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Rings of charge within the extracellular vestibule influence ion permeation of the 5-HT3A receptor.

Livesey MR, Cooper MA, Lambert JJ, Peters JA - J. Biol. Chem. (2011)

Bottom Line: 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 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.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+).

View Article: PubMed Central - PubMed

Affiliation: Centre for Neuroscience, Division of Medical Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom.

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+).

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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 E5-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 E5-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).
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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 E5-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 E5-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).

Mentions: Single channel current-voltage (i-V) relationships were generated by recording unitary currents evoked by 5-HT (10 μm) from outside-out patches at holding potentials between −100 and +100 mV in increments of 20 mV. For the 5-HT3A(QDA) receptor construct, the single channel current i-V relationship is linear at negative potentials with a slope γ of 41 pS (20). However, at positive potentials, γ increases to 46 pS, indicative of modest outward rectification quantified as an RI value (see “Experimental Procedures”) of 1.12 (20) (Fig. 2B). Under identical ionic conditions, the i-V relationships obtained for the 5-HT3A(QDA) D113N and the 5-HT3A(QDA) D113K receptor constructs reveal that both mutations enhance outward rectification, the RI values being 1.7 and 2.63, respectively (Table 1). Notably, either mutation reduced only inwardly (and not outwardly) directed single channel currents (Fig. 2B). Such results are consistent with a scheme whereby the ring of negativity formed by the Asp113 residues acts via simple coulombic attraction to raise the availability of cations within the receptor extracellular vestibule for translocation to deeper regions of the pore. Neutralization or reversal of charge at this locus would be anticipated to reduce the local concentration of cations within the extracellular vestibule, leading to the emergence of outward rectification. Notably, the effect of neutralizing or reversing the charge at the Asp113 position was increased when [Na+]o was reduced to 95 mm (see below) (see Fig. 5). In comparison with the 46 and 58% reductions in γ found for the D113N and D113K mutations, respectively, with [Na+]o set at 146 mm, the corresponding reductions in the Na+-deficient solution were 66 and 72%. The enhanced effect of the mutations at the lower ionic strength is once more consistent with a predominantly electrostatic influence of the Asp113 residue. This is due to the screening effect of oppositely charged ions upon amino acid residues that have fixed charges that would reduce the influence of the Asp113 residue at high ionic strength.


Rings of charge within the extracellular vestibule influence ion permeation of the 5-HT3A receptor.

Livesey MR, Cooper MA, Lambert JJ, Peters JA - J. Biol. Chem. (2011)

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 E5-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 E5-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).
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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 E5-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 E5-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).
Mentions: Single channel current-voltage (i-V) relationships were generated by recording unitary currents evoked by 5-HT (10 μm) from outside-out patches at holding potentials between −100 and +100 mV in increments of 20 mV. For the 5-HT3A(QDA) receptor construct, the single channel current i-V relationship is linear at negative potentials with a slope γ of 41 pS (20). However, at positive potentials, γ increases to 46 pS, indicative of modest outward rectification quantified as an RI value (see “Experimental Procedures”) of 1.12 (20) (Fig. 2B). Under identical ionic conditions, the i-V relationships obtained for the 5-HT3A(QDA) D113N and the 5-HT3A(QDA) D113K receptor constructs reveal that both mutations enhance outward rectification, the RI values being 1.7 and 2.63, respectively (Table 1). Notably, either mutation reduced only inwardly (and not outwardly) directed single channel currents (Fig. 2B). Such results are consistent with a scheme whereby the ring of negativity formed by the Asp113 residues acts via simple coulombic attraction to raise the availability of cations within the receptor extracellular vestibule for translocation to deeper regions of the pore. Neutralization or reversal of charge at this locus would be anticipated to reduce the local concentration of cations within the extracellular vestibule, leading to the emergence of outward rectification. Notably, the effect of neutralizing or reversing the charge at the Asp113 position was increased when [Na+]o was reduced to 95 mm (see below) (see Fig. 5). In comparison with the 46 and 58% reductions in γ found for the D113N and D113K mutations, respectively, with [Na+]o set at 146 mm, the corresponding reductions in the Na+-deficient solution were 66 and 72%. The enhanced effect of the mutations at the lower ionic strength is once more consistent with a predominantly electrostatic influence of the Asp113 residue. This is due to the screening effect of oppositely charged ions upon amino acid residues that have fixed charges that would reduce the influence of the Asp113 residue at high ionic strength.

Bottom Line: 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 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.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+).

View Article: PubMed Central - PubMed

Affiliation: Centre for Neuroscience, Division of Medical Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom.

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+).

Show MeSH
Related in: MedlinePlus