The oxidant thimerosal modulates gating behavior of KCNQ1 by interaction with the channel outer shell.
Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.
Kerst
G
G
University Children's Hospital, Hoppe-Seyler-Strasse 1, 72076 Tübingen, Germany. gunter.kerst@med.uni-tuebingen.de
Brousos
H
H
Schreiber
R
R
Nitschke
R
R
Hug
M J
MJ
Greger
R
R
Bleich
M
M
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004553:Electric Conductivity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects*; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D009865:Oocytes; D016877:Oxidants / Q000494:pharmacology*; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D013849:Thimerosal / Q000494:pharmacology*; D014982:Xenopus laevis
D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D016877:Oxidants; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C059983:potassium channel protein I(sk); D013849:Thimerosal; D002118:Calcium