Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

Abstract

Zhu XC, Sarker R, Horton JR, Chakraborty M, Chen TE, Tse CM, Cha B, Donowitz M. Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity. Am J Physiol Cell Physiol 308: C758 -C766, 2015. First published February 25, 2015 doi:10.1152/ajpcell.00421.2014.-Genetic determinants appear to play a role in susceptibility to chronic diarrhea, but the genetic abnormalities involved have only been identified in a few conditions. The Na ϩ /H ϩ exchanger 3 (NHE3) accounts for a large fraction of physiologic intestinal Na ϩ absorption. It is highly regulated through effects on its intracellular COOH-terminal regulatory domain. The impact of genetic variation in the NHE3 gene, such as single nucleotide polymorphisms (SNPs), on transporter activity remains unexplored. From a total of 458 SNPs identified in the entire NHE3 gene, we identified three nonsynonymous mutations (R474Q, V567M, and R799C), which were all in the protein's intracellular COOH-terminal domain. Here we evaluated whether these SNPs affect NHE3 activity by expressing them in a mammalian cell line that is null for all plasma membrane NHEs. These variants significantly reduced basal NHE3 transporter activity through a reduction in intrinsic NHE3 function in variant R474Q, abnormal trafficking in variant V567M, or defects in both intrinsic NHE3 function and trafficking in variant R799C. In addition, variants NHE3 R474Q and R799C failed to respond to acute dexamethasone stimulation, suggesting cells with these mutant proteins might be defective in NHE3 function during postprandial stimulation and perhaps under stressful conditions. Finally, variant R474Q was shown to exhibit an aberrant interaction with calcineurin B homologous protein (CHP), an NHE3 regulatory protein required for basal NHE3 activity. Taken together, these results demonstrate decreased transport activity in three SNPs of NHE3 and provide mechanistic insight into how these SNPs impact NHE3 function.

Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride CotransporterSlc12a2

“…In fact, ~10% of total NKCC1 was roughly estimated at the plasma membrane under basal conditions (Figures 4(a) – 4(c) ), mostly of the core/high-mannose N-glycan type. Although the proportion of total NKCC1 in the plasma membrane estimated in our experiments is well within the ranges recently reported for other transporters including the Na + /H + exchanger-3 (NHE3) [ 65 ], several members of the Slc26a family of transporters [ 66 ], or channels such as CFTR [ 67 ], the possibility of contamination with intracellular NKCC1 as the source of ~130 kDa core/high-mannose N-glycosylated transporters in the plasma membrane may still remain. However, GAPDH, an abundant cytoplasmic protein expressed in all cells, was minimally detected, if any, in biotinylated preparations (see Figures 4(a) , 8(c) , 9(c) , and 9(f) ).…”

Pathophysiology of IBD associated diarrhea

“…Another study reported the inhibitory impact of SNPs (single nucleotide polymorphisms) on NHE3 function. 48 Additionally, GWAS studies have established strong association between UC and SLC9A3 gene locus. 49 , 50 Alterations in NHE3 function and/or expression observed in IBD patients have further substantiated that NHE3 dysfunction contributes to IBD associated diarrhea.…”

Pathophysiology, Evaluation, and Management of Chronic Watery Diarrhea

“…7 The function and/or expression of intestinal SLC9 and SLC26 transporters are also downregulated by a variety of inflammatory cytokines, and this may contribute to diarrhea in patients with inflammatory bowel diseases (IBD). 8 Similarly, single nucleotide polymorphisms in SLC9A3 that reduce activity of the transporter could increase susceptibility to chronic diarrhea, 9 though this hypothesis should be tested in patients.…”