Fig. 1. (a) Possible hypotheses as to how NIS transports different substrates with different stoichiometries.

(i) Electrogenic transport takes place when 2 Na⁺ ions and I⁻ are translocated. Hypotheses for electroneutral transport: (ii) Hypothesis 1: Oxyanion transport is electroneutral (1 Na⁺: 1 oxyanion) because binding of the oxyanion to its transport (T) site (yellow circle) causes a partial overlap with the NaA site (light green square), thus inhibiting binding of Na⁺ to this site; according to this hypothesis, I⁻ could not be transported electroneutrally, because I⁻ binding to its T site (blue circle in panel i) would not cause the latter to partially overlap with the NaA site (light green square), (iii) Hypothesis 2: Oxyanion transport is electroneutral because binding of the oxyanion to a non-transport (NT) site (red hexagon) causes the site to partially overlap with the NaA site (light green square), thus inhibiting binding of Na⁺ to this site; according to this hypothesis, I⁻ could be transported electroneutrally in the presence of an oxyanion, but high concentrations of Na⁺ would outcompete the oxyanion at the NT site (red hexagon) and would restore electrogenic transport of I⁻; (iv) Hypothesis 3: Oxyanion transport is electroneutral because binding of the oxyanion to an NT site (red hexagon) allosterically inhibits binding of one Na⁺ to the NaA site (green) (no overlap); according to this hypothesis, I⁻ could be transported electroneutrally in the presence of an oxyanion, and high concentrations of neither Na⁺ nor of I⁻ would restore electrogenic transport of I⁻, (b) ClO₄⁻ changes the stoichiometry of NIS-mediated I⁻ transport. Initial rates (4-min time points) of I⁻ transport at different concentrations of Na⁺ (0-280 mM) and ClO₄⁻ (0-5 μM). Carrier-free ¹²⁵I⁻ was used as a tracer to determine the effect of ClO₄⁻ on the mechanism of I⁻ transport. (i) Points represent the average of duplicate or triplicate ¹²⁵I uptake data. The surface, calculated with equation 1a, represents the rate of transport (cpm/μg DNA), expressed as ${\mathrm{V}}_{\max }$ times the sum of the fraction of NIS species that can transport I⁻, i.e., the fraction of NIS molecules that are occupied by 2 Na⁺ ions and the fraction occupied by 1 Na⁺ and ClO₄⁻ at the NT site. (ii) Data from panel a, showing only the experimental points at 0 μM and 5 μM ClO₄⁻ (i.e., the sections of the surface in panel b (i) at the concentrations indicated). (c) The change in the stoichiometry of I⁻ transport from electrogenic to electroneutral brought about by ClO₄⁻ persists even at high concentrations of I⁻. Initial rates (4-min time points) of I⁻ transport at different concentrations of I⁻ (0.75-160 μM), at a constant concentration of ClO₄⁻ (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 1b.