EGF increases TRPM6 activity and surface expression

Abstract

Recent identification of a mutation in the EGF gene that causes isolated recessive hypomagnesemia led to the finding that EGF increases the activity of the epithelial magnesium (Mg2+) channel transient receptor potential M6 (TRPM6). To investigate the molecular mechanism mediating this effect, we performed whole-cell patch-clamp recordings of TRPM6 expressed in human embryonic kidney 293 (HEK293) cells. Stimulation of the EGF receptor increased current through TRPM6 but not TRPM7. The carboxy-terminal alpha-kinase domain of TRPM6 did not participate in the EGF receptor-mediated increase in channel activity. This activation relied on both the Src family of tyrosine kinases and the downstream effector Rac1. Activation of Rac1 increased the mobility of TRPM6, assessed by fluorescence recovery after photobleaching, and a constitutively active mutant of Rac1 mimicked the stimulatory effect of EGF on TRPM6 mobility and activity. Ultimately, TRPM6 activation resulted from increased cell surface abundance. In contrast, dominant negative Rac1 decreased TRPM6 mobility, abrogated current development, and prevented the EGF-mediated increase in channel activity. In summary, EGF-mediated stimulation of TRPM6 occurs via signaling through Src kinases and Rac1, thereby redistributing endomembrane TRPM6 to the plasma membrane. These results describe a regulatory mechanism for transepithelial Mg2+ transport and consequently whole-body Mg2+ homeostasis.

Figure 1.

EGF treatment of HEK293 cells stimulates TRPM6 current. (A) EGFR protein expression in HEK293 cells by immunoblot analysis. (B) Immunoblot of TRPM6, using the HA tag, in transiently transfected HEK293 cells. (C and D) Time course of the current development (pA/pF) at +80 mV (C) and −80 mV (D) of TRPM6 transfected HEK293 cells with (▴) or without (•) EGF pretreatment (10 nM, 30 min, 37°C) and empty vector–transfected HEK293 cells with (▵) or without (○) EGF pretreatment (n = 11 to 15 cells). (E) Representative current–voltage relations of TRPM6-transfected HEK293 cells with (▴) or without (•) EGF pretreatment and mock-transfected HEK293 cells with (▵) or without (○) EGF pretreatment after 200 s.

Figure 2.

EGFR stimulation by EGF mediates TRPM6 current increase. (A) Time course of the TRPM6 current development (pA/pF) at +80 mV (in transiently transfected HEK293 cells) pretreated with AG1478 (tyrphostin, 1 μM, 1 h) alone (▵) or before EGF pretreatment (▴) in comparison with nontreated (○) or EGF-treated (•) cells (n = 9 to 13 cells). (B) Time course of TRPM6 current development (pA/pF) at +80 mV in transiently transfected HEK293 cells pretreated with RG13022 (tyrosine kinase inhibitor, 50 μM, 10 min) alone (⋄) or before EGF pretreatment (♦) relative to nontreated (○) or EGF-treated (•) cells (n = 11 to 14 cells). (C) Time course of the TRPM7 current development (pA/pF) at +80 mV after transient transfection of HEK293 cells with (•; n = 21) or without (○; n = 16) EGF pretreatment and empty vector–transfected HEK293 cells with (▴) or without (⋆) EGF pretreatment (n = 9 to 14 cells).

Figure 3.

EGF mediates an Src-family kinase and MAPK-dependent activation of TRPM6 independent of the α-kinase domain. (A) Histogram summarizing the current density (pA/pF) at +80 mV and −80 mV after 200 s in mock-transfected (□) or TRPM6 Δ-kinase–transfected (▪) HEK293 cells in the presence or absence of EGF as indicated. *P < 0.05 versus cells transfected with Δ-kinase in the absence of EGF treatment (n = 13 to 19 cells). (B) Histogram summarizing the averaged current density (pA/pF) at +80 mV and −80 mV after 200 s, of TRPM6-transfected HEK293 cells, pretreated with PP2 (5 μM, 10 min), PP3 (5 μM, 10 min), U73122 (10 μM, 15 min), U17343 (10 μM, 15 min), PD98059 (15 μM, 25 min), UO126 (50 μM, 25 min), or forskolin (10 μM, 15 min) without (□) or with (▪) EGF pretreatment (10 nM, 30 min). *P < 0.05 versus TRPM6 current in the absence of EGF treatment (n = 9 to 25 cells).

Figure 4.

Stimulation of TRPM6 by EGF depends on PI3K, Rac1, and the actin cytoskeleton. (A) Time course of the current development (pA/pF) at +80 mV of TRPM6-transfected HEK293 cells pretreated with wortmannin (10 nM, 20 min; ▴) or LY492005 (50 μM, 20 min; ▾) before treatment with EGF. For comparison, cells that were not treated at all (○) or treated just with EGF (•) are included (n = 9 to 13 cells). (B) Time course of the current development (pA/pF) at +80 mV of TRPM6 and empty vector (circles), TRPM6 and Rac1-G12V (triangles), and TRPM6 and Rac1-T17N (diamonds) transfected HEK293 cells, pretreated (filled symbols) or not (open symbols) with EGF (n = 13 to 19 cells). (C) Histogram summarizing the current density (pA/pF) at +80 and −80 mV of TRPM6 and empty vector (Control), TRPM6 and Rac1-T17N (T17N), and TRPM6 and Rac1-G12V (G12V) transfected HEK293 cells pretreated or not with EGF as indicated. □, Experimental conditions in which HEK293 cells were transfected with the empty vector; ▪, HEK293 cells transfected with TRPM6 in the absence or presence of Rac1 mutants. *P < 0.05 versus TRPM6 current in empty vector–transfected, nontreated conditions (n = 13 to 19 cells). (D) Histogram summarizing the current density (pA/pF) at +80 and −80 mV of TRPM6-transfected HEK293 cells pretreated or not with EGF or cytochalasin D (CytD; 10 μM, 40 min) as indicated. □, Experimental conditions in which HEK293 cells were transfected with empty vector; ▪ HEK293 cells transfected with TRPM6. *P < 0.05 versus TRPM6 current under nontreated conditions (n = 14 to 16 cells).

Figure 5.

EGF increases the mobility of TRPM6. (A) Immunoblot of GFP-TRPM6 transiently transfected in HEK293 cells. (B) A confocal slice through a representative cell. (C) A plot of fluorescence recovery over time in the presence (▪) or absence (○) of EGF. (D) Similar curves of cells co-transfected with GFP-TRPM6 and Rac1-G12V, Rac1-T17N, or mock in the presence or absence of EGF (□, mock; ▵, mock + EGF; ×, Rac1-G12V; *, Rac1-G12V + EGF; ⋄, Rac1-T17N; ○, Rac1-T17N + EGF). (E) A histogram displaying the final recovery of TRPM6 in the presence or absence of EGF when co-transfected with mock (Control), Rac1-G12V, or Rac1-T17N as indicated. (F) A histogram displaying the final recovery of TRPM6 in the presence or absence of EGF after treatment with AG1478/tyrphostin (AG) or RG13022 (RG) as indicated. *P < 0.05 versus TRPM6 final recovery without EGF treatment in the presence of mock co-transfection (n = 14 to 21 cells).

Figure 6.

EGF increases the cell surface fraction of TRPM6. (A) Representative immunoblots of total cellular TRPM6, the input (top), and the biotinylated, cell surface fraction (bottom) after treatment of GFP-TRPM6-MDCK cells with EGF (30 min, 10 nM) or vehicle (Control). Nontransfected MDCK cells (Wild Type) and cells that were not treated with biotin (No Biotin) are included for comparison. (B) Quantification of experiment in A. *P < 0.05 versus vehicle treated condition (n = 11 per condition). (C) EGFR stimulation by EGF leads to tyrosine kinase, Src, PI3K, and Rac1 activation. The last effector alters the mobility of endomembrane TRPM6, potentially redistributing the channel from within a storage compartment (SC) to a recycling endomembrane compartment (RE). Ultimately, an increase in the plasma membrane insertion of TRPM6 is achieved and Mg²⁺ influx through the channel is thereby increased.