Cellular localization of the Kir7.1 channel.

<p><b>CHO</b> cells expressing the pEGFP-hKir7.1, pEGFP-R162W (<b>A</b>) or both pEGFP-hKir7.1+ pmCherry-R162W (<b>C</b>, <b>D</b>, <b>E</b>) were studied by live cell fluorescence microscopy using a 60X water immersion objective. Kir7.1 localized mainly to the plasma membrane (<b>A</b>. upper panel green: hKir7.1, red: ER and blue: nucleus) in the pEGFP-hKir7.1 transfected cells. pEGFP-R162W expression co-localized with ER labeling (<b>A</b>. middle panel). Control pEGFP expressing cells are shown in the lower panel (<b>A</b>). (<b>B</b>) Line scans (white arrow) of fluorescence intensity distribution of pEGFP-hKir7.1 (<b>A</b>. black trace upper panel), pEGFP-R162W (<b>A</b>. green trace middle panel), and pEGFP (<b>A</b>. dark green trace lower panel) transfected cells. Red and blue traces (<b>B</b>. upper panel and middle panel) represent ER labeling and Hoechst nucleus staining, respectively. In co-transfection experiments, the GFP fluorescence localized to the cellular membrane (<b>C</b>), whereas mCherry fluorescence shows an intracellular aggregated localization (<b>D</b>). Superposition of both red and green fluorescence (<b>E</b>) further illustrates that there is very little co-localization of the wild-type and mutant channel signals. (<b>F</b>) Fluorescence quantification of membrane vs. cytoplasmic expression from five independent co-transfections with pEGFP-hKir7.1 and pmCherry-R162W plasmids is shown in <b>F</b>, p<0.01.