PMC

External NH₄ ⁺ effect on MIC current in the presence of internal Mg²⁺. RBL cells in 2 Ca²⁺ external solution. (A) The internal solution contained 12 mM EGTA and 5 mM MgCl₂ ([Mg²⁺]_free = 3 mM). Application of external 81 mM NH₄ ⁺ reversed inhibition by internal Mg²⁺ in several successive trials; Mg²⁺ inhibition was reestablished upon washout of NH₄ ⁺. (B) NH₄ ⁺ prevents inhibition of preactivated MIC current by 2.3 mM free Mg²⁺ (12 mM EGTA + 4 mM MgCl₂).

Activation of MIC current by NH₄ ⁺ in perforated-patch recording. RBL cell in 2 Ca²⁺ external solution; amphotericin B–containing pipette solution. (A) MIC current failed to develop during recording before application of external 162 NH₄ ⁺ which resulted in the development of a robust current. I_MIC declined slowly upon removal of NH₄ ⁺ and rapidly upon addition of 40 mM propionate-containing solution. Both propionate and NH₄ ⁺ effects were reversible. (B) I-V relations obtained from the experiment shown in A in the presence of external NH₄ ⁺ and propionate.

pH dependence of TRPM7 kinase domain phosphotransferase activity. (A) TRPM7 kinase domain phosphorylation of MBP (myelin basic protein) was assessed in vitro at various pH values. The panel shows an autoradiogram of radioactive ATP-labeled MBP after phosphorylation in buffer at various pH values. (B) The kinase activity of TRPM7 was quantified by comparing the intensity of the P³² signal shown in A. Band intensity at 7.3 was set as 100%. The activity was substantially reduced at pH 4.0 and 9. The figure is representative of three separate experiments.

Neomycin inhibition of MIC and IRK1 currents. RBL cells with 10 mM EDTA-containing internal solution and 4.5 K⁺, 2 Ca²⁺ external solution. (A) Time course of development of MIC (measured at +85 mV) and IRK1 (measured at −110 mV) currents. (B) Neomycin (3 mM) inhibits preactivated MIC and IRK1 currents from inside. (C) I-V relations obtained from B at times 1–3. The plot on top is enlarged below to show that neomycin block precedes its inhibition of IRK1 current. The red trace shows blocked (but not inhibited) IRK1 current. The arrow indicates outward IRK1 current that is rapidly blocked by neomycin diffusing into the cell.

PI(4,5)P₂ and pH effects on TRPM7 current in inside-out patch. CHO cell overexpressing mTRPM7. Divalent-free external solution permits inward monovalent current recording. (A) Pipette and bath contained 1 mM HEDTA, 154 mM Cs⁺ aspartate. pH 8.4 increased current whereas pH 5.6 completely inhibited it. 5 μM PIP₂-diC8 application in pH 7.3 mimicked the effect of increased pH. 2 mM MgCl₂ was applied. (B) Bath solution contained Cs⁺ glutamate, 10 EGTA. Establishment of inside-out configuration (i-o) resulted in increased current due to Mg²⁺ washout followed by rapid rundown. Application of 5 μM PIP₂-diC8 to the cytosolic side of the membrane resulted in TRPM7 current recovery.

The pH_i dependence of recombinant TRPM7 in whole-cell and inside-out patch recordings. (A) Time course of internal pH inhibition and recovery by external NH₄ ⁺ during whole-cell recording of overexpressed mTRPM7 in a CHO cell. pH 5.6 internal solution; 2 Ca²⁺ external solution. Whole-cell recording was initiated at arrow. TRPM7 current amplitude was measured at +85 mV. (B) Individual I-V traces obtained from A. (C) Inside-out patch recording; time or patch excision indicated by arrow. Pipette and bath solutions were 1 mM HEDTA, 154 mM Cs⁺ aspartate to minimize rundown. Divalent-free external solution permits inward monovalent current recording. pH 5.6 at cytoplasmic surface inhibited TRPM7 fully and reversibly. In the same patch, 2 mM MgCl₂ also inhibited the current. (D) Currents at −100 mV and during voltage ramps to +85 mV. (E and F) Increasing the pH from 7.4 to 8.4 increased TRPM7 current amplitude in inside-out patches.

Block of outward MIC current by internal quaternary ammonium derivatives and hexamethonium. Human resting T cells (A–D) and RBL cells (E and F) with 130 mM Cs⁺ glutamate, 8 mM NaCl, 10 mM EDTA, 0.9 mM CaCl₂, 10 mM HEPES, pH 7.3; and 10 HEDTA-Cs⁺ (A–D) and 1 HEDTA-Cs⁺ (E and F) external solution. Divalent-free external solution permits inward monovalent current recording. I-V relations were obtained at break-in (red traces) and after full development of the current with internal solutions containing no blocker (A) or 20 mM NH₄ ⁺ (B), TMA (C), TEA (D), and hexamethonium (E). TMA, TEA, and hexamethonium, but not NH₄ ⁺, produced voltage-dependent block of the outward MIC current. (E) Inward MIC current in the presence and absence of 16 mM hexamethonium. The values are not statistically different (P > 0.05).

Effects of external acetate ion on MIC current in RBL cells. (A) MIC current inhibition by application of external acetate when internal solution contained 10 mM HEPES. (B) Increasing internal pH buffering with 122 mM HEPES shifts the acetate effect to higher concentrations. (C) Summary of 40 mM acetate inhibition of MIC current with 10 mM (n = 5) or 122 mM (n = 3) HEPES in the pipette (*, P < 0.005 compared with the value obtained with 10 mM HEPES). The current amplitude was measured at 360 s after the start of acetate application, divided by the amplitude immediately before acetate addition, and plotted as percent of control current. (D) MIC current fails to develop with 0 Mg²⁺ inside in the presence of external 40 mM acetate. Upon washout of acetate, the current developed normally. (E) Reversible MIC current inhibition by propionate (40 mM) and acetate (40 mM) in the same cell. After prolonged dialysis, the current ran down completely in the absence of external acetate. MIC current amplitudes were measured at + 85 mV.

TRPM7 current recovery from rundown; inhibition in cell-attached mode. CHO cells overexpressing mTRPM7 WT and TAP mutant. (A and B) Inside-out patch. Monovalent TRPM7 current was allowed to run down fully in 1 mM EGTA, pH 7.3 bath solution. Current recovered and was potentiated by applying pH 8.4 solution to the cytosolic side. Upon return to pH 7.3, the current was inhibited again (compare with C). (C and D) Cell-attached patch. Sensitivity of I_TRPM7 to internal pH changes. The basal ∼40 pS channel activity was inhibited when bath solution was switched from aspartate to acetate in order to acidify the cytosol of the intact cell. The acetate effect was reversible. (E) Sensitivity of I_TRPM7-TAP (kinase-dead mutant) to internal pH changes in the cell-attached mode. Constitutive activity of TRPM7-TAP channels was monitored in cell-attached patch in 2 Ca²⁺ bath solution. Repeated addition of 40 mM acetate to the bath inhibited channel activity. (F) Traces from experiment shown in E. The current trace in the middle represents current before acetate application.

Inhibition of MIC current by internal polyvalent cations and NH₄ ⁺. RBL cells with standard internal solution and 2 Ca²⁺ external solution. All current amplitudes were measured at +85 mV during voltage ramp stimuli. (A) Time course of I_MIC development during whole-cell recording and dialysis with 10 mM EDTA. Outward MIC current amplitude is plotted against time after break-in. The inset shows I-V relations obtained at break-in and after 10 min of recording. (B) The time course of inhibition of preactivated MIC current by La³⁺ (top). The pipette solution contained 1 mM EDTA + 3 mM La³⁺. The inset shows the I-V relation at break-in and after complete inhibition. The bottom panel shows the time course of inhibition of preactivated MIC current by spermine. The pipette solution contained 10 mM EDTA + 5 mM spermine. The inset shows I-V relations obtained at break-in and after complete inhibition. (C) Maximal current densities with control (1 mM, n = 5 cells) or 10 mM EDTA (n = 3) solutions and with solutions containing inhibitory cations. The cation concentrations in mM were: 5 Ca²⁺ (n = 5); 3 La³⁺ (n = 8); 8 putrescine (n = 4); 5 spermidine (n = 5); 4 spermine (n = 5); 6 neomycin (n = 5); 4 polylysine (n = 6); 112 NH₄ ⁺ (n = 5). *, P < 0.005 compared with controls in 1 or 10 mM EDTA. **, P = 0.16.

Effect of external NH₄ ⁺ on MIC current in the absence of internal Mg²⁺: recovery from rundown. RBL cells in 2 Ca²⁺ external solution with 12 mM EGTA internal solution. (A) MIC current was allowed to develop, and I-V plots obtained at break-in and after the current was fully developed (shown in red color). NH₄ ⁺ at varying concentrations (20 mM, 81 mM, and 162 mM) was applied subsequently and I-V plots are shown. Note the inward current activated in external NH₄ ⁺-containing solution. (B) Rapid application of 81 mM NH₄ ⁺ causes reversible slow potentiation of the MIC current. (C) MIC current development and rundown. 12 mM EGTA, 100 μM MgCl₂ internal solution, [Mg²⁺]_free = 54 μM. 81 mM NH₄ ⁺ applied after rundown resulted in recovery of I_MIC. (D) Dependence of MIC current rundown on Mg²⁺ and neomycin. Current amplitude (+85 mV) was obtained 30 min (I_{30 min}) after break-in, divided by maximal amplitude (I_max) in the same cell, and the ratio plotted for 12 mM EGTA (free Mg²⁺ = 54 μM; n = 10 cells), 12 mM EGTA + 0.2 mM EDTA (free Mg²⁺ = 2 μM, n = 5), 8 mM EGTA + 4 mM EDTA (free Mg²⁺ = 52 nM, n = 7) and 10 mM EDTA+ 500 μM neomycin (n = 3) internal solutions. *, P < 0.005 compared with the value for 2 μM free Mg²⁺.