Single-channel analysis of KCNQ K+ channels reveals the mechanism of augmentation by a cysteine-modifying reagent.
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 microm 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 (P(o)) of KCNQ3 was near unity, much higher than that of KCNQ2, KCNQ4, and KCNQ5. NEM increased the P(o) 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 P(o). Analysis of KCNQ3/4 chimeras determined the C terminus to be responsible for the differential maximal P(o), 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.
Li
Yang
Y
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio Texas 78229, USA.
Gamper
Nikita
N
Shapiro
Mark S
MS
D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003545:Cysteine / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005033:Ethylmaleimide / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006801:Humans; D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014162:Transfection
D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; C117367:KCNQ4 protein, human; C412231:KCNQ5 protein, human; C496915:Kcnq2 protein, rat; C496918:Kcnq3 protein, rat; C492166:Kcnq4 protein, rat; C501028:Kcnq5 protein, rat; D008164:Luminescent Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D049452:Green Fluorescent Proteins; D003545:Cysteine; D005033:Ethylmaleimide