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Originally published as Biophys J. BioFAST on October 29, 2004.
doi:10.1529/biophysj.104.052217
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Biophysical Journal 88:243-254 (2005)
© 2005 The Biophysical Society

A Ring of Negative Charges in the Intracellular Vestibule of Kir2.1 Channel Modulates K+ Permeation

Hsueh-Kai Chang, Shih-Hao Yeh and Ru-Chi Shieh

Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China

Correspondence: Address reprint requests to Ru-Chi Shieh, PhD, Institute of Biomedical Sciences, Academia Sinica, 128 Yen-Chiu Yuan Rd. Section 2, Taipei 11529, Taiwan, Republic of China. Tel.: 886-2-2652-3914; Fax: 886-2-2782-9143; E-mail: ruchi{at}ibms.sinica.edu.tw.

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 or Tl+ as compared to internal K+. These results suggest that 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.




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