Insulin Receptor Substrate 4 (IRS4) Contributes to Hypomagnesemia by Mediating the Insulin Effect on the Renal Magnesium Channel TRPM6

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Background: The kidney is the key regulator of magnesium (Mg2+) homeostasis in the human body. In the distal convoluted tubule (DCT), the apical epithelial Mg2+ channel TRPM6, determines how much Mg2+ is excreted. To better understand the regulation of human renal Mg2+ absorption we screened for novel TRPM6 interaction partners. We identified insulin receptor substrate 4 (IRS4) as a new TRPM6 modifier mediating the insulin effect on TRPM6. Methods: After a TRPM6-GST pulldown assay we pursued liquid chromatography/tandem mass spectrometry (LC/MS MS) sequencing and confirmed the TRPM6-IRS4 protein interaction by co-immunoprecipitation. For tubular Irs4 gene expression we performed microdissection of wild-type (WT) C57BL/6 mouse tubules. Gene expression of Irs4 and magnesiotropic genes (in kidneys of WT and Irs4 knockout (Irs4-/-) mice) was studied with qRT-PCR. 24 h urinary Mg2+ excretion and serum Mg2+ levels were tested in Irs4-/- and WT mice using metabolic cages. Glucose tolerance tests were performed. The IRS4 effect on TRPM6 was examined with whole-cell patch-clamp studies. Results: With LC/MS MS sequencing, we found IRS4 enriched with TRPM6-GST but not with GST control. We confirmed physical interaction between IRS4 and TRPM6 by co-immunoprecipitation. Micro-dissecting mouse tubules, we detected IRS4 mRNA expression mostly in the DCT and to a lower degree in the proximal tubule and thick ascending limb. Given the overall low abundance of IRS4 mRNA along the tubule, we investigated the phenotype of Irs4-/- mice. The Irs4-/- mice displayed significantly higher urinary Mg2+ losses at 3, 6, and 12 months and lower blood Mg2+ levels at 6 and 12 months than WT mice. Claudin-16, claudin-19, and Hnf1b mRNA and Claudin-16 and Trpm6 protein expression was significantly higher in kidneys of 3 month old Irs4-/- mice consistent with a compensatory mechanism to conserve Mg2+ in the body. Applying whole-cell patch clamp recording we confirmed the stimulatory role of insulin on TRPM6 and showed that IRS4 targets the two TRPM6 phosphorylation sites T1391 and S1583 to enhance TRPM6 current density. Glucose tolerance was mildly abnormal in 6 month old Irs4-/- mice. Conclusions: We show that IRS4 mediates insulin signaling towards TRPM6 by phosphorylating the TRPM6 residues T1391 and S1583. Funding: Other U.S. Government Support