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. 2011;12(12):9125-37.
doi: 10.3390/ijms12129125. Epub 2011 Dec 8.

Swelling-activated anion channels are essential for volume regulation of mouse thymocytes

Ranokhon S Kurbannazarova  1 , Svetlana V Bessonova, Yasunobu Okada, Ravshan Z Sabirov
Affiliations

Swelling-activated anion channels are essential for volume regulation of mouse thymocytes

Ranokhon S Kurbannazarova et al. Int J Mol Sci. 2011.

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 Gd(3+) 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 Gd(3+) 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.

Keywords: DIOA; anion channels; phloretin; thymocytes; volume regulation.

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Figures

Figure 1
Figure 1
Two types of whole-cell macroscopic currents activated by cell swelling in mouse thymocytes. The pipette solution contained either 3 mM ATP (A) or no ATP (B). (Top panel) Time course of whole-cell current activation in response to cell swelling. Currents were elicited by application of alternating pulses from 0 to ±25 mV (every 5 s). (Middle panel) Representative traces of current responses recorded after maximal current activation. In A, the holding potential was 0 mV; after a pre-pulse to −100 mV (500 ms), currents were elicited by application of step-pulses (1000 ms) from −100 to +100 mV in 20-mV increments. In B, the holding potential was 0 mV; currents were elicited by application of step pulses (1000 ms) from −50 to +50 mV in 10-mV increments. (Bottom panel) Instantaneous current-to-voltage relationships measured at the beginning of test pulses after maximal current activation; pipette solution contained 125 mM Cl− (circles) or 25 mM Cl− (triangles). The inset on the bottom panel in B shows single maxi-anion channel-like current fluctuations seen in whole-cell recordings.
Figure 2
Figure 2
Pharmacological profiles of whole-cell currents activated by an osmotic challenge in thymocytes. Effects of 200 μM phloretin (A), 50 μM Gd3+ (B) and 10 μM DIOA (C) on mean currents recorded at +25 mV (open columns) and −25 mV (filled columns). Data are normalized to the mean current measured before application of drugs. Each column represents the mean ± SEM (vertical bar). # Significantly different from the control current without drug at P < 0.05. * Significantly different from the current measured with ATP-containing pipette solution at P < 0.05.
Figure 3
Figure 3
Two types of microscopic currents activated in osmotically swollen thymocytes. (A) Single VSOR anion channel activity. (B) Single maxi-anion channel activity. Pipette: 100 mM TEACl-pipette solution; bath: hypotonic high-K+ solution. Dashed lines correspond to zero current level. (C) I-V relationship for the single-channel events similar to those shown in (A). Unitary currents were recorded in the cell-attached mode with standard 100 mM CsCl-pipette solution (open circles), 100 mM TEACl-pipette solution (closed diamonds) and 30 mM CsCl-pipette solution (filled triangles). Each data point represents the mean ± SEM of 5 to 29 measurements from 8–14 different patches. (D) I-V relationship for the single-channel events similar to those shown in (B). Unitary currents were recorded in the cell-attached mode with 100 mM TEACl-pipette solution and hypotonic high-K+ solution in the bath (filled triangles), or in inside-out mode with Ringer solution in the pipette and in the bath (open circles). Filled diamonds and filled squares: 135 mM NaCl in the bath solution was replaced with equimolar TEACl and Na-glutamate, respectively (inside-out, pipette filled with Ringer solution). Each data point represents the mean ± SEM of 5 to 30 measurements from 7–10 different patches. (E) Effects of phloretin (200 μM), DIOA (10 μM) and Gd3+ ions (50 μM) on the occurrence of the VSOR-type and maxi-anion-type channels in the on-cell mode (the drugs added to the pipette solution). On the top of the bars: number of channel-containing patches/total number of patches. * Significantly different from the control value at P < 0.05.
Figure 4
Figure 4
Effects of inhibitors of swelling-activated anion channels and K-Cl cotransporter on volume regulation of mouse thymocytes. (A) Time courses of cell volume change in isotonic conditions (filled circles) and in hypotonic conditions in the absence (open circles) and presence of 50 μM Gd3+ (open triangles), 50 μM phloretin (open squares) and 250 μM glibenclamide (open diamonds). (B) Time courses of cell volume change in isotonic conditions (filled circles) and in hypotonic conditions in the absence (open circles) and presence of 1 μM and 10 μM DIOA (open triangles and open squares, respectively). The cell volume was measured by Coulter-type cell sizing method.
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