Structural changes in the cytoplasmic pore of the Kir1.1 channel during pH(i)-gating probed by FRET

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(i )7.4 and open at pH(i )10.0. FRET efficiency was increased from 25% to 40% when the pH(i )was 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(i )gating such that the N- and C-termini move apart from each other at pH(i )7.4, when the channel is open.