I conducted the recovery from inactivation protocol to record INa in isolated cardiomyocytes. The very strange thing is that the sodium current couldn't return to the max value (marked 1). The attached picture shows the INa tracing. The first one is marked as 1, the other INa are marked as 2,3,4,5,6,7,8,.... The protocol is 2-s holding potential of -100mV and followed by -35mV for 20ms and the interval is varying every 5 ms. My question is: Why couldn't INa return to the max value after they are stable? Any suggestion is greatly appreciated!
Dear Xiaobo,
Can you provide the scaling in your image?
One explanation for your finding is that your series resistance has increased. The series resistance with the capacitances filters the sodium channel current.
More on this: Traynelis 1988, Article Software-based correction of single compartment series resis...
Your capacitive current now bleeds into your sodium current, indicating that you have not reached the clamp voltage when the sodium current starts.
Do you measure your series resistance before and after your experiment? And what are your pipette and cell capacitance?
Try to optimize the series resistance compensation to attain a rapid transient that is at steady-state before the sodium current starts. Be careful that the positive feedback does not kill your cell.
Good luck,
Martijn
Martijn Sierksma Thank you for your suggestion! The series resistance is pretty stable before and after recording. I ran the same protocol today in multiple cardiomyocytes and every single cardiomyocyte has the similar recovery tracing even if the holding potential is set at -120mV. The pipette resistance is about 2.5Mohms and the capacitance is about 120pF. The original recording is attached for your reading. Thank you so much for your thought!
Dear Xiaobo,
Do you mean with pipette resistance your open tip resistance (before patching) or your series resistance (when you got whole-cell)?
The time constant of your voltage-clamp is determined by the series resistance and the capacitance (tau = RsCm). If your series resistance is 5 MOhms and your cell capacitance is 120 pF, your time constant becomes 0.6 ms, meaning that it will take about 3-5 ms before you reach steady-state voltage when you perform the step.
(see Barbour’s https://www.bio.ens.psl.eu/perso/barbour/electronics_for_electrophysiologists.pdf).
Which amplifier do you use and do you use active compensation to improve your voltage-clamp?
Best,
Martijn
Martijn Sierksma Hi Martijn, the series resistance with the whole cell is from 5-7 MOhms. I will read the paper you sent to me. I am using Axopatch 200B Amplifier. I don't use active compensation. You can tell I only did series resistance compensation, which is up to 70%. The attached file is the sodium current recording. Hopefully it can help a bit. I appreciate your help. Thank you so much!
Dear Xiaobo,
I recommend you revisit the Axopatch 200B manual to see how you can improve your voltage clamp using the prediction and correction. The series resistance setting does not improve your voltage clamp.
Practice with a model cell first to get a bit of experience as minor adjustments can cause the voltage clamp to oscillate and kill your cell.
Regarding your slow recovery from inactivation, you may be interested in Featherstone et al 1996 (https://www.cell.com/biophysj/pdf/S0006-3495(96)79504-8.pdf) and for a possible model see Zhang et al 2013 (Article Kinetic Model of Nav1.5 Channel Provides a Subtle Insight in...
). Try longer intervals to see if your current returns to baseline (and keep in mind that your series resistance needs to be stable).Good luck,
Martijn
Dear Xiaobo,
The reason could be that you are loosing points in the recording, mainly in fastest phase of the currents. I opened your recording file with clampfit and your Sampling rate is 10.00 kHz. I think it is low for INa. Try to increase at least to 33.33 KHz in Clampex. In the recording file I can not see the filter you are using. You must set the low pass filter in the axopatch to 5 or 10 KHz.
Increasing your sampling rate will increase the weight of your recording files. Set off your first 2 seconds and last 100 ms steps to holding potential and the start-to-start intervals to 3 seconds.
Good Luck,
Oscar
Oscar Casis Thank you for the suggestion. I increased the sampling rate and also the filter as you suggested. The INa still cannot return to the max value. I recorded INa only at -35mV for several sweeps starting with 2-s -100mV holding, INa is pretty stable. That is so strange. I am considering if I need to change my internal and external solution reciple. My external solution has no any Cs, instead I use N-methy-D-glucamine.
It's possible that the intracellular sodium concentration changes during the voltage protocol you used. When channels open up, Na+ enters the cell and is eliminated via diffusion into the patch pipette. If the rate of Na+ inflow into the cell thorugh the channels does not match the rate of Na+ elimination through the patch pipette, Na+ accumulates in the cytosol, affecting the current size. Albertino
To enhance diffusional exchange between the pipette solution and the interior of the cell, I believe you should maintain the patch pipette resistance as low as possible. Apart from that, I believe you have to acknowledge the limitations imposed by the experimental setup.
Good luck
Albertino
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