I noticed that the resting membrane potential varies from different studies. I am wondering what's the best way to measure it.
With sharp electrode and whole cell recording, I am think that the concentration difference between the cytoplasmic and pipette solution may change the equilibrium potentials of the ions. Since we don't know the exact concentration of the ions, especially in some neurological conditions. Should we measure the resting potential as soon as the pipette break into the cell before the exchange happens between the two solution?
Will perforated patch clamp be the best approach to get the accurate value of the resting potentials?
Please share your opinion, thanks!
Jianli
Hi Jianli,
We usually get our main readings of RMP using current clamp right after breaking into the cell. This works quite nicely as long as you have a seal resistance several times higher than your input resistance (really only a problem for small cells, see Wilson et al Channels 2011...).
However, some people routinely determine RMP by looking at channel noise during a series of pulses in cell attached mode. I suspect that this method may be more resilient to changes in internal ionic composition.
Dear Jianli,
In my opinion, intracellular recording with sharp electrodes filled with potassium salts such as methylsulphanate (Resistances ~80-100 megaOhms) give the most reliable readings. Firstly, the leakage of pipette solutions is restricted due to high tip resistance, secondly the membrane damage is limited due to small tip size.
If you have to use patch-clamp, then I recommend to fill your electrodes with potassium methylsulphanate based pipette solution (see my publications below for details). Also breaking the membrane with voltage pulses provides smooth/gentle breaking of the plasma membrane by electroporation compared to suction and breaking of large piece of the membrane.
In either case, the resting membrane potential should be measured immediately or within ~30s of impailment or breaking in. Also include cells that have stable baselines and require no current injection to stabilize.
Best wishes,
Refik
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In patch-clamp recording, there’s no way to have an accurate measurement of resting potential unless the resistance of the cell membrane is much smaller than the resistance of the seal. Although the seal resistance is usually quite large (Gigaohms), in small neurons the cell membrane resistance could be also very large. In voltage-clamp mode, the membrane voltage at which the total current goes to zero (called zero-current potential, V0) is often used as an estimation of the resting potential. This is a big mistake: if the seal resistance and the cell membrane are both, say, 2 Gigaohms, then what you measure is much smaller than the true resting potential, due to charge leakage through the seal resistance.
Also, uncompensated liquid junction potentials represent a source of errors.
I suggest you to read the inspiring paper by Barry PH and Lynch JW “Liquid junction potentials and small cell effects in patch-clamp analysis” J Membr Biol. 1991 Apr;121(2):101-17.
As to intracellular recording with sharp microelectrodes, you might have, in addition to liquidi junction potential, unwanted “Tip potentials” that can be totally unpredictable. You can find more information on the possible drawbacks in measuring “true” resting potential with intracellula recording approach in this very nice book: Purves RD “Microelectrode methods for intracellular recording and ionophoresis” Academic Press 1981.
So, is it possible to measure the “true” resting potential of a neuron? My answer is no, but you can get close to it.
Thanks, Dr. Bigiani. I guess, all we can do is to get the closest and be consistent. I will try to get Barry PH's paper .
Check this https://www.physiology.org/doi/abs/10.1152/jn.1992.67.3.508?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed
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