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Drosophila TRPM channel is essential for the control of extracellular magnesium levels.

Hofmann T, Chubanov V, Chen X, Dietz AS, Gudermann T, Montell C - PLoS ONE (2010)

Bottom Line: We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules.In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality.Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+.

View Article: PubMed Central - PubMed

Affiliation: Institut für Pharmakologie und Toxikologie, Philipps-Universität Marburg, Marburg, Germany.

ABSTRACT
The TRPM group of cation channels plays diverse roles ranging from sensory signaling to Mg2+ homeostasis. In most metazoan organisms the TRPM subfamily is comprised of multiple members, including eight in humans. However, the Drosophila TRPM subfamily is unusual in that it consists of a single member. Currently, the functional requirements for this channel have not been reported. Here, we found that the Drosophila TRPM protein was expressed in the fly counterpart of mammalian kidneys, the Malpighian tubules, which function in the removal of electrolytes and toxic components from the hemolymph. We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules. In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality. Supplementation of the diet with a high concentration of Mg2+ exacerbated the phenotype, resulting in growth arrest during the larval period. Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+. We conclude that loss of Drosophila trpm leads to hypermagnesemia due to a defect in removal of Mg2+ from the hemolymph. These data provide the first evidence for a role for a Drosophila TRP channel in Mg2+ homeostasis, and underscore a broad and evolutionarily conserved role for TRPM channels in Mg2+ homeostasis.

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Impact of dietary Mg2+ on larval growth in the trpm mutant.(A) Sizes of larvae after feeding for three days on food containing 1 or 30 mM total Mg2+. (B) Fat bodies dissected from w1118 and trpm2 3rd instar larvae fed on either 1 mM or 30 mM Mg2+. (C) Summary of total larval protein per larvae after 80 hrs on media supplemented with the indicated divalent cation salts (n = 5−10). (D) Sensitivity of trpm larvae (trpm1) to the duration of Mg2+ exposure. Larvae were kept in media containing 1 or 30 mM MgCl2 for 1–3 days as indicated. (E) Hemolymph protein content in larvae exposed to low and high levels of MgCl2 (white bars, Canton S; black bars, trpm2). (F) Detection of hemolymph proteins by silver staining after by SDS-PAGE fractionation of the equivalent of 160 nl of hemolymph in each lane. Protein sizes markers are indicated to the left (kDa) and the bracket indicates the LSP proteins/hexamerins. (G) Protein content in the fat bodies microdissected from 3rd instar larvae after being maintained on day 3 on either 1 or 30 mM Mg2+. (H) Effect of 1 day of exposure to elevated Mg2+ on the locomotive performance of w1118 and trpm1 larvae. 3rd instar larvae were allowed to move freely on a 2% agar field demarcated with a projected 1 cm grid. We then assayed the number of lines crossed after 3 min (n = 5−6 each, mean +/− SEM). The level of statistical significance of the difference between groups is indicated above the bars (*, p<0.05; ***, p<0.001, n.s. not significant).
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pone-0010519-g002: Impact of dietary Mg2+ on larval growth in the trpm mutant.(A) Sizes of larvae after feeding for three days on food containing 1 or 30 mM total Mg2+. (B) Fat bodies dissected from w1118 and trpm2 3rd instar larvae fed on either 1 mM or 30 mM Mg2+. (C) Summary of total larval protein per larvae after 80 hrs on media supplemented with the indicated divalent cation salts (n = 5−10). (D) Sensitivity of trpm larvae (trpm1) to the duration of Mg2+ exposure. Larvae were kept in media containing 1 or 30 mM MgCl2 for 1–3 days as indicated. (E) Hemolymph protein content in larvae exposed to low and high levels of MgCl2 (white bars, Canton S; black bars, trpm2). (F) Detection of hemolymph proteins by silver staining after by SDS-PAGE fractionation of the equivalent of 160 nl of hemolymph in each lane. Protein sizes markers are indicated to the left (kDa) and the bracket indicates the LSP proteins/hexamerins. (G) Protein content in the fat bodies microdissected from 3rd instar larvae after being maintained on day 3 on either 1 or 30 mM Mg2+. (H) Effect of 1 day of exposure to elevated Mg2+ on the locomotive performance of w1118 and trpm1 larvae. 3rd instar larvae were allowed to move freely on a 2% agar field demarcated with a projected 1 cm grid. We then assayed the number of lines crossed after 3 min (n = 5−6 each, mean +/− SEM). The level of statistical significance of the difference between groups is indicated above the bars (*, p<0.05; ***, p<0.001, n.s. not significant).

Mentions: Given that members of the TRP superfamily are cation channels, some of which are known to be essential for divalent cation homeostasis, we wondered whether the trpm pupal lethality might be enhanced or suppressed by supplementing the food with increased levels of cations. We found that addition of 30 mM Mg2+ to the standard food (1 mM Mg2+) caused nearly complete larval lethality, as no homozygous larvae reached the pupal stage (Figure 1F). In contrast to these results, high Mg2+ had no effect on survival of trpm+ control animals (w1118; Figure 1F). Introduction of a variety of other cations (CaCl2, NaCl and KCl) to the food had no effect on larval survival of trpm1 or the rate of wild-type development (Figure 1F). When we added 10–30 mM MgCl2 to the food, the larvae were smaller and thinner than wild-type (Figure 2A), and the cells in the fat bodies were dramatically reduced in size (Figure 2B and Figure S2). However, when we reared the larvae on standard food (0.5–1 mM Mg2+), the overall dimensions of the trpm1 larvae and the size of the fat body cells were reduced only slightly relative to wild-type (Figure 2A and B).


Drosophila TRPM channel is essential for the control of extracellular magnesium levels.

Hofmann T, Chubanov V, Chen X, Dietz AS, Gudermann T, Montell C - PLoS ONE (2010)

Impact of dietary Mg2+ on larval growth in the trpm mutant.(A) Sizes of larvae after feeding for three days on food containing 1 or 30 mM total Mg2+. (B) Fat bodies dissected from w1118 and trpm2 3rd instar larvae fed on either 1 mM or 30 mM Mg2+. (C) Summary of total larval protein per larvae after 80 hrs on media supplemented with the indicated divalent cation salts (n = 5−10). (D) Sensitivity of trpm larvae (trpm1) to the duration of Mg2+ exposure. Larvae were kept in media containing 1 or 30 mM MgCl2 for 1–3 days as indicated. (E) Hemolymph protein content in larvae exposed to low and high levels of MgCl2 (white bars, Canton S; black bars, trpm2). (F) Detection of hemolymph proteins by silver staining after by SDS-PAGE fractionation of the equivalent of 160 nl of hemolymph in each lane. Protein sizes markers are indicated to the left (kDa) and the bracket indicates the LSP proteins/hexamerins. (G) Protein content in the fat bodies microdissected from 3rd instar larvae after being maintained on day 3 on either 1 or 30 mM Mg2+. (H) Effect of 1 day of exposure to elevated Mg2+ on the locomotive performance of w1118 and trpm1 larvae. 3rd instar larvae were allowed to move freely on a 2% agar field demarcated with a projected 1 cm grid. We then assayed the number of lines crossed after 3 min (n = 5−6 each, mean +/− SEM). The level of statistical significance of the difference between groups is indicated above the bars (*, p<0.05; ***, p<0.001, n.s. not significant).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865541&req=5

pone-0010519-g002: Impact of dietary Mg2+ on larval growth in the trpm mutant.(A) Sizes of larvae after feeding for three days on food containing 1 or 30 mM total Mg2+. (B) Fat bodies dissected from w1118 and trpm2 3rd instar larvae fed on either 1 mM or 30 mM Mg2+. (C) Summary of total larval protein per larvae after 80 hrs on media supplemented with the indicated divalent cation salts (n = 5−10). (D) Sensitivity of trpm larvae (trpm1) to the duration of Mg2+ exposure. Larvae were kept in media containing 1 or 30 mM MgCl2 for 1–3 days as indicated. (E) Hemolymph protein content in larvae exposed to low and high levels of MgCl2 (white bars, Canton S; black bars, trpm2). (F) Detection of hemolymph proteins by silver staining after by SDS-PAGE fractionation of the equivalent of 160 nl of hemolymph in each lane. Protein sizes markers are indicated to the left (kDa) and the bracket indicates the LSP proteins/hexamerins. (G) Protein content in the fat bodies microdissected from 3rd instar larvae after being maintained on day 3 on either 1 or 30 mM Mg2+. (H) Effect of 1 day of exposure to elevated Mg2+ on the locomotive performance of w1118 and trpm1 larvae. 3rd instar larvae were allowed to move freely on a 2% agar field demarcated with a projected 1 cm grid. We then assayed the number of lines crossed after 3 min (n = 5−6 each, mean +/− SEM). The level of statistical significance of the difference between groups is indicated above the bars (*, p<0.05; ***, p<0.001, n.s. not significant).
Mentions: Given that members of the TRP superfamily are cation channels, some of which are known to be essential for divalent cation homeostasis, we wondered whether the trpm pupal lethality might be enhanced or suppressed by supplementing the food with increased levels of cations. We found that addition of 30 mM Mg2+ to the standard food (1 mM Mg2+) caused nearly complete larval lethality, as no homozygous larvae reached the pupal stage (Figure 1F). In contrast to these results, high Mg2+ had no effect on survival of trpm+ control animals (w1118; Figure 1F). Introduction of a variety of other cations (CaCl2, NaCl and KCl) to the food had no effect on larval survival of trpm1 or the rate of wild-type development (Figure 1F). When we added 10–30 mM MgCl2 to the food, the larvae were smaller and thinner than wild-type (Figure 2A), and the cells in the fat bodies were dramatically reduced in size (Figure 2B and Figure S2). However, when we reared the larvae on standard food (0.5–1 mM Mg2+), the overall dimensions of the trpm1 larvae and the size of the fat body cells were reduced only slightly relative to wild-type (Figure 2A and B).

Bottom Line: We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules.In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality.Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+.

View Article: PubMed Central - PubMed

Affiliation: Institut für Pharmakologie und Toxikologie, Philipps-Universität Marburg, Marburg, Germany.

ABSTRACT
The TRPM group of cation channels plays diverse roles ranging from sensory signaling to Mg2+ homeostasis. In most metazoan organisms the TRPM subfamily is comprised of multiple members, including eight in humans. However, the Drosophila TRPM subfamily is unusual in that it consists of a single member. Currently, the functional requirements for this channel have not been reported. Here, we found that the Drosophila TRPM protein was expressed in the fly counterpart of mammalian kidneys, the Malpighian tubules, which function in the removal of electrolytes and toxic components from the hemolymph. We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules. In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality. Supplementation of the diet with a high concentration of Mg2+ exacerbated the phenotype, resulting in growth arrest during the larval period. Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+. We conclude that loss of Drosophila trpm leads to hypermagnesemia due to a defect in removal of Mg2+ from the hemolymph. These data provide the first evidence for a role for a Drosophila TRP channel in Mg2+ homeostasis, and underscore a broad and evolutionarily conserved role for TRPM channels in Mg2+ homeostasis.

Show MeSH
Related in: MedlinePlus