Hypomagnesemia with Secondary Hypocalcemia due to a Missense Mutation in the Putative Pore-forming Region of TRPM6

Abstract

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...

New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia

“…10,[14][15][16][17]20,31 Furthermore, five missense mutations leading to HSH have been described so far, from which only S 141 L and P 1017 R were further studied at the molecular level. 10,19 The S 141 L mutation found in the highly conserved serine 141 resulted in impaired trafficking of TRPM6 to the membrane, 10,18 while the P 1017 R mutation located in the putative pore-forming region is the only known example of a mutation affecting TRPM6-gating properties without altering assembly or trafficking events. 19 The remaining three-point mutations were found in three Polish families; however, the authors did not report whether HSH was a consequence of either TRPM6 trafficking or functional impairment.…”

“…10,19 The S 141 L mutation found in the highly conserved serine 141 resulted in impaired trafficking of TRPM6 to the membrane, 10,18 while the P 1017 R mutation located in the putative pore-forming region is the only known example of a mutation affecting TRPM6-gating properties without altering assembly or trafficking events. 19 The remaining three-point mutations were found in three Polish families; however, the authors did not report whether HSH was a consequence of either TRPM6 trafficking or functional impairment. 20 Overall, 34 different mutations have been previously described, from which only five are point mutations, thus B85% of mutations are truncating.…”

“…10,11,[14][15][16][17] So far, there are two missense mutations described previously, S 141 L and P 1017 R, resulting in either trafficking or gating impairment of TRPM6. 18,19 Another three-point mutations leading to HSH, where the underlying molecular mechanism was not disclosed, have also been published. 20 Here, we characterize a new set of five missense mutations identified in five HSH patients.…”

Regulation of the Epithelial Mg2+ Channel TRPM6 by Estrogen and the Associated Repressor Protein of Estrogen Receptor Activity (REA)

“…Previous studies demonstrated that expression of TRPM6 is regulated by dietary Mg 2ϩ and its channel activity is strongly inhibited by the intracellular Mg 2ϩ concentration ([Mg 2ϩ ] i ) (8,9). Generally, Mg 2ϩ -free solutions are used to measure the characteristic outwardly rectifying TRPM6 currents (8,10,11). Remarkably, TRPM6 contains a C-terminal ␣-kinase domain, of which the regulatory role on channel activity remains elusive (12)(13)(14).…”

Renal TRPathies

“…11,76 Functional consequences of the naturally occurring TRPM6 mutations include introduction of frame shifts, stop codons, or disruption of exon splice sites. 66,77,78 Only five missense mutations have been identified so far (Figure 2A) 66,77,78 ; their impact on TRPM6 function is discussed next.…”