1. ClC proteins are a class of voltage-dependent Cl- channels with several
members mutated in human diseases. The prototype: ClC-0 Torpedo channel is
a dimeric protein; each subunit forms a pore that can gate independently fr
om the other one. A common slower gating mechanism acts on both pores simul
taneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-
2 Cl- channel is also activated by hyperpolarization, as are some ClC-1 mut
ants (e.g. D136G;) and wild-type (WT) ClC-1 at certain pH values.
2. We studied the dependence on internal Cl- ([Cl-](i)) of the hyperpolariz
ation-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G
:, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch
clamping channels expressed in Xenopus oocytes.
3. With all these channels, reducing [Cl-](i) shifted activation to more ne
gative voltages and reduced the maximal activation at most negative voltage
s.
4. We also investigated the external halide dependence of WT ClC-2 using tw
o-electrode voltage-clamp recording. Reducing external Cl- ([Cl-](o)) activ
ated ClC-2 currents. Replacing [Cl-](o) by the less permeant Br- reduced ch
annel activity and accelerated deactivation.
5. Gating of the ClC-2 mutant K566Q in normal [Cl-](o) resembled that of TN
T ClC-2 in low [C1(-)](o), i.e. channels had a considerable open probabilit
y (P-o) at resting membrane potential. Substituting external Cl- by Br- or
I- led to a decrease in P-o.
6. The [Cl-](i) dependence of the hyperpolarization-activated gates of vari
ous ClC channels suggests a similar gating mechanism, and raises the possib
ility that the, gating charge for the hyperpolarization-activated gate is p
rovided by Cl-
7. The external halide dependence of hyperpolarization-activated gating of
ClC-2 suggests that it is mediated or modulated by anions as in other ClC c
hannels. In contrast to the depolarization-activated fast gates of ClC-0 an
d ClC-1, the absence of Cl- favours channel opening. Lysine 556 may be impo
rtant for the relevant binding site.