Oxidation of an engineered pore cysteine locks a voltage-gated K+ channel in a nonconducting state.
We report the use of cysteine-substituted mutants in conjunction with in situ oxidation to determine the physical proximity of a pair of engineered cysteines in the pore region of the voltage-gated K+ channel Kv2.1. We show that the newly introduced cysteine 1379C, located near the outer end of the narrow ion-conduction pathway, renders the K+ channel sensitive to oxidation by H2O2, but only if the native cysteine at position 394 in S6 remains in place. Conservative substitutions in S6 for cysteine 394 abolish H2O2 sensitivity in the Kv2.1 mutant 1379C. Comparative immunoblot analysis of wild-type and 1379C Kv2.1-expressing HEK293 cells demonstrates the presence of subunit dimers for 1379C, but not for wild-type Kv2.1. At the single-channel level, the probability of opening of 1379C channels, unlike wild-type, is reduced in the presence of H2O2; however, oxidation of 1379C does not alter unit current. These findings imply that cysteine 379, located near the outer end of the narrow ion-conduction pathway, participates in disulfide bridge formation, locking the channel in a nonconducting state from which it cannot undergo conformational transitions required for opening.
Zhang
H J
HJ
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9111, USA.
Liu
Y
Y
Zühlke
R D
RD
Joho
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003545:Cysteine; D051668:Delayed Rectifier Potassium Channels; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015640:Ion Channel Gating; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051669:Shab Potassium Channels; D014162:Transfection; D014982:Xenopus laevis
D051668:Delayed Rectifier Potassium Channels; C496988:KCNB1 protein, human; D046911:Macromolecular Substances; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051669:Shab Potassium Channels; D006861:Hydrogen Peroxide; D003545:Cysteine