Elimination of the slow gating of ClC-0 chloride channel by a point mutation.
The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn2+) from the extracellular side, leading to a Zn2+-induced current inhibition. To further explore the relation of Zn2+ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn2+ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn2+ inhibition. These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.
Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.
Y W
YW
Lin
C W
CW
Lin
T Y
TY
Chen
D000595:Amino Acid Sequence; D000818:Animals; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D004553:Electric Conductivity; D005260:Female; D015640:Ion Channel Gating / Q000502:physiology*; D009865:Oocytes; D017354:Point Mutation / Q000502:physiology*; D013997:Time Factors; D014101:Torpedo; D014981:Xenopus; D015032:Zinc / Q000494:pharmacology
C120501:CLC-0 chloride channel protein, Torpedo californica; D018118:Chloride Channels; D015032:Zinc