Localization of the pH gate in Kir1.1 channels.
We used cysteine-modifying reagents to localize the pH-sensitive gate in the renal inward-rectifier K(+) channel Kir1.1a (ROMK1). Cytoplasmic-side methanethiosulfonate (MTS) reagents blocked K(+) permeation in native Kir1.1 channels, expressed in Xenopus oocytes. Replacement of three cysteines in the N-terminus, C-terminus, and transmembrane domains eliminated this sensitivity to MTS reagents, as measured with inside-out macropatches. Reintroduction of one cysteine at 175-Kir1.1a in the second transmembrane domain allowed blockade of the open channel by the MTS reagents MTSEA, MTSET, and MTSES and by Ag(+). However, closure of the channel by low pH protected it from modification. Cysteine was also introduced into position G223, which is thought to line the cytoplasmic pore of the channel. MTSET blocked G223C in both the open and closed state. In contrast, MTSEA reduced G223C single-channel conductance from 40 to 23 pS but did not produce complete block. We conclude that cytoplasmic acidification induces a conformational change in the channel protein that prevents access of cysteine-modifying reagents, and presumably also K(+) ions, to the transmembrane pore from the cytoplasm. This is consistent with localization of the Kir1.1 pH gate at the helix bundle crossing near the cytoplasmic end of the transmembrane pore.
Zhang
Yu-Yang
YY
Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021,USA.
Sackin
Henry
H
Palmer
Lawrence G
LG
D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000502:physiology; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D015640:Ion Channel Gating; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
D007202:Indicators and Reagents; D008698:Mesylates; D024661:Potassium Channels, Inwardly Rectifying; C074502:methanethiosulfonate; D003545:Cysteine; D011188:Potassium