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 (Asp(113) and Asp(127)) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT(3)A) receptor (5-HT(3)A(QDA)) upon γ, modulation of the latter by extracellular Ca(2+), and the permeability of Ca(2+) with respect to Cs(+) (P(Ca)/P(Cs)). 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 Ca(2+). The P(Ca)/P(Cs) of 3.8 previously reported for the 5-HT(3)A(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 (P(Na)/P(Cl)) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT(3)A receptor that markedly influence γ, P(Ca)/P(Cs), and additionally the suppression of γ by Ca(2+).

FIGURE 1.

Locations of Asp¹¹³ and Asp¹²⁷ within a homology model of the 5-HT₃A(QDA) receptor. A and B, homology model of the human 5-HT₃A(QDA) receptor using the nACh receptor of Torpedo marmorata as a template (Protein Data Bank entry 2BG9 (9)). The locations of Asp¹¹³ (D113) and Asp¹²⁷ (D127) within the ECD are shown in side (A) and top (B) elevations. Note how the residues form two rings of charge that face into the central vestibule within the ECD. C, sequence alignment of the α1 subunit of the human adult skeletal muscle nACh receptor and human 5HT3A subunit across the relevant region of the proteins.

FIGURE 2.

Neutralization, or reversal, of charge at the Asp¹¹³ locus depresses inwardly directed single channel currents. Single channel events, elicited by 5-HT (10 μm), were recorded from outside-out patches excised from tsA-201 cells transfected with the construct of interest. The Cs⁺-based pipette solution and extracellular solution E1 were utilized. A, exemplar single channel events recorded at −80 and +80 mV from the 5-HT₃A(QDA), 5-HT₃A(QDA) D113N, and 5-HT₃A(QDA) D113K mutant receptors. B, i-V profiles for the 5-HT₃A(QDA) (solid circles), 5-HT₃A(QDA) D113N (open circles), and 5-HT₃A(QDA) D113K (inverted triangles) receptors. Note that mutation of the Asp¹¹³ residue does not impact upon outwardly directed single channel currents. Data points are the mean of a minimum of three observations made from separate patches, and error bars, where visible, indicate S.E. Data for the 5-HT₃A(QDA) receptor are from Livesey et al. (20).

FIGURE 3.

The 5-HT3A(QDA) D127N construct single channel i-V relationship. Single channel current events were recorded from excised outside-out patches in extracellular solution E1 and the Cs⁺-based pipette solution. A, exemplar currents recorded at −80 mV from the 5-HT3A(QDA), 5-HT3A(QDA) D127N, and 5-HT3A(QDA) D127K constructs. Note that the latter construct does not support resolvable single channel events in response to 5-HT. B, single channel i-V relationships for the 5-HT₃A(QDA) construct (filled squares) and the 5-HT₃A(QDA) D127N mutant receptor (open circles). Note that the 5-HT₃A(QDA) D127N receptor mediated single channel currents that rectify outwardly but are additionally substantially reduced at positive holding potentials. Data points are the mean of at least three independent determinations, and error bars, where visible, indicate S.E. Data for the 5-HT₃A(QDA) receptor are from Livesey et al. (20).

FIGURE 4.

Macroscopic I-V relationships for 5-HT₃A(QDA) receptor constructs harboring mutations to Asp¹¹³ and Asp¹²⁷ within the ECD. I-V plots were generated by the voltage ramp protocols detailed under “Experimental Procedures,” and 5-HT (10 μm)-evoked currents were recorded in an extracellular medium containing Ca²⁺ as the sole permeant ionic species. Representative leak-subtracted whole-cell currents are shown for 5-HT₃A(QDA) (A), 5-HT₃A(QDA) D113N (B), 5-HT₃A(QDA) D113K (C), 5-HT₃A(QDA) D127N (D), and 5-HT₃A(QDA) D127K (E) receptor constructs. Note that all mutations cause a negative shift in E_5-HT that is indicative of a reduced P_Ca/P_Cs ratio. Paralleling the latter is an enhancement of outward rectification of the macroscopic currents due to a progressively diminished ability to conduct Ca²⁺ inwardly.

FIGURE 5.

Mutations in the ECD of the 5-HT3A(QDA) construct attenuate suppression of γ by extracellular Ca²⁺. A, exemplar single channel currents for the 5-HT3A(QDA), 5-HT3A(QDA) D113N, 5-HT3A(QDA) D113K, and 5-HT3A(QDA) D127N constructs recorded from outside-out patches held at −80 mV using the Cs⁺-based patch pipette solution and an extracellular solution containing 95 mm [Na⁺]_o and either 0.1 mm (solution E3), 1 mm (solution E4), or 10 mm (solution E5) [Ca²⁺]_o. Note that the mutations alleviate the suppression of single channel currents by Ca²⁺ in comparison with the 5-HT₃A(QDA) receptor. The mean E_5-HT ± S.E. for each construct in such solutions are given beneath the appropriate currents with n values in parenthesis. B, single channel conductance versus Ca²⁺ activity ((Ca²⁺)_o) for the 5-HT₃A(QDA) (filled circles), 5-HT₃A(QDA) D113N (open circles), 5-HT₃A(QDA) D113K (inverted solid triangles), and 5-HT₃A(QDA) D127N (inverted open triangles) receptor constructs. Single channel conductances were calculated using the values for E_5-HT given in A. Data points indicate the mean of 3–6 single channel amplitude measurements from separate patches, and error bars depict S.E. Shown is statistical significance compared with that obtained for the 0.1 mm Ca²⁺, 95 mm Na⁺ mixture, as determined by one-way ANOVA with post hoc Dunett's test (* and ***, p < 0.05 and p < 0.001, respectively). Data for the 5-HT₃A(QDA) receptor are from Livesey et al. (20).