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Elimination of the slow gating of ClC-0 chloride channel by a point mutation.

Lin YW, Lin CW, Chen TY - J. Gen. Physiol. (1999)

Bottom Line: With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition.At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.

ABSTRACT
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.

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Related in: MedlinePlus

Single-channel recordings of (A) Wild-type ClC-0, (B) C213G, and (C) C480S. Continuous recording from excised inside-out patch at a holding potential of −50 mV. The top of each panel shows a 4-min recording in a compressed time window. The bottom is with an expanded time window starting at the position indicated by the arrow. Note that two C213G channels were present in the patch. The temperature during recording was ∼23–25°C.
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Figure 6: Single-channel recordings of (A) Wild-type ClC-0, (B) C213G, and (C) C480S. Continuous recording from excised inside-out patch at a holding potential of −50 mV. The top of each panel shows a 4-min recording in a compressed time window. The bottom is with an expanded time window starting at the position indicated by the arrow. Note that two C213G channels were present in the patch. The temperature during recording was ∼23–25°C.

Mentions: Inside-out patches (Hamill et al. 1981) were obtained with glass electrodes fire-polished to a resistance of 2–7 MΩ. Single-channel currents were recorded with an Axopatch 200B (Axon Instruments) amplifier. The output of the amplifier was filtered at 200 Hz (−3 dB corner frequency, four-pole Bessel; Dagan Corp.) and digitized at 1 kHz by a Microstar DAP 800 acquisition board (Microstar Laboratories, Inc.) installed in a Pentium computer using home-written software (Chen and Miller 1996). The external (pipette) solution contained (mM): 110 N-methyl-d-glucamine (NMDG)–Cl, 5 MgCl2, 1 CaCl2, 5 HEPES, pH 7.6. The bath solution contained (mM): 110 NaCl, 5 MgCl2, 5 HEPES, 1 EGTA, pH 7.6. To display the slow-gating transition with long single-channel trace, every 500 sampling points (equivalent to 0.5 s) were averaged and shown as one data point (see Fig. 6Fig. 7Fig. 8). In such a compressed time window, the fast-gating transition is no longer visible, but any inactivation event with duration longer than ∼200 ms can be seen as an upward deflection (e.g., see Fig. 6 A). The displays of the fast gating are the original recording traces sampled at 1 kHz.


Elimination of the slow gating of ClC-0 chloride channel by a point mutation.

Lin YW, Lin CW, Chen TY - J. Gen. Physiol. (1999)

Single-channel recordings of (A) Wild-type ClC-0, (B) C213G, and (C) C480S. Continuous recording from excised inside-out patch at a holding potential of −50 mV. The top of each panel shows a 4-min recording in a compressed time window. The bottom is with an expanded time window starting at the position indicated by the arrow. Note that two C213G channels were present in the patch. The temperature during recording was ∼23–25°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229640&req=5

Figure 6: Single-channel recordings of (A) Wild-type ClC-0, (B) C213G, and (C) C480S. Continuous recording from excised inside-out patch at a holding potential of −50 mV. The top of each panel shows a 4-min recording in a compressed time window. The bottom is with an expanded time window starting at the position indicated by the arrow. Note that two C213G channels were present in the patch. The temperature during recording was ∼23–25°C.
Mentions: Inside-out patches (Hamill et al. 1981) were obtained with glass electrodes fire-polished to a resistance of 2–7 MΩ. Single-channel currents were recorded with an Axopatch 200B (Axon Instruments) amplifier. The output of the amplifier was filtered at 200 Hz (−3 dB corner frequency, four-pole Bessel; Dagan Corp.) and digitized at 1 kHz by a Microstar DAP 800 acquisition board (Microstar Laboratories, Inc.) installed in a Pentium computer using home-written software (Chen and Miller 1996). The external (pipette) solution contained (mM): 110 N-methyl-d-glucamine (NMDG)–Cl, 5 MgCl2, 1 CaCl2, 5 HEPES, pH 7.6. The bath solution contained (mM): 110 NaCl, 5 MgCl2, 5 HEPES, 1 EGTA, pH 7.6. To display the slow-gating transition with long single-channel trace, every 500 sampling points (equivalent to 0.5 s) were averaged and shown as one data point (see Fig. 6Fig. 7Fig. 8). In such a compressed time window, the fast-gating transition is no longer visible, but any inactivation event with duration longer than ∼200 ms can be seen as an upward deflection (e.g., see Fig. 6 A). The displays of the fast gating are the original recording traces sampled at 1 kHz.

Bottom Line: With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition.At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus