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. 2019 Mar 5;116(10):4706-4715.
doi: 10.1073/pnas.1810633116. Epub 2019 Feb 15.

TRPM7 is the central gatekeeper of intestinal mineral absorption essential for postnatal survival

Affiliations

TRPM7 is the central gatekeeper of intestinal mineral absorption essential for postnatal survival

Lorenz Mittermeier et al. Proc Natl Acad Sci U S A. .

Abstract

Zn2+, Mg2+, and Ca2+ are essential minerals required for a plethora of metabolic processes and signaling pathways. Different categories of cation-selective channels and transporters are therefore required to tightly control the cellular levels of individual metals in a cell-specific manner. However, the mechanisms responsible for the organismal balance of these essential minerals are poorly understood. Herein, we identify a central and indispensable role of the channel-kinase TRPM7 for organismal mineral homeostasis. The function of TRPM7 was assessed by single-channel analysis of TRPM7, phenotyping of TRPM7-deficient cells in conjunction with metabolic profiling of mice carrying kidney- and intestine-restricted null mutations in Trpm7 and animals with a global "kinase-dead" point mutation in the gene. The TRPM7 channel reconstituted in lipid bilayers displayed a similar permeability to Zn2+ and Mg2+ Consistently, we found that endogenous TRPM7 regulates the total content of Zn2+ and Mg2+ in cultured cells. Unexpectedly, genetic inactivation of intestinal rather than kidney TRPM7 caused profound deficiencies specifically of Zn2+, Mg2+, and Ca2+ at the organismal level, a scenario incompatible with early postnatal growth and survival. In contrast, global ablation of TRPM7 kinase activity did not affect mineral homeostasis, reinforcing the importance of the channel activity of TRPM7. Finally, dietary Zn2+ and Mg2+ fortifications significantly extended the survival of offspring lacking intestinal TRPM7. Hence, the organismal balance of divalent cations critically relies on one common gatekeeper, the intestinal TRPM7 channel.

Keywords: TRP channels; TRPM7; calcium; magnesium; zinc.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Assessment of human haploid leukemia (HAP1) cells deficient in TRPM7. (A) Determination of elementary levels of Ca (Left), Mg (Center), and Zn (Right) in wild-type (WT) and TRPM7-deficient (KO) HAP1 cells. Dried cell pellets were obtained from WT and KO HAP1 cells cultured in a standard cell culture medium for 24 h and analyzed by ICP-MS. Total elementary contents were normalized to dry pellet weight and represented as mean ± SEM of n = 4 independent measurements. (B) Total elementary contents of divalent metals obtained in A were normalized to total elementary contents of sulfur (S) and represented as mean ± SEM. ***P ≤ 0.001; **P ≤ 0.01; n.s., not significant (Student’s t test). (C) Uptake of 65Zn2+ in WT and KO HAP1 cells. Cells were incubated in the presence of 1 mM Mg2+, 2 mM Ca2+, and 2 µM 65Zn2+ and time-dependent accumulation of 65Zn2+ was determined, presented as mean ± SEM for n = 6 independent measurements. Datasets were fitted using a one-phase exponential association equation followed by a statistical assessment with the extra sum-of-squares F test.
Fig. 2.
Fig. 2.
Assessment of TRPM7 channel activity in lipid bilayers. (A) Single-channel recordings of TRPM7 at +100 mV in the absence or presence of 5 µM PIP2. Representative current traces of TRPM7 were obtained from n = 5 independent experiments (n = 1,565 events analyzed). The closed and open states of TRPM7 are outlined by c and o, respectively. (B and C) Voltage sensitivity of the TRPM7 channel as assessed by analysis of mean slope conductance (B) and open probability (Po) (C) of TRPM7 currents measured in the presence of 2.5 µM PIP2. Data are presented as mean ± SEM (n = 11, n = 149,763). Experiments in AC were performed under symmetric ionic conditions (Materials and Methods). (D) Representative current traces of TRPM7 channel activity under Mg2+out−K+in or Zn2+out−K+in biionic conditions in the presence of 2.5 µM PIP2 (n = 4 for each condition, n = 10,521). The voltage-ramp recordings were obtained over a period of 90 s. Reversal potentials (Erev) were 4.74 ± 0.46 mV at Mg2+out−K+in (mean ± SEM, n = 11) and 7.58 ± 0.74 mV at Zn2+out−K+in conditions (mean ± SEM, n = 8). (E) Po (mean ± SEM) of TRPM7 calculated for measurements shown in D. (F and G) Effects of Mg2+in on TRPM7 currents. (F) Representative traces of TRPM7 currents evoked analogously to A followed by a sequential application of 0.5 mM and 1 mM Mg2+ (n = 3). (G) Time-dependent suppression of TRPM7 conductance (mean ± SEM, n = 15, n = 5,642) calculated from the measurements shown in F. G, Inset shows representative traces of single-channel recordings of TRPM7 at +100 mV obtained at 0 min, 12 min, and 24 min. ***P ≤ 0.001 (one-way ANOVA).
Fig. 3.
Fig. 3.
Characterization of mice with intestine-restricted inactivation of Trpm7. (AD) Trpm7fl/fl (control) and Trpm7fl/fl;Villin1-Cre (intestine KO) littermates were studied for overall physical appearance at P5 (A) and growth (B), survival rate (C), and total length of the intestine normalized to body weight at P5 (D). The results in B and D are represented as mean ± SEM. (EH) Assessment of Villin1-Cre–mediated deletion of WT Trpm7 transcripts in the intestine of Trpm7fl/fl;Villin1-Cre mice. (E) Representative images of purified villi isolated from the whole intestine of P5 littermates. (F and G) Relative expression levels (mean ± SEM) of Villin1 (F) and Trpm7 (G) in mRNA extracts from villi examined by qPCR using Hprt as a reference transcript. (H) Relative expression levels of Dclk1, Villin1, Lgr5, Muc2, and Lyz1 in mRNA extracts from the whole duodenum analyzed by qPCR using Hprt as a reference transcript. Results are shown as fold change (mean ± SEM) in Trpm7fl/fl;Villin1-Cre (n = 8) mice vs. Trpm7fl/fl (n = 7) mice. (I) Pathways commonly affected in the intestine of Trpm7-deficient mice and the liver of Trpm6-deficient mice (13). Whole-genome profiling of the transcriptome of villi isolated from P5 Trpm7fl/fl;Villin1-Cre (n = 3) and Trpm7fl/fl (n = 3) littermates was analyzed using ingenuity pathway analysis (IPA) (Datasets S1 and S2). Next, IPA was used to compare gene networks highly affected in Trpm7fl/fl;Villin1-Cre (Trpm7 KO) and Trpm6 null (Trpm6 KO) mice (13). (J) Relative expression levels of markers of mature and progenitor enterocytes Alpi and Mki-67 and genes representing the IPA glutathione-mediated detoxification and the NRF2-mediated oxidative stress response (Dataset S2) were examined in mRNA extracts from villi of P5 littermates using qPCR, using Hprt as a reference transcript (n = 3 mice per genotype). The results are shown as fold change (mean ± SEM) in Trpm7fl/fl;Villin1-Cre samples vs. Trpm7fl/fl samples. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; n.s., not significant (Student’s t test).
Fig. 4.
Fig. 4.
Analysis of Ca2+, Mg2+, and Zn2+ homeostasis in mice with intestine-restricted inactivation of Trpm7. (A and B) ICP-MS analysis of elementary levels of Ca (Left), Mg (Center), and Zn (Right) in serum (A) and bones (right tibia) (B) of P1, P3, and P5 Trpm7fl/fl (control) and Trpm7fl/fl;Villin1-Cre (intestine KO) littermates (n, number of mice examined per data point). In B total elementary contents were normalized to dry bone weights (mean ± SEM); n, number of mice per genotype. (C and D) P5 littermates were examined for circulating levels of PTH (C) and 1,25-(OH)2D3 (D) (mean ± SEM). Note that only two control samples contained PTH levels in the range of ELISA sensitivity, and statistical analysis could not be conducted. (E) Relative expression levels (mean ± SEM) of Cyp27b1 in mRNA extracts from whole kidneys of P5 littermates were studied using qPCR and Ywhaz as a reference transcript. (F) Relative expression levels of indicated transcripts were investigated in mRNA extracts from whole duodenums of P5 littermates using qPCR and Hprt as a reference transcript. Results are calculated as fold change (mean ± SEM) in Trpm7fl/fl;Villin1-Cre (n = 8–12) mice vs. Trpm7fl/fl (n = 7–12) mice. (GI) P5 littermates were assessed for circulating levels of IGF1 (G), T4 (H), and IL-6 (I) (mean ± SEM). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; n.s., not significant (Student’s t test); n, number of mice examined.
Fig. 5.
Fig. 5.
Effects of Ca2+, Mg2+, and Zn2+ dietary treatments on survival of mice with intestine-restricted inactivation of Trpm7. (AC) Kaplan–Meier survival distributions of Trpm7fl/fl;Villin1-Cre mice maintained on a regular diet vs. Ca- (A), Mg- (B), and Zn-supplemented and combined Ca/Mg/Zn-supplemented mice (C) as outlined in SI Appendix, Table S2. The indicated P values were calculated by comparison of each supplemented group vs. the group maintained at regular diet using a log-rank test; n, number of mice examined. (D) Representative images of a surviving P33 Trpm7fl/fl;Villin1-Cre (intestine KO) mouse and a corresponding Trpm7fl/fl (control) littermate maintained at an enriched Zn2+ diet.

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