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The Journal of Neuroscience, June 2, 2004, 24(22):5079-5090; doi:10.1523/JNEUROSCI.0882-04.2004
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Cellular/Molecular
Single-Channel Analysis of KCNQ K+ Channels Reveals the Mechanism of Augmentation by a Cysteine-Modifying Reagent
Yang Li, Nikita Gamper, andMark S. Shapiro Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio Texas 78229
The cysteine-modifying reagent N-ethylmaleimide (NEM) is known to augment currents from native M-channels in sympathetic neurons and cloned KCNQ2 channels. As a probe for channel function, we investigated the mechanism of NEM action and subunit specificity of cloned KCNQ2–5 channels expressed in Chinese hamster ovary cells at the whole-cell and single-channel levels. Biotinylation assays and total internal reflection fluorescence microscopy indicated that NEM action is not caused by increased trafficking of channels to the membrane. At saturating voltages, whole-cell currents of KCNQ2, KCNQ4, and KCNQ5 but not KCNQ3 were augmented threefold to fourfold by 50 µM NEM, and their voltage dependencies were negatively shifted by 10–20 mV. Unitary conductances of KCNQ2 and KCNQ3 (6.2 and 8.5 pS, respectively) were much higher that those of KCNQ4 and KCNQ5 (2.1 and 2.2 pS, respectively). Surprisingly, the maximal open probability (Po) of KCNQ3 was near unity, much higher than that of KCNQ2, KCNQ4, and KCNQ5. NEM increased the Po of KCNQ2, KCNQ4, and KCNQ5 by threefold to fourfold but had no effect on their unitary conductances, suggesting that the increase in macroscopic currents can be accounted for by increases in Po. Analysis of KCNQ3/4 chimeras determined the C terminus to be responsible for the differential maximal Po, channel expression, and NEM action between the two channels. To further localize the site of NEM action, we mutated three cysteine residues in the C terminus of KCNQ4. The C519A mutation alone ablated most of the augmentation by NEM, suggesting that NEM acts via alkylation of this residue.
Key words: N-ethylmaleimide; K+ channel; patch clamp; ion channel modulation; M current; KCNQ channel; signaling; gating; biotinylation
Received Jan 19, 2004; revised April 7, 2004; accepted April 8, 2004.
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