Extracellular protease trypsin activates amiloride‐insensitive sodium channels in human leukemia cells

Abstract

Sodium influx is tightly regulated in the cells of blood origin. Amiloride-insensitive sodium channels were identified as one of the main sodium-transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na channel (ENaC)-like channels, we utilized leukemia K562 cells as a unique model to examine single channel behavior in a whole-cell patch-clamp experiments. We have shown for the first time that extracellular serine protease trypsin directly activates sodium channels in plasma membrane of K562 cells. The whole-cell single current recordings clearly demonstrate no inhibition of trypsin-activated channels by amiloride or benzamil. Involvement of proteolytic cleavage in channel opening was confirmed in experiments with soybean trypsin inhibitor. More importantly, stabilization of F-actin with intracellular phalloidin did not prevent trypsin-induced channel activation indicating no implication of cytoskeleton rearrangements in stimulatory effect of extracellular protease. Our data reveals a novel mechanism modulating amiloride-insensitive ENaC-like channel activity and integral sodium permeability in leukemia cells.

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis

“…However, in detached cells in suspension, the Na + content was increased so much that it was comparable to the intracellular K + content and the cell K/Na ratio approached 1 (Figure 1A). We asked whether the elevated Na + content in detached eMSCs was a consequence of cell treatment with trypsin solution which is used to remove cells from adhesive plates and which is able to increase the permeability of cell membrane to Na + [36]. To answer We asked whether the elevated Na + content in detached eMSCs was a consequence of cell treatment with trypsin solution which is used to remove cells from adhesive plates and which is able to increase the permeability of cell membrane to Na + [36].…”

“…We asked whether the elevated Na + content in detached eMSCs was a consequence of cell treatment with trypsin solution which is used to remove cells from adhesive plates and which is able to increase the permeability of cell membrane to Na + [36]. To answer We asked whether the elevated Na + content in detached eMSCs was a consequence of cell treatment with trypsin solution which is used to remove cells from adhesive plates and which is able to increase the permeability of cell membrane to Na + [36]. To answer this question, we maintained the detached eMSCs in suspension in growth medium by soft rocking up to 2 h in a humidified chamber with 5% CO 2 at 37 • C. It turned out that under these conditions, an increased content of Na + (and high K + content) in eMSCs persisted as long as the cells were in suspension (Figure 1A).…”

Piezo1 Activation Prevents Spheroid Formation by Malignant Melanoma SK-MEL-2 Cells

“…A detailed description of the whole-cell mode of the patch-clamp technique used to record single ion channels in the plasma membrane is available in our publications [6,12]. Cytosollike solution contained (in mM) 140 KAsp, 5 NaCl, 1 MgCl 2 , 2 EGTA, 20 HEPES/TrisOH and CaCl 2 to establish intracellular Ca 2+ concentration ([Ca 2+ ] i ) at 10 nM.…”

Piezo1 Activation Prevents Malignant Melanoma Spheroid Formation

“…The detailed description of the whole-cell mode of patch-clamp technique used to record single ion channels in the plasma membrane is available in our publications [8,9]. Cytosol-like solution contained (in mM): 140 KAsp, 5 NaCl, 1 MgCl2, 2 EGTA, 20 HEPES/TrisOH and CaCl2 to establish intracellular Ca2+ concentration ([Ca 2+ ]i) at 10 nM.…”