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Hypomagnesemia with secondary hypocalcemia (HSH) is characterized by generalized convulsions and neurological damage due to the lack of magnesium. Several point mutations in the TRPM6 gene have been found to be responsible for the development of the disease. Thus, TRPM6 is the first member of the TRPM channel subfamily to be involved in magnesium reabsorption in the human intestine and kidney. In the present study, a new mutation found in a patient with HSH (P1017R) was analyzed. In contrast to other mutations, which were not able to reach the cell surface, the TRPM6P1017R protein was localized in the cell membrane after coexpression with TRPM7. Measurements of TRPM6P1017R in the presence of TRPM7, which is necessary for the formation of functional channel complexes, revealed that the heteromeric channels containing the mutant protein were not capable of generating any currents. P1017R even supressed the current of the heterologously expressed TRPM7 protein, as well as endogenous cation currents in HEK293 cells, thus exhibiting a dominant negative effect. As the homologous mutation in TRPM7 (P1040R) decreased currents to an even higher degree, it can be concluded that this proline plays an important role in the pore of TRPM6 and TRPM7, and is essential for their physiological role.
Further studies focussed on the generation of pore mutants to gain insight into the molecular background determining divalent permeation properties in the TRPM subfamily. By performing site directed mutagenesis, single amino acids of the divalent selective TRPM7 channel were exchanged for the corresponding amino acids of the non selective TRPM2. The substitution of glutamate 1047 by glutamine almost abolished divalent permeation through the TRPM7 channel. At the same time, the inhibition of channel activity by extracellular, but not intracellular magnesium ions was profoundly affected. The additional substitution of a proline for a tyrosine (E1047Q/Y1049P) resulted in a current voltage relationship which strongly resembled that of TRPM2. In line with this observation, the reciprocal exchange of both amino acids in TRPM2 to those from TRPM7 resulted in a higher permeation of divalent cations. Hence, we identified two residues in the pore region that determine divalent cation permeability in the TRPM subfamily. In addition, a phylogenetic analysis revealed that these two amino acids and together with them the permeation properties of TRPM channels have changed during evolution. The ancestral TRPM channel was a calcium-permeable, TRPM2-like protein with a NUDT9-like domain and the ’EVY’-motif. The ’QIP’-motif only appeared in more recent species. Therefore, human TRPM2 is an ancient channel with a ’modern’ pore.
In conclusion, the present study offers valuable clues to the relationship between structure and permeation properties in cation channels. In a phylogenetic context, these results allow conclusions about the evolution of the pore region in TRPM proteins.