TRPM7 channels in hippocampal neurons detect levels of extracellular divalent cations

Abstract

Exposure to low Ca(2+) and/or Mg(2+) is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca(2+) overload and cell death. This phenomenon has been called the "Ca(2+) paradox" of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca(2+) and Mg(2+) is "detected" and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca(2+) and Mg(2+) that mimic the initial condition of the Ca(2+) paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg(2+), (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)), and (v) can be inhibited by Galphaq-linked G protein-coupled receptors linked to phospholipase C beta1-induced hydrolysis of PIP(2). Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons "detect" reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia.

Fig. 1.

Cultured hippocampal neurons demonstrate graded responses to reductions in extracellular divalents. (A–D) Graded increases in action potential firing frequency are detected when sequentially applying solutions containing 1.3 mM Ca²⁺ and 0.9 mM Mg²⁺ (standard solution) (A), 0.2 mM Ca²⁺ and 0.9 mM Mg²⁺ (low Ca²⁺) (B), 0 mM Ca²⁺ and 0.9 mM Mg²⁺ (Ca²⁺-free) (C), or 0.2 mM Ca²⁺ and 0.2 mM Mg²⁺ (low divalent) (D). (E) For each of these conditions, the change in action potential firing frequency is plotted for a series of six cells and shows the graded excitation. *, P < 0.05; **, P < 0.01. (F and G) Whole-cell patch-clamp recordings of currents generated at −60 mV in a cultured (F) and isolated (G) neuron in response to a change to low Ca²⁺ (a), low divalent (b), low Ca²⁺, no Mg²⁺ (c). (H) In HEK293 cells expressing TRPM7 (Tet induction), a graded increase in cell death is observed when extracellular divalents are progressively reduced. No similar increase is detected in HEK293 cells that do not express TRPM7 (no-Tet induction). Increased cell death is indicated by an increase in the fluorescent ratio F/F_max, which represents the estimated fraction of total HEK293 cells that took up propidium iodide.

Fig. 2.

Divalent-free solutions enhance currents in isolated and cultured neurons as well as TRPM7 currents in HEK293 cells expressing this protein. (A) A voltage ramp (±100 mV) was applied for 500 ms from a holding potential of −60 mV to an isolated neuron. The extracellular solution was switched from one containing 2 mM extracellular Ca²⁺ (no Mg²⁺) (a) to a divalent-free solution (b). (B) On switching to the divalent-free solution, large inward currents were induced and outward rectification was diminished (b) in an isolated neuron. (C and D) A similar response is seen in a cultured hippocampal neuron (C) and in a HEK293 cell expressing homomeric recombinant TRPM7 channels (D) (also previously shown in ref. 12).

Fig. 3.

The time-dependent regulation of responses to lowered divalent cation by intracellular Mg²⁺and PIP₂. Voltage ramps (±100 mV) were used to assess the current amplitudes of isolated neurons in the presence of 1.3 mM extracellular Ca²⁺ (no Mg²⁺). (A and B) The amplitude of inward and outward currents increased (n = 6) over a period of 5 min using a low concentration of Mg²⁺ (0 MgCl₂, 5 mM ATP) in the patch electrode. (B) In contrast, when using a relatively high concentration of Mg²⁺ (6 MgCl₂, 2 mM ATP), these currents progressively decreased over the first 5 min (▵; n = 8). (C) Responses to applications of low-Ca²⁺ solution to cultured neurons also decreased over this time period (−60 mV) when the high intracellular Mg²⁺ was used. The upper trace shows an example recording of the response after breakthrough, and the lower trace illustrates the response to the same application after 5 min of recording. (D) Including PIP₂ (20 μM, n = 6) in the patch pipette leads to a time-dependent increase in the inward leak current relative to control recordings without PIP₂ (n = 6). (E) Responses of isolated neurons to a low-calcium, no Mg²⁺ solution were greater after 10 min of recording with PIP₂ in the patch pipette. There was an increase in inward leak current. (F) This current was −105 ± 12 pA in control (n = 6) versus −445 ± 64 pA in PIP₂-treated neurons (n = 8) (*, difference, P > 0.01, Student's t test). (G) In isolated neurons from WT mice, CHPG inhibited responses to low Ca²⁺, no Mg²⁺ (applied where indicated by the bar; ○; n = 10). This inhibition was blocked by the mGluR5 receptor antagonist MPEP (▴; n = 6). A control series of recordings is also shown (no CHPG applied; ●; n = 7). The CHPG-induced inhibition was also absent in isolated neurons from PLCβ1 knockout mice (□; n = 5). (H) Summary graph illustrating inhibition of responses by CHPG (±SEM; *, difference, P < 0.01, Student's t test), blockade by MPEP, and lack of inhibition in PLCβ1 knockout mice.

Fig. 4.

Immunocytochemistry confirmed the depression of TRPM7, but not TRPM2, protein in neurons treated with adenovirus shRNA_TRPM7. (A) Confocal images at high magnification (×63) show an shRNA_control/GFP-infected (GFP-expressing; green; Center) cultured neuron immunostained for TRPM7 (red; Left); merged image is shown (Right). (B) A similar neuron infected with shRNA_TRPM7/GFP is shown imaged for the TRPM7 (red; Left), GFP (green; Center), and merged (Right). There was a demonstrable reduction in the level of expression of TRPM7. GFP-positive cells were confirmed as neurons by triple immunostaining the cultures with a neuronal marker, anti-NeuN antibody (blue; not shown). (C) No suppression in protein level of TRPM2 was observed in the shRNA_TRPM7 group. TRPM2 (red; Left) colocalized with GFP (green; Center) and the merged images (Right) indicated a strong overlap of signals. (D) A comparison of fluorescence intensities of antibody staining from cell bodies of WT neurons (GFP negative) taken from the same field for each treatment group and neurons infected with shRNA_control or shRNA_TRPM7. Numbers of neurons for each group are as indicated on the top of each bar. A.U., arbitrary unit. *, difference, P < 0.05, ANOVA; P < 0.01, multiple comparison test (Fisher's least significant difference test). (E) TRPM2 fluorescence intensity was unaltered by treatment with shRNA_TRPM7.

Fig. 5.

Infection with shRNA_TRPM7 reduces the level of TRPM7 message and the responses to low-Ca²⁺, no Mg²⁺ solutions in cultured neurons. Responses were recorded from 26 neurons and subsequently harvested for single-cell RT-PCR. TRPM7 message was detected in 75% of the shRNA_control-treated neurons but only 28% of the shRNA_TRPM7 cells. (A) Results from eight representative cells. Control cells from lanes 7 and 8 demonstrate a robust response to divalent-free solution, whereas the six shRNA_TRPM7 treated cells (lanes 1–6) showed reduced responses to lowering extracellular divalents. (B) Group data demonstrating a decrease in current density of these responses in shRNA_TRPM7-treated neurons versus control (n = 10 for shRNA_control, n = 13 for shRNA_TRPM7). (C) Enhancement of responses with PIP₂ in the patch pipette from neurons infected with shRNA_TRPM7 versus shRNA_control. Individual cells were not subjected to single-cell PCR for these recordings. In shRNA_control neurons, the low Ca²⁺, no Mg²⁺ current density was increased from −8.2 ± 1.3 pA/pF to −17.8 ± 1.1 pA/pF (n = 10) by PIP₂, whereas in shRNA_TRPM7 neurons, the current density was increased from −4.9 ± 0.8 pA/pF to −8.9 ± 1.8 pA/pF (n = 12) (Student's t test, P < 0.01). *, difference, P < 0.05; **, difference, P < 0.01.