Rings of Charge within the Extracellular Vestibule Influence Ion Permeation of the 5-HT3A Receptor

Abstract

The determinants of single channel conductance (γ) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that γ is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp113 and Asp127) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT3A) receptor (5-HT3A(QDA)) upon γ, modulation of the latter by extracellular Ca2+, and the permeability of Ca2+ with respect to Cs+ (PCa/PCs). Mutations neutralizing (Asp → Asn), or reversing (Asp → Lys), charge at the 113 locus decreased inward γ by 46 and 58%, respectively, but outward currents were unaffected. The D127N mutation decreased inward γ by 82% and also suppressed outward currents, whereas the D127K mutation caused loss of observable single channel currents. The forgoing mutations, except for D127K, which could not be evaluated, ameliorated suppression of inwardly directed single channel currents by extracellular Ca2+. The PCa/PCs of 3.8 previously reported for the 5-HT3A(QDA) construct was reduced to 0.13 and 0.06 by the D127N and D127K mutations, respectively, with lesser, but clearly significant, effects caused by the D113N (1.04) and D113K (0.60) substitutions. Charge selectivity between monovalent cations and anions (PNa/PCl) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT3A receptor that markedly influence γ, PCa/PCs, and additionally the suppression of γ by Ca2+.

Copper and protons directly activate the zinc-activated channel

“…This characteristic is very similar to the monovalent cation permeability of 5-HT 3 A receptors [17]. In contrast to the Ca 2+ and Mg 2+ permeability of 5-HT 3 A and nACh α7 receptors [24,25], our data indicate that ZAC is not only impermeable to both divalent cations, but that the ions also inhibit the channel. In previous studies several residues in the outer extracellular (20′) and cytoplasmic rings (−4′) of transmembrane domain 2 (TM2) in 5-HT 3 A and nACh α7 receptors as well as the Asp 127 in the extracellular vestibule of 5-HT 3 A have been identified as determinants of the divalent cation permeability of these receptors [24,26–28].…”

“…In contrast to the Ca 2+ and Mg 2+ permeability of 5-HT 3 A and nACh α7 receptors [24,25], our data indicate that ZAC is not only impermeable to both divalent cations, but that the ions also inhibit the channel. In previous studies several residues in the outer extracellular (20′) and cytoplasmic rings (−4′) of transmembrane domain 2 (TM2) in 5-HT 3 A and nACh α7 receptors as well as the Asp 127 in the extracellular vestibule of 5-HT 3 A have been identified as determinants of the divalent cation permeability of these receptors [24,26–28]. An alignment of the amino acid sequences of ZAC, 5-HT3A and α7 subunits predicts the corresponding residues in ZAC to be Gln (TM2 20′: Asp 293 in 5-HT 3 A, Glu 281 in nAChα7), Arg (Asn 269 in TM2 −4′ ring in 5-HT 3 A, Asp 257 in nAChα7) and Trp (Asp 127 in 5-HT 3 A, Asp 119 in nAChα7), and we propose that these substantial differences in the physicochemical properties of the residues at these positions of ZAC are likely to contribute to the impermeability of Ca 2+ and Mg 2+ .…”

A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability

“…As Asp 42 was found to form an electrostatic interaction with Lys 46 from the adjacent subunit, this interaction either hinders the ability of Asp 42 to interact with calcium ions, or instead this interaction may be necessary for the normal function of the α7 nAChR as the ligand-binding domain of nAChRs establishes a major communication link with the pore domain [24]. Interestingly, two aspartate residues in the ECD of the 5-HT 3 A receptor were shown through mutagenesis to influence calcium permeability, supporting the idea that the ECD plays an important role in the ionic permeability of ligand gated ion channels [25]. …”

Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels

“…In particular, the two rings of Cs + bound to Asp86 and Asp88 and the ring of sulphates bound to Lys38 and Arg109 are all exposed to the lumen of the channel and might have functional implications in ion permeation or selectivity. Important residues for ion permeation have already been identified in a similar region of the ECD from AchBP structures bound to ions (Hansen et al , 2008; Brams et al , 2011), and by site‐directed mutagenesis studies carried out on the nACh (Hansen et al , 2008) or 5‐HT3A (Livesey et al , 2011) receptors.…”