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J Biomed Sci
2009 Mar 06;16:29. doi: 10.1186/1423-0127-16-29.
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Structural changes in the cytoplasmic pore of the Kir1.1 channel during pHi-gating probed by FRET.
Lee JR
,
Shieh RC
.
???displayArticle.abstract??? 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 (pHi)-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 pHi < 7.4 where pHi-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 pHi 7.4 and open at pHi 10.0. FRET efficiency was increased from 25% to 40% when the pHi 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 pHi gating such that the N- and C-termini move apart from each other at pHi 7.4, when the channel is open.
Figure 1. Effect of the pHi on the fluorescence intensities of ECFP or EYFP fused to the Kir2.1 channel. A. Images of HEK293T cell plasma membrane sheets expressing ECFP-Kir2.1 or Kir2.1-EYFP exposed to pHi 10.0 or 6.0. For clarity of presentation (but not during data analysis), the contrast of photos was increased by 20% with Photoshop CS3. B. Averaged effect of the pHi on the florescence intensities of ECFP-Kir2.1 or Kir2.1-EYFP in plasma sheets (C, n = 15). The fluorescence intensities were normalized to those obtained at pHi 10.0. *** indicates p < 0.005 compared to the pHi 10.0 value.
Figure 2. pHi-gating of Kir1.1 channels. A. Currents were recorded at pHi 8.0 and 6.0 in an inside-out oocyte patch expressing ECFP-Kir1.1-EYFP. Currents were recorded by holding the membrane potential at -80 mV and stepping to +50 mV for 50 ms every 1 Hz. B. Normalized current-pHi relationship. Currents were normalized to those recorded at pH 8.0 for the wild-type Kir1.1 and ECFP-Kir1.1-EYFP construct and to those recorded at pH 10.0 for the S219R mutant and ECFP-S219R-EYFP construct. The smooth curves are the best fit of the data to the Hill equation: 1/{1 + [H+]/Kd]n}. n = 3 – 10. C. Currents were recorded at pHi 10.0 and 7.4 in an inside-out oocyte patch expressing ECFP-S219R-EYFP using the same protocol as in A. C-Kir1.1-Y and C-S219R-Y stand for ECFP-Kir1.1-EYFP and ECFP-S219R-EYFP, respectively.
Figure 3. Effects of the pHi on the FRET efficiency. A. Bright field image of a pipette containing a oocyte membrane patch (right panel) and currents recorded at +50 mV from a holding potential of -80 mV at pHi 7.4 (left panel) or pHi 10.0 (center panel). B. Fluorescence images acquired with the ECFP, EYFP, and FRET filter cubes at the indicated pHi. Fluorescence intensities were measured in the area of the membrane patch indicated by the dotted circle. Scale bar, 20 μm. C. Averaged FRET efficiency of ECFP-S219R-EYFP in the open state (pHi 10.0, n = 6) and closed state (7.4, n = 8) and FRET efficiency of ECFP + EYFP. n = 6 – 8. *** indicates p < 0.005 compared to the pHi 10.0 value.
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