Molecular characterization of V59E NIS, a Na+/I- symporter mutant that causes congenital I- transport defect

Abstract

I(-) is actively transported into thyrocytes via the Na+/I(-) symporter (NIS), a key glycoprotein located on the basolateral plasma membrane. The cDNA encoding rat NIS was identified in our laboratory, where an extensive structure/function characterization of NIS is being conducted. Several NIS mutants have been identified as causes of congenital I(-) transport defect (ITD), including V59E NIS. ITD is characterized by low thyroid I(-) uptake, low saliva/plasma I(-) ratio, hypothyroidism, and goiter and may cause mental retardation if untreated. Studies of other ITD-causing NIS mutants have revealed valuable information regarding NIS structure/function. V59E NIS was reported to exhibit as much as 30% of the activity of wild-type NIS. However, this observation was at variance with the patients' phenotype of total lack of activity. We have thoroughly characterized V59E NIS and studied several amino acid substitutions at position 59. We demonstrated that, in contrast to the previous report, V59E NIS is inactive, although it is properly targeted to the plasma membrane. Glu and all other charged amino acids or Pro at position 59 also yielded nonfunctional NIS proteins. However, I(-) uptake was rescued to different degrees by the other substitutions. Although the Km values for Na+ and I(-) were not altered in these active mutants, we found that the structural requirement for NIS function at position 59 is a neutral, helix-promoting amino acid. This result suggests that the region that contains V59 may be involved in intramembrane helix-helix interactions during the transport cycle without being in direct contact with the substrates.

Figure 1

Schematic representation of the NIS secondary structure model. The current secondary structure model of NIS depicts 13 TMS (labeled with Roman numerals) with an extracellular N and a cytosolic C terminus. The 12 ITD-causing NIS mutations are indicated by the single-letter amino acid code, showing the WT residue followed by the position number and the substituted residue. FS, Frame shift; X, premature stop codon; Δ, deletion.

Figure 2

Characterization of activity and expression of V59E NIS in COS-7 cells. A, Steady-state I⁻ uptake analysis in nontransfected (NT) COS-7 cells or cells transfected with either WT or V59E NIS was determined. Cells were incubated for 1 h in HBSS containing 140 mm Na⁺ and 20 μm ¹²⁵I⁻ (black bars) or HBSS containing 140 mm Na⁺ and 20 μm ¹²⁵I⁻ and 40 μm ClO₄⁻ (white bars); accumulated I⁻ was measured in a γ-counter and standardized by micrograms of DNA. NT cells represent background levels of I⁻ transport. Values are expressed as a percentage of the activity of WT NIS (typically 80–100 pmol I⁻/μg DNA). Values represent the average of at least three different experiments; in each experiment, transport measurements were performed in triplicate. B, Immunoblot analysis. Cell lysates (2 μg) from nontransfected (NT) cells or cells transfected with either WT NIS or V59E NIS (E) were electrophoresed, electrotransferred, and immunoblotted with 2 nm anti-rNIS. C, Cell surface biotinylation. Transfected cells were incubated with the membrane-impermeable sulfo-NHS-SS-biotin reagent (1 mg/ml) and precipitated overnight with streptavidin beads. Streptavidin beads and bound biotinylated proteins were heated in sample buffer at 75 C, and the cell surface biotinylated proteins were immunoblotted with 2 nm anti-rNIS. The α-subunit of the Na⁺/K⁺ ATPase was used as a loading control (B and C, bottom panels).

Figure 3

NIS is inactive with charged amino acids or Pro at position 59 despite proper expression and plasma membrane targeting. COS-7 cells were transfected with WT, V59D, V59E, V59K, V59P, or V59R NIS and analyzed as follows. A, Steady-state I⁻ transport in cells incubated with 20 μm I⁻ and 140 mm Na⁺ (black bars) and in cells incubated with 20 μm I⁻, 140 mm Na⁺, and 40 μm ClO₄⁻ (white bars). Values are expressed as a percentage of WT NIS activity (typically 80–100 pmol I⁻/μg DNA). B, Immunoblot analysis. C, Cell surface biotinylation. The α-subunit of the Na⁺/K⁺ ATPase was used as a loading control (B and C, bottom panels).

Figure 4

NIS is active with neutral amino acids other than Pro at position 59. COS-7 cells were transfected with V59A, I, L, M, N, Q, T, or WT NIS cDNAs and analyzed as follows. A, Steady-state I⁻ transport in cells incubated with 20 μm I⁻ and 140 mm Na⁺ (black bars) or 20 μm I⁻, 140 mm Na⁺, and 40 μm ClO₄⁻ (white bars). Stars indicate the amino acid substitutions that are branched at the β-carbon (as Val is), which yield the proteins that mediate the highest levels of I⁻ accumulation compared with WT NIS. B, Immunoblot analysis. C, Cell surface biotinylation. The α-subunit of the Na⁺/K⁺ ATPase was used as a loading control (B and C, bottom panels).

Figure 5

A, I⁻-dependent kinetic assays. cDNA constructs encoding WT NIS or NIS with amino acids at position 59 that were conducive to activity (V59A, I, L, M, N, Q, and T) were assayed for initial I⁻ transport rates (2 min) as a function of different I⁻ concentrations, ranging from 0.6125–80 μm, at a constant Na⁺ concentration of 140 mm Na⁺. V_max and K_m values were calculated with the equation v([I⁻]) = (V_max× [I⁻])/(K_m + [I⁻]) + A × [I⁻] + B and the gnuplot program. B, Na⁺-dependent kinetic assays. Cells were assayed to determine the initial rates of I⁻ transport as a function of the external [Na⁺]. Cells were assayed for initial I⁻ transport rates (2 min) as a function of different Na⁺ concentrations, ranging from 0–140 mm, at a constant I⁻ concentration of 20 μm. Results were analyzed using the gnuplot program and applied to the equation v([Na⁺]) = (V_max× [Na⁺]²)/(K_m² + [Na⁺]²) − A × [Na⁺] + B to determine V_max and K_m. C, Average K_m (I⁻ and Na⁺) and V_max (I⁻ and Na⁺) values as determined by kinetic analysis. The table lists the average K_m and V_max values from six I⁻-dependent kinetic assays (left) and six Na⁺-dependent kinetic assays (middle); in each experiment, activity was analyzed in triplicate. On the right, the position of the amino acid side chain of each amino acid is listed. The variation in V_max and the lack of significant variation of K_m values indicate that the decreased levels of I⁻ accumulation are not due to a change in the apparent affinity of the mutant NIS proteins for Na⁺ or I⁻. Interestingly, the amino acids that are branched at the β-carbon (such as Ile or Thr), i.e. equal to Val, accumulated the highest levels of I⁻. Graphs in A and B correspond to a representative experiment.

Figure 6

Expression, plasma membrane targeting, and activity of V59E/T354P NIS. A, Immunoblot analysis. Cell lysates (2 μg) from COS-7 cells cotransfected with 2 μg each of V59E and T354P or transfected with 4 μg WT NIS were subjected to SDS-PAGE and immunoblotted with 2 nm anti-rNIS. B, Cell surface biotinylation. C, I⁻ uptake. V59E and T354P NIS cDNA (2 μg each) were cotransfected into COS-7 cells, and I⁻ accumulation was compared with that of V59E, T354P, or WT NIS cDNA (4 μg). Shown is I⁻ transport in cells incubated with 20 μm I⁻ and 140 mm Na⁺ (black bars) or with 20 μm I⁻, 140 mm Na⁺, and 40 μm ClO₄⁻ (white bars). Values are a percentage of WT NIS activity. Values represent the average of at least three different experiments; in each experiment, activity was analyzed in triplicate.