Figure 4.

PIP₂ facilitates Kir2.1 cell surface expression and Kir2.1 movement at the cell membrane. (A) Localization and distribution of PIP₂ and Kir2.1 channels on the cell membrane. Hela cells were transfected with GFP-PLCdelta-PH and mCherry-fused Kir2.1 channels. PIP₂ was traced by PLC delta fused with GFP. Scale bar: 10 μm. (B) Quantification of PIP₂ -Kir channel co-localization by Pearson’s correlation coefficient. For n = 18–24 cells pooled across three independent experiments. *** p < 0.001. (C) Kir2.1 distribution in Hela cells before and after PIP₂ depletion. Hela cells were transfected with mCherry-fused Kir2.1. Scale bar: 25μm. The quantification is shown below. n = 25–33 cells pooled across three independent experiments. ** p < 0.01. (D) Kir2.1-K188Q fused mCherry distribution in Hela cells cultured with or without spermin (100uM). Hela cells were transfected with mCherry-fused Kir2.1-K188Q. Spermin is a Kir2.1-PIP₂ binding enhancer. Scale bar: 25 μm. The quantification is shown below. n = 19 cells pooled across three independent experiments. * p < 0.05. (E) Movement patterns of Kir2.1 before and after PIP₂ depletion. Kir2.1 was labeled with the different pseudo colors at different time frames, and co-localization parts in the merged images represent the stillness of Kir2.1. Scale bar: 5 μm.