Hou, J. S. | Search bioRxiv

bioRxiv2022

Insulin receptor substrate 4 deficiency mediates the insulin effect on the epithelial magnesium channel TRPM6 and causes hypomagnesemia

The kidney is the key regulator of magnesium (Mg2+) homeostasis in the human body. In the distal convoluted tubule (DCT), the apical epithelial magnesium (Mg2+) channel TRPM6, determines how much Mg2+ is excreted in the urine. To better understand the regulation of human renal Mg2+ absorption we identified novel, potential interaction partners of TRPM6 by pursuing a liquid chromatography - tandem mass spectrometry (LC-MS/MS) proteomics approach. We found insulin receptor substrate 4 (IRS4) enriched with TRPM6 tagged to glutathione S-transferase (TRPM6-GST) but not GST control. Physical interaction between IRS4 and TRPM6 was confirmed by co-immunoprecipitation. Applying microdissection of mouse tubules, we detected Irs4 mRNA expression mostly in the DCT and to a lower degree in the proximal tubule and thick ascending limb of Henle. Given the overall low abundance of Irs4 mRNA along the tubule we investigated the phenotype of Irs4 knockout mice (Irs4-/-). These Irs4-/- mice displayed significantly higher urinary and fecal Mg2+ losses and lower blood Mg2+ levels than wild-type (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+. Applying whole-cell patch-clamp recording we confirmed the stimulatory role of insulin on TRPM6 channel activity and showed that IRS4 targets the two TRPM6 phosphorylation sites T1391 and S1583 to enhance TRPM6 current density. To test the effect of Mg2+ deficiency on metabolism, we performed glucose and insulin tolerance studies, which were mildly abnormal in Irs4-/- mice. SIGNIFICANCE STATEMENTMagnesium (Mg2+) is the second most abundant intracellular cation but the regulation of Mg2+ homeostasis is not well understood. The kidney is the key organ for regulating Mg2+ homeostasis. Insulin is a known stimulator of the apical epithelial Mg2+channel TRPM6. We present a novel modifier of Mg2+ absorption with insulin receptor substrate 4 (IRS4) which illuminates further, how insulin activates the TRPM6 channel and modifies Mg2+ homeostasis. Applying protein biochemistry, tubular microdissection, whole mouse physiology, and patch-clamp recording, we demonstrate that IRS4 mediates the stimulatory effect of insulin by enhancing phosphorylation of two specific TRPM6 residues. Irs4-/- mice develop increased urinary and stool Mg2+ losses, lower serum Mg2+ concentration, and display mild impairment in glucose and insulin tolerance.

physiology↗

bioRxiv2019

Encephalitic alphaviruses exploit caveolae-mediated transcytosis at the blood-brain barrier for CNS entry

Venezuelan and Western equine encephalitis viruses (VEEV and WEEV) invade the CNS early during infection, via neuronal and hematogenous routes (1, 2). While viral replication mediates host-shut off, including expression of type I interferons (IFN) (3, 4), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin (Cav)-1-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and non-replicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1-association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS, and that IFN differentially restricts this process at the BBB.\n\nImportanceVEEV, WEEV and EEEV are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the blood-stream, however the underlying molecular mechanisms have remained largely unknown. Here we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPAses within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

microbiology↗