A common NHE3 single-nucleotide polymorphism has normal function and sensitivity to regulatory ligands

Abstract

Na⁺/H⁺ exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene (SLC9A3) has more than 400 single-nucleotide polymorphisms (SNPs) but few nonsynonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5-57.6% in Asian populations, 11.1-23.6% in European populations, and 10.2-22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but a higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC₅₀ values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease.NEW & NOTEWORTHY This study reports results on the functional significance of human NHE3-799C under basal conditions and in response to regulatory ligands, including a novel NHE3 inhibitor called tenapanor. We demonstrate that NHE3-799C is a common variant of NHE3 that is enriched in Asian populations; however, in contrast to our previous studies using rabbit NHE3, its presence seems to have limited clinical significance in humans and is not associated with compromised function or abnormal transport regulation.

Keywords: NHE3; SNP; diarrhea; phenotype; polymorphism.

Fig. 1.

NHE3-799R is highly conserved among mammals, whereas NHE3-799C is a nonconservative variant. Phylogenetic analysis was performed by searching the Ensembl genome database (accessed May 2016). NHE3-799R (corresponding to allele G) was highly conserved among mammals, whereas NHE3-799C (corresponding to allele A) was a nonconservative variant and was probably exclusive to humans.

Fig. 2.

Homogeneous expression of NHE3 among cell clones of transfected cell lines. PS120/FLAG-NHERF2 cells were transfected with human NHE3-799R or NHE3-799C, followed by selection by antibiotics and regular acid challenge. For each stably transfected cell line, mixed cell clones were used for experiments. Homogeneity of NHE3 expression among mixed clones was verified by immunofluorescence. NHE3 was expressed in almost all cells with slight variation between cell clones within each cell line. Scale bar, 50 µm.

Fig. 3.

NHE3-799C-transfected cells have lower total protein/mRNA expression but a higher percentage of surface protein expression than NHE3-799R-transfected cells. Total protein expression and mRNA expression were studied by immunoblotting and quantitative real-time PCR, respectively. Surface protein expression was determined by cell surface biotinylation and immunoblotting. A and B: the total protein expression of NHE3 was significantly lower in the NHE3-799C-transfected cells than in the NHE3-799R-transfected cells (55.0% reduction, P < 0.05, n = 4). C: quantitative real-time PCR revealed lower mRNA expression in the NHE3-799C-transfected cells compared with the NHE3-799R-transfected cells (28.4% reduction, P < 0.05, n = 3). D and E: the percentage of surface expression of NHE3 was significantly higher in the NHE3-799C-transfected cells than in the NHE3-799R-transfected cells (means ± SE: 12.4 ± 0.4% vs. 8.1 ± 1.4%, P < 0.05, n = 3). IF, intracellular fraction; SF, surface fraction; TL, total lysate. *P < 0.05.

Fig. 4.

Association of NHE3 genotype and mRNA expression across human tissues. Expression quantitative trait loci (eQTL) analysis was performed by searching the Genotype-Tissue Expression (GTEx) project database (accessed March 2017). NHE3-799C (corresponding to allele A) was associated with lower mRNA expression compared with NHE3-799R (corresponding to allele G) across human tissues, including but not limited to thyroid, testis, and esophagus mucosa, which were the top three tissues ranked by P value.

Fig. 5.

NHE3-799C-transfected cells have similar basal activity to NHE3-799R-transfected cells and are similarly stimulated or inhibited, by serum or forskolin, respectively. A: basal NHE3 activity was not significantly different between cells stably expressing human NHE3-799C and NHE3-799R (means ± SE: V_max: 694.9 ± 61.7 vs. 891.4 ± 75.2 µM/s, P = 0.11, n = 4). B: the NHE3-799C-transfected cells and NHE3-799R-transfected cells both responded normally by upregulating activity following exposure to 10% dialyzed serum (means ± SE: +32.4 ± 10.0% and +26.4 ± 7.8%, n = 4), and by downregulating activity following exposure to 10 µM forskolin (means ± SE: −35.7 ± 8.7% and −31.2 ± 8.9%, n = 4). ns, not significant; V_max, maximum velocity. *P < 0.05.

Fig. 6.

Tenapanor dose-dependently inhibits the activity of human NHE3-799C and NHE3-799R in the nanomolar range. A: representative data showing the dose-dependent inhibitory effect of tenapanor on the basal activity of human NHE3-799R. B: representative data showing the estimation of IC₅₀ of tenapanor for the two NHE3 variants using a logistic regression model. The IC₅₀ value of tenapanor was significantly higher for human NHE3-799C than for NHE3-799R (means ± SE: 2.41 ± 0.69 vs. 0.87 ± 0.34 nM, P < 0.05, n = 5).