Activation of K+ and Cl? channels in MDCK cells during volume regulation in hypotonic media

Abstract

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.

Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice.

“…Volume-regulated K+ conductances have been found in different tissues. Their activation may be Ca21-dependent (27)(28)(29)(30), triggered by the entry of Ca2+ via stretchactivated cation channels (31,32), or Ca2+-independent (33,34). Although the precise identity of these channels and the mechanisms by which they are activated in the small intestinal crypts remain to be elucidated, a basolateral K+ conductance responding to Ca2+ mobilization has been described in small intestinal crypts of guinea pig (35) and colonic crypts of rabbit (36) and rat (37).…”

Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl‐ channels.

“…On the other hand, small conductance Cl-channels activated by HTS have been described in many cell types (< 4 pS; Kunzelmann, Kubitz, Grolik, Warth & Greger, 1992;Lewis, Ross & Cahalan, 1993;Nilius et al 1994a, b). Marunaka & Eaton (1990) (Christensen & Hoffmann, 1992), in MDCK cells (Banderali & Roy, 1992a) and also in proximal tubule cells from Necturus kidney (Fillipovic & Sackin, 1992 will shift the membrane potential to a value close to the equilibrium potential of the other ion. This will prevent a net efflux of osmolytes via channels, and hence RVD.…”

A Novel Function of BCL-2 Overexpression in Regulatory Volume Decrease

“…brane (23,24). Further studies confirmed that this was because of a transient activation of K ϩ channel and a sustained activation of Cl Ϫ channel (25,26), indicating that the swelling-activated Cl Ϫ channel plays a critical role in the control of RVD in MDCK cells. Activation of swelling-activated Cl Ϫ channel requires the Ca 2ϩ entry in MDCK cells.…”