New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia

Abstract

Despite recent progress in our understanding of renal magnesium (Mg(2+)) handling, the molecular mechanisms accounting for transepithelial Mg(2+) transport are still poorly understood. Mutations in the TRPM6 gene, encoding the epithelial Mg(2+) channel TRPM6 (transient receptor potential melastatin 6), have been proven to be the molecular cause of hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014). HSH manifests in the newborn period being characterized by very low serum Mg(2+) levels (<0.4 mmol/l) accompanied by low serum calcium (Ca(2+)) concentrations. A proportion of previously described TRPM6 mutations lead to a truncated TRPM6 protein resulting in a complete loss-of-function of the ion channel. In addition, five-point mutations have been previously described. The aim of this study was to complement the current clinical picture by adding the molecular data from five new missense mutations found in five patients with HSH. To this end, patch-clamp analysis and cell surface measurements were performed to assess the effect of the various mutations on TRPM6 channel function. All mutant channels, expressed in HEK293 cells, showed loss-of-function, whereas no severe trafficking impairment to the plasma membrane surface was observed. We conclude that the new TRPM6 missense mutations lead to dysregulated intestinal/renal Mg(2+) (re)absorption as a consequence of loss of TRPM6 channel function.

Figure 1

Family pedigrees and clinical data of patients with HSH. Panel a shows the five family pedigrees. Affected family members are indicated by black circles (girls) and squares (boys). The double horizontal line in the diagram for family 37 indicates parental consanguinity. Mutation analysis is shown for patients as well as for parents in families F37, F38 and F40. Panel b shows levels of serum Mg²⁺ and Ca²⁺ at manifestation, the dosage of oral Mg²⁺ supplements during maintenance therapy as well as the serum Mg²⁺ levels measured during follow-up. The black circles indicate the values measured in the five index patients of this study. White circles indicated individual values obtained in a previously published cohort of patients with truncating TRPM6 mutations (Schlingmann et al) to allow for a better comparison of phenotypes. The reference ranges for serum magnesium and calcium are indicated by gray shading, the recommended daily allowance for oral Mg²⁺ intake (0.25 mmol/kg/day) is indicated by a dashed line.

Figure 2

Functional analysis of WT and mutant TRPM6 channels transiently expressed in HEK293 cells. (a) Averaged current density at +80 mV after 200 s of mock, WT TRPM6, and mutant TRPM6 (≥10 cells per condition; n=3 experiments). *P<0.05 compared with WT. (b) Current–voltage (I/V) relationship from representative traces of WT TRPM6 (1), Q¹⁶⁶³R SNP (2), E⁸⁷²G or S¹⁷⁵⁴N mutants (3), and mock or the other mutants (4). (c) Cell surface biotinylation of mock, WT TRPM6, and mutant/SNP TRPM6 expressing HEK293 cells. TRPM6 expression was analyzed by immunoblotting for plasma membrane fraction (lower panel) and input from the total cell lysates (upper panel). Representative immunoblot of three independent experiments is shown. (d) Cell surface biotinylation of cytosolic proteins in HEK293 cells. Left panel shows TRPM6 expression and membrane-binding control, whereas the middle and left panels show no biotin bound to cytosolic proteins (GFP and Akt in this case). This control confirms that only membrane proteins are detected in our biotinylation assays.

Figure 3

Functional effect of the E⁸⁷²G mutation. (a) Representative traces of the time course of the current density at +80 mV of TRPM6/TRPM6 (solid circles), TRPM6/E⁸⁷²G (open squares), and Q¹⁶⁶³R/E⁸⁷²G SNP and mutant (open triangles), n≥15 cells per condition; n=3 experiments. (b) Averaged current density at +80 mV after 200 s of the indicated conditions. *P<0.05 compared with WT. (c) Current–voltage relationship from representative traces of TRPM6/TRPM6 (solid line), TRPM6/E⁸⁷²G (dotted line), and Q¹⁶⁶³R/E⁸⁷²G SNP and mutant (dashed line), n≥15 cells per condition; n=3 experiments. (d) Cell surface biotinylation of mock, TRPM6/TRPM6, TRPM6/E⁸⁷²G, and Q¹⁶⁶³R/E⁸⁷²G coexpressing HEK293 cells. TRPM6 expression was analyzed by immunoblotting for plasma membrane fraction (lower panel) and input from the total cell lysates (upper panel). Representative immunoblot of three independent experiments is shown.

Figure 4

Topological illustration of a monomeric subunit of TRPM6. TRPM6 is exhibiting six putative transmembrane segments as well as intracellular N-terminal and C-terminal domains. It also contains its characteristic α-kinase domain at the C-terminus. The missense mutations and SNP found in the HSH patients are indicated in red. The full colour version of this figure is available at European Journal of Human Genetics online.