Evolutionary determinants of divergent calcium selectivity of TRPM channels.

The mammalian TRPM gene family can be subdivided into distinct categories of cation channels that are either highly permeable for Ca(2+) (TRPM3/6/7), nonselective (TRPM2/8), or even Ca(2+) impermeable (TRPM4/5). TRPM6/7 are fused to alpha-kinase domains, whereas TRPM2 is linked to an ADP-ribose phosphohydrolase (Nudix domain). At a molecular level, the evolutionary steps that gave rise to the structural and functional TRPM channel diversity remain elusive. Here, we provide phylogenetic evidence that Nudix-linked channels represent an ancestral type of TRPMs that is present in various phyla, ranging from protists to humans. Surprisingly, the pore-forming segments of invertebrate TRPM2-like proteins display high sequence similarity to those of Ca(2+)-selective TRPMs, while human TRPM2 is characterized by a loss of several conserved residues. Using the patch-clamp technique, Ca(2+) imaging, and site-directed mutagenesis, we demonstrate that restoration of only two "ancient" pore residues in human TRPM2 (Q981E/P983Y) significantly increased (approximately 4-fold) its permeability for Ca(2+). Conversely, introduction of a "modern" sequence motif into mouse TRPM7 (E1047Q/Y1049P) resulted in the loss of Ca(2+) permeation and a linear TRPM2-like current-voltage relationship. Overall, our findings provide an integrative view on the evolution of the domain architecture and the structural basis of the distinct ion permeation profiles of TRPM channels.

Hypomagnesemia with secondary hypocalcemia due to a missense mutation in the putative pore-forming region of TRPM6.

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.