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Biophys. J. BioFAST: First Published October 29, 2004. doi:10.1529/biophysj.104.052217
© 2004 by the Biophysical Society.

A more recent version of this article appeared on January 1, 2005.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

A Ring of Negative Charges in the Intracellular Vestibule of Kir2.1 channel modulates K+ Permeation

Hsueh-Kai Chang 1, Shih-Hao Yeh 2 and Ru-Chi Shieh 1*

1 Academia Sinica IBMS
2 Acadmia Sinica IBMS

* To whom correspondence should be addressed. E-mail: ruchi{at}ibms.sinica.edu.tw.

Submitted on September 1, 2004
Revised on September 30, 2004
Accepted on 22 October 2004


  Abstract
The glutamate at the 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 are not observed in either the wild type or an E224D mutant. Introducing a negatively charged MTS reagent to the E224C mutant irreversibly increases channel conductance and eliminates 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 the 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.

Key Words: ion gating, ion-ion interaction, open-channel fluctuations, scanning cysteine accessibility method,, substate




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Copyright © 2004 by the Biophysical Society.