5-HT3A receptors. We introduced a D293A mutation into the 5-HT3A(QDA) receptor (yielding the 5-HT3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca2+. The D293A mutation decreased the PCa/PCs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca2+. The D293A mutation also reduced PCa/PCs when engineered into the wild-type 5-HT3A receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence PCa/PCs and additionally directly demonstrated a depression of single channel conductance by Ca2+.
Highlights
On macroscopic currents mediated by 5-HT3A receptors
The 5-hydroxytryptamine type 3 (5-HT3)4 receptor is a cation-selective ligand-gated ion channel of the Cys-loop superfamily with structural and functional homology to the family of nicotinic acetylcholine (ACh) receptors [1,2,3]. 5-HT3 receptor activation elicits rapidly activating and desensitizing inward current responses mediated by the flux of both mono- and divalent cations [2]. 5-HT3 receptor stimulation contributes to fast excitatory synaptic transmission in the central nervous system (4 –7) and modulates the release of several neurotransmitters including acetylcholine, cholecystokinin, dopamine, glutamate, norepinephrine, and ␥-aminobutyric acid, the exocytosis of which is enhanced by direct Ca2ϩ influx through the ionophore of presynaptic 5-HT3 receptors [8, 9]
Residues in the MA helix of the 5-HT3A Receptor Influence Ca2ϩ Permeability—We recently demonstrated that arginine residues 432, 436, and 440 within the MA helix motif of the
Summary
5-HT, 5-hydroxytryptamine; ACh, acetylcholine; MA, membrane-associated; TM, transmembrane; pS, picosiemens; HEK, human embryonic kidney; I-V, whole-cell current-voltage relationship; i-V, single channel current-voltage relationship. Arginine residues engineered into the corresponding locations of both subunit species of the nicotinic ACh ␣42 receptor depress single channel conductance [22]. Such findings have been rationalized in the light of the 4 Å resolution model of the nicotinic ACh receptor of Torpedo marmorata in which the five MA-stretch helices manifest as an inverted pentagonal cone that projects from the plasma membrane into the cytoplasm forming the intracellular vestibule of the channel [26]. The introduction of a D293A mutation into the 5-HT3A(QDA) receptor (yielding the 5-HT3A(QDA D293A) construct) markedly suppressed PCa/PCs and reduced inwardly directed single channel conductance
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