| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 87 === The chloride channel from Torpedo electric organ, CLC-0, is the best studied member of the CLC channel family. Two gates with opposite voltage-dependence control the opening of CLC-0 chloride channel. The fast gate opens at depolarized voltages with a gating relaxation time constant on a millisecond time scale. The slow gate, on the other hand, opens at negative voltages on a time scale of tens or hundreds of seconds.
Previous studies have shown that slow gating is highly temperature sensitive. Besides, applying extracellular Zn2+ can reversibly inhibit the CLC-0 channel, perhaps by facilitating the inactivation of the slow gate. To investigate the relationship of Zn2+ inhibition and the inactivation process of the slow gate, we first explored the Zn2+ effect on 12 single-point cysteine mutants. With this approach, we found that nine out of the twelve cysteine mutants behaved like the wild-type (WT) channel. On the other hand, C213G and C480S altered the property of the slow gating. Nevertheless, the slow gate of the channels still displayed the characteristics of voltage and temperature dependence. At the same time, both mutants were Zn2+ sensitive with a K1/2 smaller than 10 mM, a phenomenon similar to WT.
The most interesting result came from the C212S mutant. The macroscopic current measured from whole oocyte recording of C212S revealed neither voltage dependence nor temperature dependence, as if the slow gating process of this mutant was absent. The channel's sensitivity to Zn2+ inhibition was also greatly reduced. To further explore the slow-gating process in C212S mutants, we compared the extracellular chloride effect and the pH sensitivity of the channel gating between the WT and C212S. The results together suggest that the slow-gating transition appeared to be absent in C212S mutant. Single-channel recordings indeed showed that the inactivation probability of C212S was very small, almost close to zero (Lin et al., 1999). The correlation between the zinc sensitivity and the slow gating behavior suggests that the inhibition of the channel by zinc ion is indeed due to an effect on the slow gating of the channel.
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