Abstract
Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR) chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyl)oxy] alkanoic acid (DIOA), a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD) phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels.
Highlights
All living cells constantly experience osmotic challenges due to fluctuations in the concentration of the osmotically active constituents of the cytosol caused by intensive metabolic activity and of the extracellular fluids because of the intensive gas, water, nonelectrolyte and ion exchange at the epithelial surfaces along the airways, in kidney, brain, primary and secondary lymphoid organs.In order to survive, the cells must actively regulate their volume and maintain it near the level optimal for normal life [1,2,3]
In our previous pharmacological study, the Regulatory Volume Decrease (RVD) process in rat thymocytes was completely abolished by blockers of potassium channels and swelling-activated anion channels [14] suggesting involvement of ion channels rather than the coupled K-Cl cotransporter in thymocyte volume regulation under hypotonicity
We provide direct evidence that cell swelling activates two types of the volume-regulated anion channels, the volume-sensitive outwardly rectifying (VSOR) Cl− channel and the maxi-anion channel; and only the VSOR channel is responsible for the anionic efflux during RVD in thymocytes and is sensitive to DIOA
Summary
All living cells constantly experience osmotic challenges due to fluctuations in the concentration of the osmotically active constituents of the cytosol caused by intensive metabolic activity and of the extracellular fluids because of the intensive gas, water, nonelectrolyte and ion exchange at the epithelial surfaces along the airways, in kidney, brain, primary and secondary lymphoid organs. Most of the cells first swell and actively restore their volume by extruding osmolytes (K+, Cl−, taurine and some metabolites) from the cytoplasm, a process called Regulatory Volume Decrease (RVD). In our previous pharmacological study, the RVD process in rat thymocytes was completely abolished by blockers of potassium channels and swelling-activated anion channels [14] suggesting involvement of ion channels rather than the coupled K-Cl cotransporter in thymocyte volume regulation under hypotonicity. We provide direct evidence that cell swelling activates two types of the volume-regulated anion channels, the volume-sensitive outwardly rectifying (VSOR) Cl− channel and the maxi-anion channel; and only the VSOR channel is responsible for the anionic efflux during RVD in thymocytes and is sensitive to DIOA
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