Figure 4. Kcnk5b channel activity regulates developmental gene transcription in mammalian cells.

(A) FRET-FLIM images after measuring the life time of CFP of the K⁺ FRET reporter KIRIN (Shen et al., 2019). The color images indicate the differences in CFP fluorescence lifetime of the K⁺ FRET reporter KIRIN in HEK293T (HEK) cells. Assigned rainbow of colors in the delineated cytoplasm depict the range of numeric values of nanoseconds (ns) of the detected fluorescent lifetime for CFP. Red represents longer lifetime values. Blue represents shorter lifetime values, and the other colors represent intermediary lifetime values. (a) Composite image of all lifetime values of the KIRIN K⁺ reporter in control cells transfected with mCherry. (b) Image of low lifetime values in a control cell. (c) Image of high lifetime values in a control cell. (d) Composite image of all lifetime values of the KIRIN K⁺ reporter in cells expressing kcnk5b-mCherry. (e) Image of low lifetime values in cells expressing kcnk5b-mCherry. (f) Image of high lifetime values in cells expressing kcnk5b-mCherry. (g) Compared to GFP-transfected HEK cells, cells transfected with kcnk5b-mCherry show an increase in CFP lifetime due to reduction in intracellular K⁺. (Ba) qRT-PCR for SHH and LEF1 in HEK cells. (Bb) qRT-PCR for ALDH1a2, PEA3 and MSX1 in HEK cells. (C) qRT-PCR for indicated genes in HEK cells expressing GFP, kcnk9-GFP or kcnk10-GFP 24 hr after transfection. (D) qRT-PCR results in HeLa cells expressing either GFP or kcnk5b-GFP 24 hr after transfection. (E,F) qRT-PCR results in N2A cells expressing either GFP or kcnk5b-GFP 24 hr after transfection. (G) qRT-PCR results in Mcf7 cells expressing either GFP or kcnk5b-GFP 24 hr after transfection. (H,I) qRT-PCR measurement of indicated gene after 24 hr transfection of Kcnk9 in Hela cells. (J) qRT-PCR measurement of indicated gene after 24 hr transfection of Kcnk10 in HeLa cells. (K) qRT-PCR measurement of indicated gene after 24 hr transfection of Kcnk9 in N2A cells. (L) qRT-PCR measurement of indicated gene after 24 hr transfection of Kcnk10 in N2A cells. The data represent three or more experiments, The data points show all technical replicates. Student’s T-test was used for tests of significance and the levels of significance are indicated between the experimental groups.

Figure 4—figure supplement 1. Expression and activity of Kcnk5b-GFP in transfected HEK293T cells.

(A) Confocal image of through an immunocytochemical staining of a HEK293T cell transfected with zebrafish kcnk5b-GFP transgene (B) DAPI staining of the same cell in panel A. (C) Merged panels A and B. (D–L) Serial confocal cross sections for GFP in a live HEK293T cell from its surface (D) through to a mid-section of the cell (L) after transfection with the zebrafish kcnk5b-GFP transgene. (M) FRET-FLIM image after measuring the life time of CFP of the K⁺ FRET reporter KIRIN in a cell transfected with a control plasmid only expressing mCherry. The inset image of the confirmation of the FRET reporter depicting how FRET occurs. (N) FRET-FLIM image after measuring the life time of CFP of the K⁺ FRET reporter KIRIN in a cell transfected with a plasmid expressing kcnk5b-mCherry. The inset image of the confirmation of the FRET reporter depicting how FRET do not occur. (O) The graphed assessments of the changes in CFP lifetime in the control mCherry-expressing cells and the cells expressing kcnk5b-mCherry. The diagrams to the right of the graph depict the conformational changes that result in the values of the lifetimes of CFP. The data for each experiment represents three or more experiments. The graphed data points show all technical replicates.

Figure 4—figure supplement 2. Comparison of Kcnk5b with Knck9 or with Kcnk10.

(A,B) qRT-PCR measurement of the indicated gene after 24 hr transfection of Kcnk5b in HEK293T cells. (C,D) Comparison between zebrafish amino acid sequences from Kcnk5b (blue) and Kcnk9 (C, black) and Kcnk10b (D, black) show conserved (red letter) and similar properties (red plus sign) amino acids. Yellow-highlighted letters indicate calcineurin binding site, and green-highlighted letters indicate the site of post-translational modification by calcineurin. The data for each experiment represent three or more experiments. The graphed data points show all technical replicates.