Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

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.

Volume-Sensitive Anion Channels Mediate Osmosensitive Glutathione Release from Rat Thymocytes

“…As in our previous study of mouse thymocytes [24], we induced cell swelling by employing the hypertonic pipette solution (made by adding mannitol) rather than the hypotonic bath solution, since under this experimental design the whole-cell configuration was more stable and reproducible, and thereby swelling-induced macroscopic currents could be consistently observed. As shown in a representative recording, the macroscopic current developed after attaining the whole-cell configuration using the hypertonic pipette solution, and gradually increased with cell swelling reaching a steady-state level after about 3 min (Fig.…”

“…Cell isolation was performed as described previously [24]–[26]. Briefly, the 6–8 weeks old rats were anaesthetized with halothane or diethyl ether and painlessly euthanized by cervical dislocation; the thymi were dissected and carefully washed with an ice-cold Ringer solution.…”

Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis

“…It has been shown that many cells and, among them, immunocells are able to slowly down regulate their volume after a rapid swelling under hyposmotic conditions [59]. It is well recognized that potassium and chloride transport [38] typically contribute to RVD in several cell types and specifically in immunocells, such as thymocytes from rats [59] and mice [60] as well as lymphoblastic leukemia cells [44]. Interestingly it has also been suggested that in Mytilus galloprovincialis digestive cells [35] and in Mytilus californianus gill cells [61] K + and Cl _ cooperate in RVD by the efflux of these two ions followed by an obliged efflux of water from the cell.…”

Chloride Channel Blockers Suppress Formation of Engulfment Pseudopodia in Microglial Cells

“…Figures 8A and B visualize representative cells with blebbing DCPIB (20 µM). Since DIOA blocks Cl - -channels [3,22] as well as KCC [3,22,23,24,25,26], these may cause an imbalance in Cl - or intracellular pH homeostasis, because Cl - -channels and transporters are also permeable to HCO 3 - [9,27]. …”

“…Na + /H + -exchangers, Cl - /HCO 3 - -exchangers and Na + /K + /2Cl - -cotransporters are sorted to the leading edge. Because blockers of I Cl, swell may block Cl - -channels as well as KCCs, as DIOA does [3,22,26,37], and Cl - -free conditions affects several Cl - -dependent transport proteins like KCCs, NKCCs [22,38], the contribution of these different ion channels and transporters to engulfment pseudopodium formation remains to be characterized. Since all blockers used in the present study suppress at least Cl - -channel activity [3,22,37,39] and lamellipodium formation, we conclude that Cl - -channels contribute significantly in lamellipodium formation.…”