A small-conductance Cl- channel in the mouse thick ascending limb that is activated by ATP and protein kinase A.

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.