I am looking at K+ currents using whole-cell patch clamping and Clampex, with positive and negative voltage steps under voltage clamp, from a holding potential. For this I apply channel blockers (TEA, 4-AP etc) and the same voltage steps, and then use Clampfit (from Axon Instruments) to subtract the currents obtained under channel blocking from those obtained in control. Presumably the difference is then the K+ currents that were blocked by the blocker. This is done under "analyze" in Clampfit, and seems to work OK, except the polarity of the subtracted currents look odd, and in some cases opposite to what to expect. Does anyone have any suggestions as to what I may be doing wrong?
Dear Thor,
This Problem might occure if their is leak current: the resulting (substracted) values can reach values below Zero (steady state current especially).
I don´t think it is leak currents. If there is a reduction in outward currents as compared to control, then the polarity of the subtracted difference is reversed, as if it were inward currents. But of course leakage currents would make this more complex.
Thor,
You may try to build the IV plot of your unblocked K+ current and see where the reversal potential is for K ions. Then you calculate where it should be based on your pipette solution and expected K+ concentration inside the cell. If they are more or less the same, then you do not have a leak. You may deal with inward or outward rectification of potassium channel, if you have both population in membrane. AFAIK, both TEA and 4-AP block both of them
Do you use conventional or perforated whole-cell? Did you check the access resistance and cell capacitance during and after recording?
The change in resistance can cause an error. The changes in resistance and capacitance also tell whether you lost the perfect patch.
May i ask you why you don't try to isolate K+ currents pharmacollogically in order to record them?
Thanks everyone for your inputs. Evgeny, there is clearly inward and outward rectification in these cells, i.e. more than one population of K+ channels (which I am trying to find out about), which is part of the problem. I am trying to modify the protocol of pulses to activate these separately, but tough. The reversal potentials are comparable so I don´t think the problem is leaks (except sometimes...). Wenkuan, I am using conventional whole-cell. I discard recordings from cells that have any significant changes in resistance, but you are certainly right about that. Isabelle, that is precisely what I am trying to do using TEA, 4-AP, Ba+ etc, but I wish to be able to see and measure the blocked currents, using subtraction.
Why not trying to block other types of current (Ca2+ and Na+ with Cadmium and TTX)?
I think that the problem is that you have to block calcium and sodium current before applying your potassium blocker and after that make the subtraction. I also wonder: why are you using barium as you won't be abble to see SK/BK which are potassium currents?
If you think that a problem occurs with Clampfit only, it would be useful if you can share an illustration with the current traces before and after K+ currents blockade and also the traces resulting from the subtraction? It could also help to know the HP value that you apply during the stimulation protocol.
Dear Thor. Are you blocking all other inward conductances (Na and Ca) as Isabelle and Fabian pointed out? I hope you are, because if not...well they have a point: Your substracted current may indeed be exactly as you described i.e.,: "look odd, and in some cases opposite to what to expect".
Dear Fouad. When I posted the question I had not blocked other inward conductances. After reading the comments, in particular from Isabelle and Fabian, it hit me that this needed rethinking, and different experiments than what i was doing. So I haven´t been active in this discussion recently...:) But thanks everyone, this has been very helpful. I am currently saying away from the subtraction feature and just looking at the currents before and after applying blockers, to get a picture of what sort of channels are involved. I was using a HP of -45 mV, but am varying that as well.
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