Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation.
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 x 40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- > or = Br- >> I- > or = 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-2 delta NT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2 delta NT-injected oocytes (-5.9 +/- 0.4 microA), followed by ClC-2-injected oocytes (-4.3 +/- 0.6 microA), and smallest in water-injected oocytes (-0.2 +/- 0.2 microA), 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-2 delta NT-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 Xenopus oocytes appear to act for volume regulation when exposed to a hyposmolar environment.
Furukawa
T
T
Department of Autonomic Physiology, Tokyo Medical and Dental University, Japan.
Ogura
T
T
Katayama
Y
Y
Hiraoka
M
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000090007:CLC-2 Chloride Channels; D002463:Cell Membrane Permeability / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D004594:Electrophysiology; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000502:physiology*; D009579:Nitrobenzoates / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D009994:Osmolar Concentration; D011817:Rabbits; D051381:Rats; D013267:Stilbenes / Q000494:pharmacology; D014882:Water-Electrolyte Balance / Q000502:physiology*; D014981:Xenopus
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D009419:Nerve Tissue Proteins; D009579:Nitrobenzoates; D013267:Stilbenes; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid