Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg 2؉ -permeable cation channel TRPM7, resulting in the assembly of functional TRPM6͞TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6͞ TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.I nvestigations on Drosophila flies with impaired vision due to mutations in the transient receptor potential gene (trp) initiated a search for homologous proteins in mammals, leading to the discovery of three subfamilies of cation channels: TRPCs (canonical or classical TRPs), TRPVs (vanilloid receptor and related proteins), and TRPMs (melastatin and related proteins) (1, 2). TRPC channels mediate cation entry in response to phospholipase C activation, whereas TRPV proteins respond to physical and chemical stimuli, such as temperature, pH, and mechanical stress (3, 4). Within their respective subfamilies, TRPCs and TRPVs form homo-and heterotetramers displaying novel pore properties when compared to their homomultimeric counterparts (1, 5-9). The eight TRPM family members differ significantly from the aforementioned TRP channels in terms of domain structure, cation selectivity, and activation mechanisms (3, 10). Two TRPM proteins, TRPM6 and TRPM7, harbor serine͞threonine kinase domains in their C termini (11-16). Furthermore, TRPM7 displays unusual permeation properties by conducting a range of divalent metal ions including Mg 2ϩ and Mn 2ϩ (13,17,18).It was recently shown that autosomal recessive hypomagnesemia with secondary hypocalcemia (HSH) is caused by mutations in the TRPM6 gene (15,16). HSH is characterized by low serum Mg 2ϩ levels due to defective intestinal absorption or͞and renal wasting of Mg 2ϩ . Here we demonstrate that TRPM6 requires assembly with TRPM7 to form channel complexes in the cell membrane and that disruption of multimer formation by a mutated TRPM6 variant, TRPM6(S141L), results in human disease. MethodsMolecular Biology and Generation of TRPM6 Polyclonal Antisera. The cloning procedure of human TRPM6 isoforms (Table 1) as well as amplification of other TRPM cDNAs is described in detail in Supporting Methods, which is published as supporting information on the PNAS web site. For TRPM proteins C-terminally fused to cyan (CFP) or yellow (YFP) fluorescent proteins, STOP codons in TRPMs were replaced by XhoI restrictio...

The TRPM subfamily of mammalian TRP channels displays unusually diverse activation mechanisms and selectivities. One member of this subfamily, TRPM5, functions in taste receptor cells and has been reported to be activated through G protein-coupled receptors linked to phospholipase C. However, the specific mechanisms regulating TRPM5 have not been described. Here, we demonstrate that TRPM5 is a monovalent-specific cation channel with a 23 pS unitary conductance. TRPM5 does not display constitutive activity. Rather, it is activated by stimulation of a receptor pathway coupled to phospholipase C and by IP(3)-mediated Ca(2+) release. Gating of TRPM5 was dependent on a rise in Ca(2+) because it was fully activated by Ca(2+). Unlike any previously described mammalian TRP channel, TRPM5 displayed voltage modulation and rapid activation and deactivation kinetics upon receptor stimulation. The most closely related protein, the Ca(2+)-activated monovalent-selective cation channel TRPM4b, also showed voltage modulation, although with slower relaxation kinetics than TRPM5. Taken together, the data demonstrate that TRPM5 and TRPM4b represent the first examples of voltage-modulated, Ca(2+)-activated, monovalent cation channels (VCAMs). The voltage modulation and rapid kinetics provide TRPM5 with an excellent set of properties for participating in signaling in taste receptors and other excitable cells.

Significance We report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system. These cells exhibit structural markers of respiratory chemosensory cells (“brush cells”). They use the classical taste transduction cascade to detect potential hazardous compounds (bitter, umami, uropathogenic bacteria) and release acetylcholine in response. They lie next to sensory nerve fibers that carry acetylcholine receptors, and placing a bitter compound in the urethra enhances activity of the bladder detrusor muscle. Thus, monitoring of urethral content is linked to bladder control via a previously unrecognized cell type.

Background: A growing number of TRP channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. TRPM5 is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. A role of TRPM5 in other sensory systems, however, has not been studied so far.

Human magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems - next to passive paracellular magnesium flux - involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism.

BACKGROUND AND PURPOSETransient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an a-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells. EXPERIMENTAL APPROACHWe developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments. KEY RESULTSTRPM7 currents were inhibited by modulators of small conductance Ca 2+ -activated K + channels (KCa2.1-2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC50 1.6 mM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg 2+ -dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. CONCLUSIONS AND IMPLICATIONSThis study reveals a tight overlap in the pharmacological profiles of TRPM7 and KCa2.1-2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7. Abbreviations

Hypomagnesemia with secondary hypocalcemia is an autosomal recessive disorder caused by mutations in the TRPM6 gene. Current experimental evidence suggests that TRPM6 may function in a specific association with TRPM7 by means of heterooligomeric channel complex formation. Here, we report the identification and functional characterization of a new hypomagnesemia with secondary hypocalcemia missense mutation in TRPM6. The affected subject presented with profound hypomagnesemia and hypocalcemia caused by compound heterozygous mutation in the TRPM6 gene: 1208(؊1)G > A affecting the acceptor splice site preceding exon 11, and 3050C > G resulting in the amino acid change (P1017R) in the putative pore-forming region of TRPM6. To assess the functional consequences of the P1017R mutation, TRPM6 P1017R and wild-type TRPM6 were co-expressed with TRPM7 in Xenopus oocytes and HEK 293 cells, and currents were assessed by two-electrode voltage clamp and whole cell patch clamp measurements, respectively. Co-expression of wild-type TRPM6 and TRPM7 resulted in a significant increase in the amplitude of TRPM7-like currents. In contrast, TRPM6 P1017R suppressed TRPM7 channel activity. In line with these observations, TRPM7, containing the corresponding mutation P1040R, displayed a dominant-negative effect upon co-expression with wild-type TRPM7. Confocal microscopy and fluorescence resonance energy transfer recordings demonstrated that the P1017R mutation neither affects assembly of TRPM6 with TRPM7, nor co-trafficking of heteromultimeric channel complexes to the cell surface. We conclude that a functional defect in the putative pore of TRPM6/7 channel complexes is sufficient to impair body magnesium homeostasis. Mg2ϩ plays a vital role in virtually all cellular pathways as a cofactor of enzymes, an essential structural element of proteins and nucleic acids, and a modulator of receptors and ion channels (1-4). At present, the molecular mechanisms controlling wasting (3, 9, 10). Hypocalcemia results from a secondary insufficiency of the parathyroid glands in the presence of profound hypomagnesemia. Supplementation with high Mg 2ϩ doses compensates for the Mg 2ϩ deficiency of HSH patients (3, 9, 10). TRPM6 belongs to the melastatin-related group of the TRP ion channel family (11,12). Like other TRP channels, TRPM proteins contain six transmembrane helices (S1-S6) flanked by cytoplasmic N and C termini; TRPMs most likely function as tetrameric channel complexes (12). Hydrophobic segments located between the S5 and S6 helices of four channel subunits are thought to contribute to a channel pore. In contrast to other known ion channels, TRPM6 and its closest family member, TRPM7, display the unique structural feature of being cation channels fused to Ser/Thr kinase domains at their C termini (13).TRPM7 is a ubiquitously expressed protein, which is essential for Mg 2ϩ homeostasis. Disruption of the TRPM7 gene in DT40 chicken lymphocytes and the zebrafish Danio rerio, resulted in Mg 2ϩ deficiency (14, 15). Functional characterization of he...

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