title: A small-conductance Cl- channel in the mouse thick ascending limb that is activated by ATP and protein kinase A.
abstract: 1. Chloride channels were identified in the basolateral membrane of isolated cortical thick ascending limbs (CTALs) of the mouse nephron by the patch-clamp technique. A channel with a conductance of 45 pS, previously shown to be Cl- selective, was detected in 21% of cell-attached patches when CTAL fragments were pre-incubated with 10 mumol l-4 forskolin for at least 15 min. The same channel was found in only 8.5% of cell-attached patches formed on unstimulated tubules. 2. Another channel with a smaller conductance (7-9 pS) was found in 42.8% of cell-attached patches and 57% of inside-out patches in unstimulated CTAL tubules, but in 82-87% of patches from forskolin-treated tubules. 3. The small channels was Cl- selective (Cl(-)-to-Na+ permeability ratio, PCl/PNa = 9.8) with the permeability sequence: NO3- > Br- > Cl- > F- > gluconate. Channel activity decreased (Br-) or disappeared (NO3-) at negative voltages. At 140 mmol l-1, I- completely inhibited channel activity at all voltages, but a PI/PCl ratio of 1.6 was estimated using a low I- concentration (10 mmol l-1). 4. Internal adenosine triphosphate (ATP) increased normalized current (nPo) in 48% of inside-out patches containing Cl- channels from unstimulated tubules and in 63% of patches from forskolin-treated CTAL tubules. The non-hydrolysable ATP analogue, adenosine 5'-adenylyl imidodiphosphate (AMP-PNP) did not increase channel activity. 5. Adding the catalytic subunit of protein kinase A to the bath in the presence of ATP increased the activity of the small channel in 58% of inside-out patches from unstimulated tubules, but it had no effect on the 45 pS channel. 6. The Cl- channel blockers 5-nitro-2-(3-phenylpropylamine)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or glibenclamide, all at 0.1 mmol l-1, and diphenylamine-2-carboxylic acid (DPC), at 1 mmol l-1, inhibited the small channel activity by 80-100% in inside-out patches. 7. These results indicate that two Cl- channels with contrasting properties mediate the basolateral step of NaCl absorption in the thick ascending limb of the loop of Henle.
authors.affiliation: INSERM U.323, Paris, France.
authors.forename: R
authors.initials: R
authors.lastname: Guinamard
authors.forename: A
authors.initials: A
authors.lastname: Chraïbi
authors.forename: J
authors.initials: J
authors.lastname: Teulon
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001711:Biotransformation / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D018118:Chloride Channels / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D008138:Loop of Henle / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice
chemical_list: D018118:Chloride Channels; D005576:Colforsin; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid