A single channel characterization of the Cl- channels in distal nephron was
undertaken using Vesicles prepared from plasma membranes of isolated rabbi
t distal tubules. The presence in this vesicle preparation of ClC-K type Cl
- channels was first established by immunodetection using an antibody raise
d against ClC-K isoforms. A ClC-Kl based functional characterization was ne
xt performed by investigating the pH and external Ca2+ regulation of a smal
l conductance Cl- channel which we identified previously by channel incorpo
ration experiments. Acidification of the cis (external) solution from pH 7.
4 to 6.5 led to a dose-dependent inhibition of the channel open probability
Po Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold d
ecrease of the channel Po with no effect on the channel conductance. Channe
l activity also appeared to be regulated by cis (external) Ca2+ concentrati
on, with a dose-dependent increase in channel activity as a function of the
cis Ca2+ concentration. It is concluded on the basis of these results that
the small conductance Cl- channel present in rabbit distal tubules is func
tionally equivalent to the ClC-Kl channel in the rat. In addition, the pres
ent work constitutes the first single channel evidence for a chloride chann
el regulated by external Ca2+. (C) 2000 Elsevier Science B.V. All rights re
served.