Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl^– channels in human osteoblasts

Abstract

The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl- channels expressed in these cells as described previously. The reversal potential of the induced Cl- current is more negative when activated through hypotonic NaCl solution (-47 +/- 5 mV; n = 6) compared with activation through isotonic high-KCl solution (-35 +/- 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl-] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl- current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1-KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl- channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling.

Chloride Conductance Is Required for the Protein Kinase A and Rac1-dependent Phosphorylation of Moesin at Thr-558 by KCl in PC12 Cells

“…More recently, this same group suggested that volume-sensitive chloride channel is involved in KCl-induced chloride conductance in human osteoblasts, which is activated by volume increases induced by K ϩ and Cl Ϫ influx through K-Cl co-transporter (22). In the present study, we also observed high KCl-induced chloride conductance in PC12 cells.…”

“…DISCUSSION This study demonstrates that 60 mM KCl induces the phosphorylation of moesin at Thr-558 in PC12 cells PKA and Rac1 dependently. To our surprise, this signaling pathway was activated not by the depolarization-induced Ca 2ϩ influx, but rather by K ϩ -induced chloride conductance, which was only recently reported and has not been implicated in the activation of intracellular signaling pathways (16,22). It is well known that KCl induces depolarization of the plasma membrane and Ca 2ϩ influx into PC12 cells and subsequently activates diverse calcium-dependent signaling pathways, such as Ras-ERK.…”

“…Combined treatment with TEA and Ba 2ϩ did not induce moesin phosphorylation, but it did induce strong calcium-dependent (data not shown) MAPK phosphorylation in PC12 cells (Fig. 5C) (16,22). Therefore, we investigated whether 60 mM KCl changes chloride conductance in PC12 cells by using the whole cell patch clamp technique.…”

“…In contrast, considering the partial permeability of aspartate to Cl Ϫ channels (P aspartate / P Cl ϭ 0.1, Ref. 22), the net equilibrium potential for permeable anion would be 58 mV ϫ log{[6 mM ϩ (0.1 ϫ 155 mM)]/170.5}, approximately Ϫ52 mV. The high K ϩ -induced membrane current in PC12 cells reversed its direction at Ϫ40 mV, i.e.…”

Cystic fibrosis and bone disease: are we missing a genetic link?

“…Like epithelial cells, they exhibit polarised secretory properties. Osteoblasts express at least three families of Cl -channels, including Na + -Cl -cotransporters, potassium (K)-Cl -cotransporters and Na [18]. Activation of the osteoblast can be achieved through cotransporter functions.…”