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

Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a beta-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.

Abstract. Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a GϾA nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein.The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.The aquaporin-2 (AQP2) water channel plays an important role in reabsorption of water in the kidney collecting duct and consequently in concentrating urine (1). Binding of arginine vasopressin (AVP) to its V2 receptor (AVPR2) at the basolateral side of principal cells of collecting ducts leads to an increase of intracellular cAMP levels, resulting in phosphorylation of AQP2 and possibly other proteins, by protein kinase A and subsequent redistribution of AQP2 from subapical storage vesicles to the apical plasma membrane. Driven by the interstitial hypertonicity, water reabsorption and urine concentration is thereby initiated. This process is reversed after dissociation of AVP from its receptor (2,3).Several mutations in the AVPR2 and AQP2 genes have been reported to cause congenital nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to AVP. Mutations in the AVPR2 gene result in NDI that is inherited as an X-linked recessive trait, whereas mutations in the AQP2 gene cause NDI that is inherited as either an autosomal recessive or a dominant trait (1,4 -6,7). Expression studies in oocytes showed that an AQP2 mutant in dominant NDI, AQP2-E258K, was a functional water channel but was retained in the region of the Golgi complex (7). In coexpression studies with wild-...

. Oocytes coexpressing wild-type TRPV5 and TRPV5⌬N or TRPV5⌬C showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5⌬N⌬C displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5⌬N or TRPV5⌬C but not with TRPV5⌬N⌬C. TRPV5 channel assembly signals were refined between amino acid positions 64 -77 and 596 -601 in the N-tail and C-tail, respectively. Pull-down assays and co-immunoprecipitations demonstrated that N-or C-tail mutants lacking these critical assembly domains were unable to interact with tails of TRPV5. In conclusion, two domains in the N-tail (residues 64 -77) and C-tail (residues 596 -601) of TRPV5 are important for channel subunit assembly, subsequent trafficking of the TRPV5 channel complex to the plasma membrane, and channel activity.TRPV5 and TRPV6 constitute the Ca 2ϩ influx pathway in 1,25-dihydroxyvitamin D 3 -responsive epithelia, including small intestine, kidney, and placenta, and play a vital role in the process of Ca 2ϩ (re)absorption (1-4). Both channels belong to a distinct subfamily (TRPV) within the superfamily of transient receptor potential channels (TRP). 1 The TRP family consists of a diverse group of non-voltage-gated cation channels, including TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies, which varies significantly in their selectivity and mode of activation (5). The understanding of the function, gating, regulation, and structure assembly of the TRP family is developing rapidly. Initially, it was demonstrated that the Drosophila TRP and TRPL members form heteromultimeric channels associated in a supramolecular signaling complex with specific receptors and regulators (6). Moreover, it has been identified that there are many channel compositions within the TRPC family, e.g. TRPC1/3, TRPC1/5, TRPC4/5, and TRPC3/ 6/7 (7-9). Within the TRPV family, the oligomeric structure of TRPV1 was studied by biochemical cross-linking, and the predominant existence of tetramers was suggested (10). More recently, it has been reported that TRPV5 and TRPV6 form homo-or heterotetramers in order to generate a pleiotropic set of functional channels with different Ca 2ϩ transport (11-13). TRPV5 and TRPV6 share 75% sequence homology at the amino acid level (11-13) and display several similar functional properties, including the permeation profile for monovalent and divalent cations (14) and regulation by calciotropic hormones (15-21). However, detailed sequence comparison of the N-and C-tails of the TRPV5 and TRPV6 channels reveals significant differences, which may account for the unique electrophysiological properties including differences of inactivation, kinetic properties, and affinity for the blocker ruthenium red between these two homologous channels (22).A considerable amount of information in channel subunit assembly has been accumulated by studies on voltage-gated K ϩ (K v ) channels that are structurally related to t...

The Ca 2؉ channels ECaC1 and ECaC2 (TRPV5 and TRPV6) share several functional properties including permeation profile and Ca 2؉ -dependent inactivation. However, the kinetics of ECaC2 currents notably differ from ECaC1 currents. The initial inactivation is much faster in ECaC2 than in ECaC1, and the kinetic differences between Ca 2؉ and Ba 2؉ currents are more pronounced for ECaC2 than ECaC1. Here, we identify the structural determinants for these functional differences. Chimeric proteins were expressed heterologously in HEK 293 cells and studied by patch clamp analysis. Both channels retained their phenotype after exchanging the complete N termini, the C termini, or even both N and C termini, i.e. ECaC1 with the ECaC2 N or C terminus still showed the ECaC1 phenotype and vice versa. The substitution of the intracellular loop between the transmembrane domains 2 and 3 of ECaC2 with that of ECaC1 induced a delay of inactivation. Three amino acid residues (Leu-409, Val-411 and Thr-412) present in this loop determine the fast inactivation behavior. When this intracellular loop between the transmembrane domains 2 and 3 of ECaC1 was exchanged with the TM2-TM3 loop of ECaC2, the ECaC1 kinetics were analogous to ECaC2. In conclusion, the TM2-TM3 loop is a critical determinant of the inactivation in ECaC1 and ECaC2.

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.

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