Na Sulfate transporter

Introduction

The featured image was created after the post was written. The closeup of arinine at position237 was created at a UniProt viewer of the predicted structure. Arrows point to cysteines that might bind to heavy metals.

Atlas Antibodies sells antibodies to the Na⁺ dependent sulfate transporter Slc13A1. This offers us a glimpse as to how intense the protein levels can be in the proximal tubule of the human kidney. Protein Atlas reports lower protein levels in the small intestine and just messenger RNA transcripts in the gallbladder.

Images are from ProteinAtlas.org for the NaSi transporter Slc13A1

Heavy Metal Inhibition of NaSi-1 [1]

In 1998 Markovich and Brown at the University of Queensland expressed human Slc13A1 in Xenopus laevis (frog) oocytes with the goal of testing the hypothesis that the heavy metals Hg²⁺, Cd²⁺, Pb²⁺, and Cr⁶⁺ inhibit this transporter as a means of explaining renal toxcity of these metals. Uptake into the oocytes was measured with the radioactive isotope of sulfur, ³⁵S.

Fig. 1. A: effects of various heavy metals on NaSi-1-induced transport in Xenopus oocytes.
HgCl2, Pb(NO3)2, CdCl₂, and CrO₃ (each at 0.1 mM final concentration)

Uptake values (as %control) are HgCl₂ (6.9 6 0.2%), Pb(NO₃)₂ (40.9 6 1.0%), CdCl₂ (87.9 6 11.2%), and CrO₃ (60.6 62.6%). B: heavy metals, then washout
r*P < 0.05.

A panels compare mock Slc13A1 transfections to oocytes that are transfected with the genes for Slc13A1. This is to get an idea on endogenous sulfate transport in these oocytes and the ability of the heavy meta l to inhibit it.
B panels show the 100% of control at the indicated concentration of heavy metal as a function of time. The insets show actual amounts of sulfate in the control and heavy metal treated oocytes.
C panels Increasing the sulfate concentration will naturally increase the rate of transport until the Slc13A1 transporter becomes saturated. The affinity of the transporter for sulfate can be calculated from these concentration dependent rates. Adding the indicated concentration of heavy metal decreases the affinity of the transporter for sulfate.

Mercury

Cadmium

A Ki inhibitory dose for 50% inhibition, could not be calculated because close to 1mM only resulted in a little over 10% inhibition.

Lead

Chromium

parameters	Hg²⁺	Cd²⁺	Pb²⁺	Cr⁶⁺
Ki	7 µM	—	21 µM	20 µM
Sulfate Km (from 0.12mM)	0.85 mM	0.48mM	0.22 mM	2.42 mM

approximate values take from the above graphs…

Going back to the featured image, we have a lot of Ks (lysines) and Rs near a trans membrane alpha helix. Is this a selectivity filter? Also note the Cs, cysteines. We cannot say for sure, but we can speculate that this is where the inhibition is happening.

The Markovich Lab take on the NaSi-1 and Sat1 genes [2]

This publication reports the NaSi-1 having a Km for SO42- ∼0.1 mM) Sat-1, encodes a high affinity Na+-independent SO₄^2-– anion exchanger (Km for SO₄²-∼0.1 mM) which is strongly inhibited by DIDS and oxalate. Knocking out NaSi 1 results in increased urinary SO₄² secretion. This post will not discuss the rather

Mouse and pig transcription factor binding sites was edited out of this table. Slight rearrangements were made to get the transcription factors that control the expression of both required renal re absorption transporters in about the same position.

The thought is that if heavy metal poisoning is thought to be the source of autistic like symptoms, agoniss of C/EBP might bump up the expression.

Slc13A1 nonsense and Slc26A1 missense mutations.

Tise and coauthors were working with a a group of Amish who carried nonsense mutations in the Slc13A1 gene (R12X and W48X) associated with hypo sulfatemia (P = 9 × 10^− 20). A nonsense mutation results in an amino acid coding codon being replaced with the stop codon. R12X would translate into a 12 amino acid peptide and W480 as much of the 600 amino acid Na⁺/Sulfate exchanger. A missense mutation is a single nucleotide change that results in an amino acid substitution that may or may not affect protein function. Asparagine 174 is a surface exposed glycosylation site. Mutation to serine may impede N-linked glycosylation

NaSiT/Slc13A1 apical nonsense

Consult the featured image for the location of Arginine R237 to cysteine. This mutation results in the loss of a positive charge and the gain of another cysteine in a cysteine rich region. We are going to assume that the Amish population is eating healthy and not exposed to heavy metals. What if they moved to the city?

Serum sulfate concentration by *SLC13A1* genotype. Box and whisker plots: box represents 2nd and 3rd quartiles (IQR, interquartile range); horizontal band represents median value; whiskers represent 1st and 4th quartiles; ends of whiskers represent minimum and maximum values excluding outliers; open circles represent outliers (data point >1.5*IQR below the 1st quartile or above the 3rd quartile). Association analyses between genotypes and serum sulfate were conducted using a regression-based method that models variation of the trait of interest as a function of measured covariates, measured genotypes, and a polygenic component that accounts for phenotypic correlation due to relatedness. (A) Serum sulfate by *SLC13A1* N174S genotype. (B) Serum sulfate by *SLC13A1* R237C genotype. (C) Serum sulfate by *SLC13A1* nonsense SNV genotype. The P-value for the R12X or W48X heterozygotes (dark green box and whisker plot) results from the model including carrier status of an *SLC13A1* nonsense SNV (R12X or W48X) as a covariate. The P-values for the R12X heterozygotes and the W48X heterozygotes (light green box and whisker plots) result from the conditional model including both R12X and W48X as covariates.

Argine 237 of Slc13A1 is predicted to be in a transmembrane helix towards the surface. Argine is a positively charged amino acid and can be predicted to be part of the selectivity filter. Substitution with a Cys might make this allelic variation more susceptible to heavy metals like mercury.

Basolateral membrane Sat1/SLC26A1 missense

Leucine 348 of Slc26A1 is predicted to be part of a transmembrane alpha helix. Prolines have a reputation of being helix breakers.

UniProt has this to say; “Can transport other anions including bicarbonate, thiosulfate and oxalate by mediating sulfate-thiosulfate, sulfate-hydrogen carbonate and sulfate-oxalate anion exchange. Mediates oxalate-hydrogen carbonate anion exchange (By similarity)”

Remember that Slc26A1 is an exchanger bring in oxalate. Oxalate and calcium are the makings of kidney stones. The calcium homeostasis and bond marrow density were also impacted.

These data are being presented so that we do not start thinking that Slc13A1 is the only way sulfate gets into our bodies and that that other molecules are not impacted.

Gender and DHEA sulfate in Slc13A1 nonsense mutations [4]

This paper is being explored in response to male prevalence of autism spectrum disorders. This five year old post explores possibilities that do not include sulfate described in the next section.

Serum sulfate and DHEA and DHEA-S in normal men and women

Associations between DHEA hormone levels and serum sulfate concentration in men (n = 92) and women (n = 111).

a) Ratio_Log, In normal males, but not females, the higher the serum sulfate, the lower the ratio of DHEA=3/DHEA.

b) DHEA-S_Log, In females there is a borderline significant trend for DHEA-S to increase with serum sulfate.

c) DHEA_Log. Serum sulfate has no influence on dHEA normal males or females.

P represents significance from model including age, age-squared, and gender as covariates, and accounting for phenotypic correlation due to relatedness.

It would appear that the serum DHEA-S to DHEA ratio increases in men with these nonsense mutations in one of two copies of their SCL13A1 gene. Tise and coautors discussed the paradox of this finding. It should also be noted that these nonsense mutations result in a decrease in testosterone levels.

The clinical variation viewer allows us to understand how heterogeneous the human population is.

Resolving the paradox

The paradox is how mutations in Slc13A1 that decrease serum result in more SDHEA-s. Let’s take the UniProt.org subcellular annotation of three key enzymes in this story: Slc13A1, sterylsulfatase, and SULT2A1.

Going back to autism[5]

This 2000 study by Waring and Klovzra was a follow up on the observation that autistic children have lower serum phosphate.

Dehydroepiandrosterone sulfate, or DHEA-sulfate, is the sulfated version of DHEA. According to Wikipedia authors that while DHEA sulfate does not bind to steroid hormone receptors, it can modulate

TrkA,
GABA_A,
NMDA, and other receptors.

This post is not going to explore these potential targets of DHEA sulfate, that may be increased when Slc13A1 is compromised via mutations or heavy metals.

References

Markovich D, Knight D. Renal Na-Si cotransporter NaSi-1 is inhibited by heavy metals. Am J Physiol. 1998 Feb;274(2):F283-9.
Lee A, Dawson PA, Markovich D. NaSi-1 and Sat-1: structure, function and transcriptional regulation of two genes encoding renal proximal tubular sulfate transporters. Int J Biochem Cell Biol. 2005 Jul;37(7):1350-6.
Tise CG, Perry JA, Anforth LE, Pavlovich MA, Backman JD, Ryan KA, Lewis JP, O’Connell JR, Yerges-Armstrong LM, Shuldiner AR. From Genotype to Phenotype: Nonsense Variants in SLC13A1 Are Associated with Decreased Serum Sulfate and Increased Serum Aminotransferases. G3 (Bethesda). 2016 Sep 8;6(9):2909-18. PMC free article
Tise CG, Anforth LE, Zhou AE, Perry JA, McArdle PF, Streeten EA, Shuldiner AR, Yerges-Armstrong LM. Sex-specific effects of serum sulfate level and SLC13A1 nonsense variants on DHEA homeostasis. Mol Genet Metab Rep. 2017 Jan 27;10:84-91. PMC free article
Waring RH, Klovrza, LV Sulfur metabolism in autism J nutrition and environmental medicine (2000) 10, 25-32

Na Sulfate transporter

Introduction