Allosteric regulation of mammalian Na+/I symporter activity by perchlorate

Abstract

The Na+/I symporter (NIS), the plasma membrane protein that actively transports I (stoichiometry 2Na+:1I) in thyroid physiology and radioiodide-based thyroid cancer treatment, also transports the environmental pollutant perchlorate (stoichiometry 1Na+:1ClO4), which competes with I for transport. Until now, the mechanism by which NIS transports different anion substrates with different stoichiometries has remained unelucidated. We carried out transport measurements and analyzed these using a statistical thermodynamics–based equation and electrophysiological experiments to show that the different stoichiometry of ClO4 transport is due to ClO4 binding to a high-affinity non-transport allosteric site that prevents Na+ from binding to one of its two sites. Furthermore, low concentrations of ClO4 inhibit I transport not only by competition but also, critically, by changing the stoichiometry of I transport to 1:1, which greatly reduces the driving force. The data reveal that ClO4 pollution in drinking water is more dangerous than previously thought.

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Fig. 1: ClO4 changes the mechanism of NIS-mediated I transport.
Fig. 2: Differential effects of ClO4 on the transport stoichiometry of WT and G93T NIS and on their KM values for I.
Fig. 3: ClO4 at low concentrations is transported electrogenically by NIS.

Data availability

The source data for Figs. 1b,c,2,3, and Extended Data Figs. 1,2 are available in the online version of the paper.

Change history

  • 29 May 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank H. Wade and A. Lau and the members of the Carrasco laboratory for critical reading of the manuscript and insightful discussions. This study was supported by the National Institutes of Health (grants GM-114250 to N.C. and L.M.A. and R01DK041544 to N.C. and G.W.A.).

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Authors

Contributions

A.Ll.-E., R.W.M., A.R.-N., G.W.A., L.M.A., and N.C. designed experiments, performed experiments and analyzed and fitted the data. L.M.A. derived the equations. A.Ll.-E., G.W.A., L.M.A. and N.C. wrote the paper.

Corresponding authors

Correspondence to L. Mario Amzel or Nancy Carrasco.

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The authors declare no competing financial interests.

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Peer review information Katarzyna Marcinkiewicz and Ines Chen were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 ReO4 changes the stoichiometry of NIS-mediated I transport from electrogenic to electroneutral.

Initial rates of I transport at different concentrations of Na+ (0-280 mM) and ReO4 (0-5µM). Carrier-free 125I was used as a tracer to determine the effect of ReO4 on the mechanism of I transport. a, Points represent experimental data. The surface, calculated with Equation 3A, represents the rate of transport (cpm/μg DNA), expressed as νmax times the sum of the fraction of NIS species that can transport I, that is, the fraction of NIS molecules that are occupied by 2 Na+ ions and the fraction occupied by 1 Na+ and ReO4 at the NT site.). Points represent the mean of duplicate or triplicate 125I uptake experiments. b, Data from panel a, showing only the experimental points at 0 μM and 5 μM ReO4 (that is, the sections of the surface in panel a at the concentrations indicated). Points represent the mean of duplicate or triplicate 125I uptake experiments, error bars represent S.D. Data for all graphs are available as source data. Source data

Extended Data Fig. 2 High concentrations of I do not displace ReO4 bound to the NT site.

Initial rates of I transport at different concentrations of I (0.75-160 µM), at a constant concentration of ReO4 (5 µM), and as a function of the Na+ concentration (0-280 mM). Data are expressed as pmol of I/μg DNA. The surface was calculated using Equation 3B. Points represent the mean of duplicate or triplicate 125I uptake experiments. Data are available as source data. Source data

Supplementary information

Source data

Source Data Fig. 1

Experimental raw data, analysis, and statistical calculations

Source Data Fig. 2

Experimental raw data, analysis, and statistical calculations

Source Data Fig. 3

Experimental raw data, analysis, and statistical calculations

Source Data Extended Data Fig. 1

Experimental raw data, analysis, and statistical calculations

Source Data Extended Data Fig. 2

Experimental raw data, analysis, and statistical calculations

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Llorente-Esteban, A., Manville, R.W., Reyna-Neyra, A. et al. Allosteric regulation of mammalian Na+/I symporter activity by perchlorate. Nat Struct Mol Biol 27, 533–539 (2020). https://doi.org/10.1038/s41594-020-0417-5

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