Hello everyone,
I am trying to record calcium currents from my HEK cells.
Problem is that I don´t have much experience with series resistance compensation that is needed for such large currents.
I am using a Multiclamp 700B and using Multiclamp commander settings to adjust. You can check in the screenshot (Fig 1) I attach one example of a cell where I tried (unsuccesfully) to compensate Rs. Can you identify something I am doing wrong in the Commander settings?
I also attached another image showing how Ca currents appeared in this same cell after such compensation. Currents are obviously not right, as they are very slow and look strange (Fig 2, voltage-step from -80 to 10 mV, 400 ms).
I want to note that in my coverslips, cells are normally forming clumps together, so I normally do not have isolated cells to patch. So space-clamp might be also part of a problem here..
I appreciate your help!
Best regards,
Diego Fernández
Dear Diego: You may need to test different depolarizing steps to determine if there is a V (or inverted-dome) shape in current versus voltage relationship for peak Ca current.
First, I think you need to practice setting up series resistance compensation with the model cell. Have a play with one and make sure you get the known values (10 Mohm, 33 pF)
Second, when you're in a real cell, before you perform whole-cell correction/series resistance compensation make a note of the predicted series resistance from the height of the capacitive transient in response to the test pulse. The value of Rs you should use will not be more than 20% from that (and probably less than 10%).
The value you have set on the amplifier (2-3 MOhm) is pretty low. Are you using 1-1.5 MOhm pipettes? If not, it's unlikely your series resistance is this low.
If these are your real values, you hardly need to bother with series resistance compensation: The time constant of your voltage clamp is < 100 us, and your voltage error is < 1 mV.
However, as I say, I'm dubious that your access resistance is that low.
So, look at the response to your test pulse, estimate your series resistance by the size of your capacitive transient. Set Rs to this, the alter Cm to get a nice, close to monophasic test pulse response. Fine tune Rs and Cm. Then turn on Rs compensation. You really don't need your band width and percentages as high as you've got them. You're only trying to voltage clamp calcium channels, not sodium channels.
But ultimately, I don't think this has anything to do with how slow your currents are, unless your Rs is actually 40/50 MOhm. There isn't a peltier connected to the rig, cooling the cells? You don't have some kind of strange mutant? I can't think of anything that would make the gating that slow.
If you want to be really certain, get a second pipette, and dual patch the cell, have one electrode in voltage clamp, performing your steps, and the other electrode in current clamp I = 0, to check your voltage response,
hi Diego:
From the first trace I think there's already unstable oscillations. From the panel you showed I guessed your prediction and correction value are both higher than recommended.
I have no much experience in compensating this. But I usually use both 50%, which will hopefully reduce my Rs to a value acceptable. Typically if Rs is smaller than 20Mohm that is already good enough. If you still worry about bad clamping, you could put pharmacology in your internal solutions to increase Rm so that voltage can be distributed more on the cell.
Also please check manual of AXON amplifier to see the instructions for Rs compensation. I remember successful compensation needs a few steps.
Best regards
Yao
Thanks a lot for all your responses. Following William M Connelly advice, I have been practicing Rs compensation with the model cell, and slowly I am gbetting more hands on doing this faster. It is true though, that some of my cells are harder to compensate before oscillation, I guess this is because fast and slow capacitance are not well compensated themselves.
Thank you very much for your help.
Best regards,
Diego
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