Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

Abstract

In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of ×40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl− ≥ Br− ≫ I− ≥ F−. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at −160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2ΔNT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2ΔNT-injected oocytes (−5.9 ± 0.4 μA), followed by ClC-2-injected oocytes (−4.3 ± 0.6 μA), and smallest in water-injected oocytes (−0.2 ± 0.2 μA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2ΔNT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to a hyposmolar environment.

A Novel Voltage-dependent Chloride Current Activated by Extracellular Acidic pH in Cultured Rat Sertoli Cells

“…The authors concluded that gating may be controlled by a Cl Ϫ -binding site accessible only from the exterior and possibly by modification of this site by external protonation (25). ClC-2 activity has been shown to be dependent on acidic pH e (26,27). The regulation of ClC-2 remains, however, puzzling because other (apparently unrelated) mechanisms of activation exist, including cell swelling (see Ref.…”

ClC-3 Is a Fundamental Molecular Component of Volume-sensitive Outwardly Rectifying Cl− Channels and Volume Regulation in HeLa Cells and Xenopus laevis Oocytes

“…Cell volume was estimated in oocytes by diameter measurements (46). Oocyte diameters were measured using a digital video line measurement system (Ionoptix, Milton, MA) with contrast analysis of digitized image data.…”

Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes

“…Increased current amplitudes by hypotonicity and EC acidification Thiemann et al, 1992;Grunder et al, 1992;Jordt and Jentsch, 1997;Furukawa et al, 1998;Schwiebert et al, 1998 ClC-3 Guibert et al, 1998 …”