Dear Sourajit,

the holding potential in electrophysiology is defined by the potential set by your amplifier in voltage-clamp mode in between your ramp protocols. This potential will be relative to your reference electrode.

The reversal potential of a channel/exchanger is defined as the potential at which the currents through this channel/exchanger cancel each other (aka I=0).

Using your ramp protocol you are changing the holding potential dynamically, which may alter the currents that run from your recording electrode to your reference electrode. To identify the current related to your channel/exchanger, you probably have to perform an experiment under control conditions versus a blocker. This difference in current may reverse at a particular potential in your ramp protocol, and this potential (correcting for series resistance, etc.) should be the reversal potential.

Alternatively, if there are no other currents present in your system except those from your channel/exchanger, then the ramp protocol can indicate directly what the reversal potential is.

Take a look at Hamill et al. (1981, doi:10.1007/BF00656997).

Good luck.

Dear Sourajit Mukherjee ,

I will strongly back Martijn Sierksma in his answer.

Your holding potential will never alter in any way your reversal potential. Keep in mind that the reversal potential relies solely in the internal and external concentrations of an ion, dictated by the Nernst equation. To illustrate it in your context, see Fig 3B of the paper you mentioned, where the voltage ramps alter the holding potential (-100 to +60) and, what actually changes the reversal potential (i.e. the point where the current trace crosses the x axis [y=0] in each of the curves), is the external sodium concentration used.

Best,

Andre

Dear Martijn Sierksma and Andre Dagostin thank you for your wonderful responses.

Ideally the reversal potential never alter the reversal potential of ion channels/exchangers- fully agreed on this! Also, the reversal potential depends on the concentration of ions across membrane. However, I have been surprised to see no real shift in the reversal potential even if I vary the ion concentrations across the membrane. I am working with a Calcium/proton exchanger and kindly do have a look at my data where I vary Ca2+ and H+ ions.

The first picture shows the I-V plot where the EXTERNAL pH is 5.5 and Ca is 1mM and INTERNAL pH is 7.5 and Ca is 30nM.

The second picture shows the I-V plot where the EXTERNAL pH is 5.5 and Ca is 1mM and the INTERNAL pH is 7.5 and Ca is 1uM.

Essentially, between the two experiments I have everything constant but varied the internal Ca. Therefore the reversal potential should change from one figure to the other one right? But unfortunately there is no such appreciable shift in reversal potential.

Can you please help in interpreting what is happening?

Dear Sourajit,

I notice that your currents are much larger than in the paper. You have a slope resistance of about 130 MOhm, while from the paper you mention I calculate a slope resistance of 2 GOhm. Perhaps you have a leak current which reverses at 0 mV and obscures the exchanger current.

Can you pharmacologically block your exchanger? Or perhaps run your protocols on HEK cells transfected by a dead/non-functional exchanger or without the exchanger?

I have not attempted to calculate the expected reversal potential or how much it would shift under your new conditions. Have you?

Lastly, you can try your experiment with your normal conditions followed by a reduced extracellular calcium concentration (say 30 uM, 1 uM). Your control of the extracellular calcium concentrations is better than of the cytoplasmic concentrations.

Good luck with your experiments!

Dear Sourajit,

In the paper, another characteristic is that the -100mV initial hyperpolarization brings the Vm to around -20 pA, while yours, it goes to more than -500pA. Are you performing any leak subtraction? If not, I advise you to, otherwise the leak current could be too big and just mask all your results. In the paper they do it pharmacologically using LiCl and a post hoc subtraction:

"The currents observed due to voltage ramps in LiCl/EGTA bath solution (conditions preventing the exchanger from operating in either direction) at the start of the experiment were digitally subtracted from subsequent currents obtained under various conditions, and the resulting I-V traces are shown in Fig. 3 B"

Btw, using conventional p/n leak subtraction will be ineffective if your exchanger isn't voltage dependent.

Dear Martijn Sierksma I think there is a bit of misunderstanding here as the paper (a different exchanger than the one I am studying) reports current from -80mV to +40mV whereas I report from -100mV to +100mV and hence the current seems larger as it is rectifying in an outward manner. If you closely have a look, the current response at around +40mV is almost the similar magnitude as the paper.

However, I would admit that I have not considered anything about the leak current in my recordings. Also, I would like to mention that this is my first-hand experience doing whole-cell recordings and it would be extremely helpful if you could let me know how to tackle the leak current contribution (through the amplifier??)

Although there are no known pharmacological blocker of the exchanger, I have tried to transfect exchanger-dead mutant. In that scenario, the magnitude of the outward current decreases as expected.

Yes I have calculated the expected reversal potentials. When the internal Ca is 30nM (1st pic), it should be 86.4mV and when the internal Ca is 1uM (2nd pic), it should be 175.5mV.

Sure, I will definitely try to vary the external Ca and perform the experiments.

Thanks again for taking time out to help.

Dear Andre Dagostin

Thank you for your response. To be honest I have not done any leak subtraction and you are right in pointing out that to me. I would be doing that and perform the experiments again.

Hope this resolves my concern. Thanks!

Dear Andre Dagostin and Martijn Sierksma

How valid is it to subtract the current response from the untransfected cells (similar ramp protocol from -100 to +100 mV with a holding of 0mV) vs the transfected cells to get rid of the leak currents that both of you have rightly pointed might be interfering with my recordings?

Thanks!

Dear Sourajit,

My recommendation was for you to use non-transfected cells to check that the current that you would say is the exchange current, is absent in this preparation. If you have similar currents in non-transfected cells, then the exchange current will be challenging to retrieve from your recordings without some specific blockers.

I do not recommend to subtract the response from untransfected cells, unless you can prove that the only difference comes from your exchanger. Transfected cells with a inactive exchanger may be better.

If you have significant leak current, you may try to block all potassium currents using cesium in your intracellular solution to eliminate most of your leak currents. But that assumes that your exchanger is not sensitive to cesium. Similarly, this block can be first checked in your control condition to see if any currents remain.

Question: is 0 mV in your ramp 0 mV or does your amplifier make the ramp relative to a constant voltage-clamp potential?

Good luck!

Dear Martijn Sierksma

Thank you again for such an elaborate reply. I am currently trying to check the current level in the non-transfected cells to have an idea about the leak currents present in the HeLa cells which might be functioning (if at all) in the range of -100 to +100mV.

Your suggestion for the use of blocker of exchanger is very well noted. But, in literature I have not found a specific blocker for my exchanger. However, use of Caesium might be one of the option that I can try for sure (hoping it doesn't affect the exchanger as it is not supposed to transport K+).

I am not sure if I understand your question correct but I am using a protocol where the holding potential is 0mV and then the ramp goes from -100mV to +100mV. I do not apply any holding potential/constant voltage-clamp potential from the amplifier. I am attaching the protocol I am using for my experiment.

Thank you for your time Martijn!

Dear Sourajit,

Sorry I missed the last posting! I agree with Martijn Sierksma . I wouldn't try to simply subtract currents, it's not going to be good method. What is the exchanger you're studying (if you may share this information)?

Dear Andre Dagostin

Thanks for your response. I agree too with you and Martijn Sierksma

How about getting the whole-cell output from the cell line which has a knockout of the exchanger I am studying (we already have the knock out) and compare that with the exchanger over expressed cell line (same cell line)

I am studying a calcium/proton exchanger.

Sourajit Mukherjee , this is doable, however, you'll need a good amount of cells to make an average and see something - considering it's there. Also, different cells may have the protein knocked-out in different levels (I assume there could still be some residual expression in there).

I saw an old paper (Proton fluxes associated with the Ca pump in human red blood cells) where they used DIDS (100µM) to reduce the Ca-H transporter activity. I don't know your cell line, but assuming it does not have chloride currents, this is something worth trying.

BTW, this Ca-H is being knocked out from a cell line, right? Any chance you could overexpress it in HEK/CHO or something to be able to, at least, figure out some good/decent blockers?

Andre Dagostin Yes you got it right. The exchanger is knocked out from HeLa cells and the data that I had posted was from the over expressed HeLa cells.

The difference in reversal potential for a particular set of external and internal conditions among the KO vs over expressed cells are 6-8mV only and which is far off of the theoretical reversal potentials.

Dear Sourajit Mukherjee,

If you run your protocols on HeLa cells that do not express your exchanger of interest, which currents do you record? Try to eliminate these currents as much as possible using blockers or perhaps consider a different cell line with less leak currents.

If you then run your protocols on HeLa cells that are not knocked out for your exchanger, can you record the exchanger current? This may be too small, but that would argue that you need overexpression. If it is not too small, then it may be better to avoid overexpression altogether.

Then, try the overexpression, but you have to be careful that the overexpression is not inducing other, compensatory changes to the cell physiology.

If you now record some current (not too large, say