R. Guinamard et al., A SMALL-CONDUCTANCE CL- CHANNEL IN THE MOUSE THICK ASCENDING LIMB THAT IS ACTIVATED BY ATP AND PROTEIN-KINASE A, Journal of physiology, 485(1), 1995, pp. 97-112
1. Chloride channels were identified in the basolateral membrane of is
olated cortical thick ascending limbs (CTALs) of the mouse nephron by
the patch-clamp technique. A channel with a conductance of 45 pS, prev
iously shown to be Cl- selective, was detected in 21% of cell-attached
patches when CTAL fragments were pre-incubated with 10 mu mol l(-1) f
orskolin for at least 15 min. The same channel was found in only 8.5%
of cell-attached patches formed on unstimulated tubules. 2. Another ch
annel with a smaller conductance (7-9 pS) was found in 42.8% of cell-a
ttached patches and 57% of inside-out patches in unstimulated CTAL tub
ules, but in 82-87% of patches from forskolin-treated tubules. 3. The
small channel was Cl- selective (Cl--to-Na+ permeability ratio, P-Cl/P
-Na = 9.8) with the permeability sequence: NO3- > Br- > Cl- > F- > glu
conate. Channel activity decreased (Br-) or disappeared (NO3-) at nega
tive voltages. At 140 mmol l(-1), I- completely inhibited channel acti
vity at all voltages, but a P-I/P-Cl ratio of 1.6 was estimated using
a low concentration (10 mmol l(-1)). 4. Internal adenosine triphosphat
e (ATP) increased normalized current (nP(o)) in 48% of inside-out patc
hes containing Cl- channel from unstimulated tubules and in 63% of pat
ches from forskolin-treated CTAL tubules. The non-hydrolysable ATP ana
logue, adenosine 5'-adenylyl imidodiphosphate (AMP-PNP) did not increa
se channel activity. 5. Adding the catalytic subunit of protein kinase
A to the bath in the presence of ATP increased the activity of the sm
all channel in 58% of inside-out patches from unstimulated tubules, bu
t it had no effect on the 45 pS channel. 6. The Cl- channel blockers 5
-nitro-2-(3-phenylpropylamine)-benzoic acid (NPPB), 4,4'-diisothiocyan
atostilbene-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 patche
s. 7. These results indicate that two Cl- channels with contrasting pr
operties mediate the basolateral step of NaCl absorption in the thick
ascending limb of the loop of Henle.