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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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The pHi 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.
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fig9: The pHi 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.

Mentions: Like native MIC channels, heterologously expressed TRPM7 is inhibited by internal Mg2+ (Nadler et al., 2001). To examine whether the recombinant channel is also sensitive to pHi, we used whole-cell and inside-out patch recording and compared the pH dependence of channel activity with that of its kinase activity. Whole-cell dialysis using a Mg2+-free solution at pH 7.3 would normally greatly enhance the TRPM7 current. However, the current was completely inhibited by dialysis with acidic pH (Fig. 9, A and B). In control or transfected CHO cells, dialysis with acidic solutions (pH below 6) activated a small (∼150 pA at +85 mV) outward current that peaks at ∼100 s in Fig. 9 A. This current ran down 3–4 min after break-in and most probably represents a voltage-activated proton conductance present in the CHO cell line (Cherny et al., 1997). The inhibition of TRPM7 channel activity by acidic internal pH was overcome by external NH4+. External NH4+ did not alter the TRPM7 I-V shape in the presence of external divalent cations. The effects of pHi and NH4+o on the expressed TRPM7 current are therefore similar to effects on the endogenous MIC current.


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)

The pHi 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266608&req=5

fig9: The pHi 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.
Mentions: Like native MIC channels, heterologously expressed TRPM7 is inhibited by internal Mg2+ (Nadler et al., 2001). To examine whether the recombinant channel is also sensitive to pHi, we used whole-cell and inside-out patch recording and compared the pH dependence of channel activity with that of its kinase activity. Whole-cell dialysis using a Mg2+-free solution at pH 7.3 would normally greatly enhance the TRPM7 current. However, the current was completely inhibited by dialysis with acidic pH (Fig. 9, A and B). In control or transfected CHO cells, dialysis with acidic solutions (pH below 6) activated a small (∼150 pA at +85 mV) outward current that peaks at ∼100 s in Fig. 9 A. This current ran down 3–4 min after break-in and most probably represents a voltage-activated proton conductance present in the CHO cell line (Cherny et al., 1997). The inhibition of TRPM7 channel activity by acidic internal pH was overcome by external NH4+. External NH4+ did not alter the TRPM7 I-V shape in the presence of external divalent cations. The effects of pHi and NH4+o on the expressed TRPM7 current are therefore similar to effects on the endogenous MIC current.

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