Mg2؉ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Recently, a positional candidate screening approach in consanguineous families with hypomagnesemia with secondary hypocalcemia (HSH) revealed a critical region identified on chromosome 9q21.13 (2, 3). Individuals suffering from HSH display neurologic symptoms including seizures and tetany during infancy. These symptoms can be suppressed by life-long dietary magnesium supplementation, but, untreated, the disease may be fatal or result in neurological damage. The pathophysiology of HSH is largely unknown, but physiological studies have shown that there are defects in both intestinal Mg 2ϩ absorption and renal Mg 2ϩ reabsorption. Subsequent analysis of the critical region pointed to a gene, TRPM6, which was mutated in patients with HSH (2, 3). The TRPM6 protein is a member of the transient receptor potential channel (TRP) family (4).Based on the structural and sequence similarities between individual TRP proteins, three subgroups are distinguished, namely the canonical TRPC-, the vanilloid-like TRPV-, and the melastatin-like TRPM subfamilies. Most members of the TRPC-and TRPV-subfamilies have been characterized as Ca 2ϩ -permeable cation channels playing a role in Ca 2ϩ homeostasis and signaling (4). However, the functional characterization of TRPM proteins is much more incomplete. TRPM6 shows 50% sequence homology with TRPM7 (also known as TRP-PLIK), which forms a Ca 2ϩ and Mg 2ϩ -permeable cation channel. Unlike other members of the TRP family, TRPM6 and TRPM7 contain long carboxyl-terminal domains with similarity to the ␣-kinases (5). The identification of TRPM6 as the gene mutated in HSH represents the first case in which a human disorder has been attributed to a channel kinase.The aim of the present study was to functionally characterize TRPM6 as the first molecularly identified protein involved in active Mg 2ϩ (re)absorption. To this end, the (sub)localization of TRPM6 was investigated by immunohistochemical analysis of kidney and duodenum sections. Subsequently, human TRPM6 cDNA was cloned, transfected into human embryonic kidney

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Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25-dihydroxyvitaminD3 (1,25(OH)2D3). Mice in which the 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene was inactivated presented the same clinical phenotype as patients with PDDR and were used to study renal expression of the epithelial Ca2+ channel (ECaC1), the calbindins, Na+/Ca2+ exchanger (NCX1), and Ca2+-ATPase (PMCA1b). Serum Ca2+ (1.20+/-0.05 mM) and mRNA/protein expression of ECaC1 (41+/-3%), calbindin-D28K (31+/-2%), calbindin-D9K (58+/-7%), NCX1 (10+/-2%), PMCA1b (96+/-4%) were decreased in 1alpha-OHase-/- mice compared with 1alpha-OHase+/- littermates. Feeding these mice a Ca2+-enriched diet normalized serum Ca2+ levels and expression of Ca2+ proteins except for calbindin-D9K expression. 1,25(OH)2D3 repletion resulted in increased expression of Ca2+ transport proteins and normalization of serum Ca2+ levels. Localization of Ca2+ transport proteins was clearly polarized in which ECaC1 was localized along the apical membrane, calbindin-D28K in the cytoplasm, and calbindin-D9K along the apical and basolateral membranes, resulting in a comprehensive mechanism facilitating renal transcellular Ca2+ transport. This study demonstrated that high dietary Ca2+ intake is an important regulator of the renal Ca2+ transport proteins in 1,25(OH)2D3-deficient status and thus contributes to the normalization of blood Ca2+ levels.

Abstract. The family of epithelial Ca 2ϩ channels consists of two highly homologues members, TRPV5 and TRPV6, which constitute the apical Ca 2ϩ entry mechanism in active Ca 2ϩ (re)absorption in kidney and small intestine. In kidney, TRPV5 expression has been extensively studied, whereas TRPV6 localization and regulation has been largely confined to the small intestine. The present study investigated the renal distribution of TRPV6 and regulation by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). In mouse kidney, TRPV6 was detected by immunohistochemistry at the apical domain of the distal convoluted tubules (DCT2), connecting tubules (CNT), and cortical and medullary collecting ducts (CD). Furthermore, several putative vitamin D-responsive elements were detected upstream of the mouse TRPV6 start codon, and 1,25(OH) 2 D 3 treatment significantly increased renal TRPV6 mRNA and protein expression. In DCT2 and CNT, TRPV6 co-localizes with the other known Ca 2ϩ transport proteins, including TRPV5 and calbindin-D 28K . Together, these data suggest a role for TRPV6 in 1,25(OH) 2 D 3 -stimulated Ca 2ϩ reabsorption in these segments. Interestingly, distribution of TRPV6 extended to the CD, where it localized to the apical domain of principal and intercalated cells, which are not generally implicated in active Ca 2ϩ reabsorption. In addition, TRPV6 mRNA levels were quantified in a large set of tissues, and in the order of decreasing expression level were detected: prostate Ͼ stomach, brain Ͼ lung Ͼ duodenum, kidney, bone, cecum, heart Ͼ colon Ͼ skeletal muscle Ͼ pancreas. Therefore, additional physiologic functions for TRPV6 are feasible. In conclusion, TRPV6 is expressed along the apical domain of DCT2, CNT, and CD, where TRPV6 expression is positively regulated by 1,25(OH) 2 D 3 .Active Ca 2ϩ absorption plays a key role in Ca 2ϩ homeostasis and takes place in Ca 2ϩ

The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17beta-estradiol (17beta-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17beta-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17beta-E2. Treatment with 17beta-E2 upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 +/- 0.10 to 2.03 +/- 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1alpha-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.