Chloride dependence of hyperpolarization-activated chloride channel gates


Corresponding author M. Pusch: Istituto di Cibernetica e Biofisica, CNR, Via de Marini 6, I-16149 Genova, Italy. Email:


  • 1ClC 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 from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values.
  • 2We studied the dependence on internal Cl ([Cl]i) of the hyperpolarization-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.
  • 3With all these channels, reducing [Cl]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages.
  • 4We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl ([Cl]o) activated ClC-2 currents. Replacing [Cl]o by the less permeant Br reduced channel activity and accelerated deactivation.
  • 5Gating of the ClC-2 mutant K566Q in normal [Cl]o resembled that of WT ClC-2 in low [Cl]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl by Br or I led to a decrease in Po.
  • 6The [Cl]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl.
  • 7The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl favours channel opening. Lysine 556 may be important for the relevant binding site.