Abstract
Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.
Highlights
Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops
The single-channel conductance (␥) of human and mouse homomeric 5-hydroxytryptamine type 3A (5-HT3A) receptors is below the limit of direct resolution by single-channel recording and has been inferred by fluctuation analysis to be within the range 0.3–1.3 pS, dependent, at least in part, upon the concentration of divalent cations within the extracellular medium (27, 28, 34, 45– 49)
By using a combination of alanine- and arginine-scanning mutagenesis, SCAM, and single-channel recording from outside-out membrane patches, we have identified residues within the intracellular MA helix of the human 5-HT3A receptor that influence ion permeation
Summary
5-HT3A Receptor Constructs and Transfection of Subunit cDNAs—cDNAs encoding human 5-HT3A(QDA) and mutant subunit constructs thereof were cloned into prk[5]. Electrophysiology—The outside-out patch configuration was used to record single-channel currents from patches excised from transfected tsA-201 cells. Patch electrodes were filled with a solution comprising (in mM): 130 potassium gluconate, 5 NaCl, 2 MgCl2, 5 EGTA, and 10 HEPES (pH 7.2 adjusted with KOH) and had resistances within the range 4 –10 megaohms. MTS reagents were diluted in the electrode solution for outside-out patch experiments to yield a final concentration of 200 M. Sections of data recorded from outside-out patches, in which unitary events predominated, were selected for analysis. Tionship ␥ ϭ i/(Vm Ϫ Erev), in which i is the current amplitude of single-channel events, Vm is the holding potential (Ϫ74 mV), and Erev is the experimentally determined reversal potential. Analysis was performed during portions of the recording when unitary events predominated. Data sets were compared using one-way analysis of variance with a post hoc Tukey’s test. p Ͻ 0.05 was considered significant
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