Hi,
I am trying to express mouse Kir4.1 (and Kir5.1) in cell culture for whole cell patch clamp electrophysiology. I have tried Chinese hamster ovary (CHO) cells, which we normally use, and TSA201 cells. In both cases, the cells are very unstable after transfection of 0.5-1mg of plasmid with Lipfectamin in small dishes. I am using a IV protocol with voltage steps from -120 to +60 but I don't really get nice currents as seen in the literature. I mostly see Kir channels expressed in Oocytes, but we don't really have the facility to do that, I thought it would be easier in mammalian cell lines.
Does anyone have experience to share with patching Kir channels?
Cheers!
Hello Laura-Nadine,
It's not quite clear which particular problem you have with patch-clamping, but it sounds like it is all related to general health of cell culture and efficiency of transfection. You may want to get another strand of CHO cells and play with transfection conditions. In general, CHO cells are reliable host cells widely used for transfections with various ion channels. You also may want to think of HEK-293 cells as host cells alternative.
I personally do not have experience with Kir4.1, but rather with Kir2.1 and GIRK1/2 inward rectifier channels expressed in HEK-293 and CHO cells. However, in terms of electrophysiological measurements it should not matter too much. Two standard protocols are as follows: (1) a series of voltage steps negative and positive to Ek (reversal potential for K+ currents), i.e. similar to what you use in your lab; (2) voltage ramp protocol scanning similar voltage range. In case of good expression of Kir one sees much more inward current (voltage range negative to Ek) than outward current (voltage range positive to Ek). In our hands, cells lasted well in acute recordings.
RBL cells are considered to be "Gold standard" for endogenous Kir current recordings. It should be available from cell culture collections. You may want to try them to optimize your patch-clamp recording conditions with small mammalian cells and then switch to cells of interest for you.
First, I think you need a good reporter for see if you tranfection working, the CHO not have endogoneus Kir, but HEK have, maybe you can see in HEK and observed at increase of the basal current
Now in you IV protocol try to a prepulse, maybe to 0 or 20 mV for increase the driver forces of your cannel
and finally yo can change the external solution fo see the Kir more stronger if change again the Peq of the potassium, but must to careful wiht the other Kv current
cheers
I see you already received some good advice. Let me add a little to that.
Do you know whether you use CHO-K1 or CHO dhfr- cells? In our experience CHO dhfr- cells respond better to transfection. Also, I would consider a different transaction reagent. For CHO we mainly use jetPEI. This yields good transfection efficiencies and has little cell toxicity.
Regards,
Christian
We routinely use Kir4.1 and other Kir plasmids. I would guess you have to start with plasmid preparation. Use Qiagen or 5Prime kits to obtain transfection ready plasmids. As suggested earlier use a marker or even better establish a stable cell line with selection. Also check on passage number for your CHO cells. CHO and HEK cells are easy to transfect so you should have no problem in seeing current between -120 to +60. I would suggest try going from -150 to +50 as you will see mostly inward current and for Kir4.1 its characteristic saturation after -130 mV.
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