Structural changes in the cytoplasmic pore of the Kir1.1 channel during pH_i-gating probed by FRET

• Main Authors: Shieh Ru-Chi, Lee Jay-Ron
• Format: Article
• Language: English
• Published: BMC 2009-03-01
• Series: Journal of Biomedical Science
• Online Access: http://www.jbiomedsci.com/content/16/1/29
• ISSN: 1021-7770; 1423-0127

<p>Abstract</p> <p>Kir1.1 channels are important in maintaining K⁺homeostasis in the kidney. Intracellular acidification reversibly closes the Kir1.1 channel and thus decreases K⁺secretion. In this study, we used Foster resonance energy transfer (FRET) to determine whether the conformation of the cytoplasmic pore changes in response to intracellular pH (pH_i)-gating in Kir1.1 channels fused with enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (ECFP-Kir1.1-EYFP). Because the fluorescence intensities of ECFP and EYFP were affected at pH_i< 7.4 where pH_i-gating occurs in the ECFP-Kir1.1-EYFP construct, we examined the FRET efficiencies of an ECFP-S219R-EYFP mutant, which is completed closed at pH_i7.4 and open at pH_i10.0. FRET efficiency was increased from 25% to 40% when the pH_iwas decreased from 10.0 to 7.4. These results suggest that the conformation of the cytoplasmic pore in the Kir1.1 channel changes in response to pH_igating such that the N- and C-termini move apart from each other at pH_i7.4, when the channel is open.</p>