Regulation of the Cardiac Potassium Channel Kir2.1 by the alpha-arrestins
posterposted on 20.04.2020 by Natalie A. Hager, Ceara K. McAtee, Marcel Bruchez, Adam V. Kwiatkowski, Jeffrey L Brodsky, Allyson F. O'Donnell
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1. Kir2.1 is a mammalian potassium channel that is linked to numerous cardiac arrhythmias when it is unable to function or be trafficked correctly.
2. To study this channel we employed a yeast model system where we deleted the endogenous potassium channels, trk1 and trk2, and ectopically express Kir2.1. We know the yeast cells are able to use this mammalian potassium channel because we can measure potassium-dependent growth via serial dilution assays. We also measured the amount of potassium within cells using ICP-MS and saw that expressing Kir2.1 allows for a great intake of potassium compared to control.
3. We then used this assay to see if a family of protein trafficking adaptors, known as the alpha-arrestins, controlled the trafficking of Kir2.1. The alpha-arrestins play a role in trafficking membrane proteins, typically nutrient permeases, to and from the plasma membrane. When we over expressed the alpha-arrestins (Aly1, Aly2, Ldb19, and Rod1) we saw that 3 out of these 4 were able to increase growth on low potassium media, suggesting that they promote Kir2.1 localization to the plasma membrane thus allowing more potassium into the cell. This can also be seen using a sucrose gradient, where we see more Kir2.1 in the plasma membrane fractions. You may also notice that there is a significant amount of Kir2.1 in the ER. This led us to employ the fluorescent activating protein (FAP)
4. This microscopy technique allows us to see only the pools of Kir2.1 located at the plasma membrane. There are two components of FAP, the single chain antibody, which is fused to the protein of interest (Kir2.1 in our case) and the fluorgen dyes. Separate, there is no fluorescence, but when bound there is a 20,000 fold increase in fluorescence. There are two types of dyes that we can use, a permeant dye that can fluoresce anywhere in the cell and an impermeant dye that, because of a polar side chain, can only fluoresce outside the plasma membrane, thus our pool of Kir2.1 at the cell surface.
5. Using this technique, we saw that when we overexpressed out alpha-arrestin we see increased fluorescence signal, therefore more Kir2.1 is at the cell surface. (Using max z-projections so that we are seeing the entirety of the cell).
6. Moving forward we wanted to see if the mammalian alpha-arrestin could also promote Kir2.1 localization to the cell surface in our yeast model. The mammalian alpha-arrestins are also known to work in protein trafficking and are known as ArrDC1-5 and TXNIP. We see that each mammlian a-arrestin is able to improve growth on low potassium media and ArrDC2 and ArrDC3 can robustly increase growth.
7. We then expressed the mammalian alpha-arrestin in HEK293T cells and assessed their localization via IF.
8. We then moved to primary cardiomyocytes. These cells were extracted from neonatal mice, isolated, and then transfected with Kir2.1 and the mammlian a-arrestins. We did live cell-imaging but here are the IF images. We assessed the co-localization between the alpha-arrestins and Kir2.1.