A ring of negative charges in the intracellular vestibule of Kir2.1 channel modulates K+ permeation.
The glutamate at site 224 of a Kir2.1 channel plays an important role in K+ permeation. The single-channel inward current flickers with reduced conductance in an E224G mutant. We show that open-channel fluctuations can also be observed in E224C, E224K, and E224Q mutants. Yet, open-channel fluctuations were not observed in either the wild-type or an E224D mutant. Introducing a negatively charged methanethiosulfonate reagent to the E224C mutant irreversibly increased channel conductance and eliminated open-channel fluctuations. These results suggest that although the negatively charged residue 224 is located at the internal vestibule, it is important for smooth inward K+ conduction. We identified a substate in the E224G mutant and showed that open-channel fluctuations are mainly attributed to rapid transitions between the substate and the main state. Also, we characterized the voltage- and ion-dependence of the substate kinetics. The open-channel fluctuations decreased in internal NH4+ or Tl+ as compared to internal K+. These results suggest that NH4+ and Tl+ gate the E224G mutant in a more stable state. Based on an ion-conduction model, we propose that the appearance of the substate in the E224G mutant is due to changes of ion gating in association with variations of ion-ion interaction in the permeation pathway.
Chang
Hsueh-Kai
HK
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China.
Yeh
Shih-Hao
SH
Shieh
Ru-Chi
RC
D000818:Animals; D001665:Binding Sites; D002586:Cesium / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D018698:Glutamic Acid / Q000737:chemistry; D015640:Ion Channel Gating; D007477:Ions; D007700:Kinetics; D008698:Mesylates / Q000494:pharmacology; D009154:Mutation; D011188:Potassium / Q000737:chemistry; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013223:Statistics as Topic; D013696:Temperature; D013997:Time Factors; D014981:Xenopus
D007477:Ions; C489461:Kir2.1 channel; D008698:Mesylates; D024661:Potassium Channels, Inwardly Rectifying; D002586:Cesium; D018698:Glutamic Acid; C074502:methanethiosulfonate; D011188:Potassium