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Charge screening by internal pH and polyvalent cations as a mechanism for activation, inhibition, and rundown of TRPM7/MIC channels.

Kozak JA, Matsushita M, Nairn AC, Cahalan MD - J. Gen. Physiol. (2005)

Bottom Line: By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition.Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches.We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA 92697, USA.

ABSTRACT
The Mg2+-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg2+ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels the number of charges. Neomycin and poly-lysine also potently inhibited MIC current in the absence of Mg2+. These same positively charged ions inhibited IRK1 current in parallel with MIC current, suggesting that they probably act by screening the head group phosphates on PIP2 and other membrane phospholipids. In agreement with this hypothesis, internal protons also inhibited MIC current. By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition. We show that inhibition by internal polyvalent cations can be relieved by alkalinizing the cytosol using externally applied ammonium or by increasing pH in inside-out patches. Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches. We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH.

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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 IMIC 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.
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fig1: 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 IMIC 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.

Mentions: Fig. 1 A shows the typical development of MIC current in an RBL cell when internal Mg2+ is reduced during whole cell recording and dialysis using a pipette solution that contained EDTA to chelate divalent ions. After an initial delay, IMIC increased slowly and reached a maximum ∼6 min after break-in. The inset shows the current–voltage relation obtained at 10 min. Fig. 1 B illustrates preactivated MIC currents, occasionally present in RBL cells immediately following break-in to achieve whole cell recording. Inclusion of internal La3+ (top traces) in the pipette resulted in a gradual reduction of the current, inhibiting it completely after ∼120 s of dialysis. La3+ also prevented the slower development of MIC current that would normally occur. In addition to divalent metal cations tested previously (Kozak and Cahalan, 2003), Ca2+ also inhibited preactivated MIC current and its development in RBL cells (averaged data summarized in Fig. 1 C), similar to its inhibition of expressed recombinant TRPM7 currents (Matsushita et al., 2005).


Charge screening by internal pH and polyvalent cations as a mechanism for activation, inhibition, and rundown of TRPM7/MIC channels.

Kozak JA, Matsushita M, Nairn AC, Cahalan MD - J. Gen. Physiol. (2005)

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 IMIC 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.
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Related In: Results  -  Collection

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fig1: 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 IMIC 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.
Mentions: Fig. 1 A shows the typical development of MIC current in an RBL cell when internal Mg2+ is reduced during whole cell recording and dialysis using a pipette solution that contained EDTA to chelate divalent ions. After an initial delay, IMIC increased slowly and reached a maximum ∼6 min after break-in. The inset shows the current–voltage relation obtained at 10 min. Fig. 1 B illustrates preactivated MIC currents, occasionally present in RBL cells immediately following break-in to achieve whole cell recording. Inclusion of internal La3+ (top traces) in the pipette resulted in a gradual reduction of the current, inhibiting it completely after ∼120 s of dialysis. La3+ also prevented the slower development of MIC current that would normally occur. In addition to divalent metal cations tested previously (Kozak and Cahalan, 2003), Ca2+ also inhibited preactivated MIC current and its development in RBL cells (averaged data summarized in Fig. 1 C), similar to its inhibition of expressed recombinant TRPM7 currents (Matsushita et al., 2005).

Bottom Line: By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition.Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches.We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA 92697, USA.

ABSTRACT
The Mg2+-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg2+ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels the number of charges. Neomycin and poly-lysine also potently inhibited MIC current in the absence of Mg2+. These same positively charged ions inhibited IRK1 current in parallel with MIC current, suggesting that they probably act by screening the head group phosphates on PIP2 and other membrane phospholipids. In agreement with this hypothesis, internal protons also inhibited MIC current. By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition. We show that inhibition by internal polyvalent cations can be relieved by alkalinizing the cytosol using externally applied ammonium or by increasing pH in inside-out patches. Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches. We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH.

Show MeSH
Related in: MedlinePlus