I am new to whole cell patch clamp recordings. In several manuscripts, I noticed several experiments are done with a K+-gluconate solution within the recording electrode, while for other experiments within the recording electrode is a solution with a Ce-SO3 or CeCl solution.
Can someone explain the purposes of these solutions?
K-gluconate would hyperpolarize the membrane, while CsCl would also hyperpolarize the membrane. In each case, due to the molecular nature the ions would not be able to be transported out of the cell.
I am having trouble conceptualizing this phenomena.
Any clarity helps.
Thanks all
Solutions with Gluconate (or other anions larger than Cl-) are used to maintain the osmolarity of the intracellular solution whilst avoiding to have high Cl- inside the cell (physiologically, intracellular Cl- is much lower than extracellular Cl-).
If using KCl or CsCl-based solutions, the Cl- concentrations in/out of the cell are almost the same, Cl- mediated currents such as those carried by GABA(A) receptor channels reverse at 0 mv which is unnatural. But this can be advantageous for recording large GABA(A) currents clamping at -70 mV (which keeps the cells "happier") whilst blocking everything else.
Cs+ solutions are used for voltage clamp recordings: to avoid contribution from K+ channels and allow better voltage clamp. Cs+ does flow through cation permeable channels such as AMPARs and NMDARs so these currents can be nicely recorded with Cs-based.
One thing to note is that when using Gluconate there will be a junction potential between recording pipette and extracellular solution, for most physiological solutions this is approx 10mV. As a rule of tumb if using gluconate based intracellular, the membrane potential will be about -10mV lower than what is measured. Eg. if clamping at 0mV, one is clamping at -10mV. This can also be measured experimentally .
Mariana Vargas-Caballero Thank you for your time and the clarity. This was very helpful.
I think that the main reason to use CsCl or CsSO3 or also Cs-Gluconate in normal whole cell recordings is simply to abolish the K+ inward current. For the voltage gated currents involved in spike generation, for example, the K+ repolarizing current starts before the inward Na+ current is arrived at peak. If K+ current is not subtracted by the total current, the real peak of Na+ cannot be correctly measured. Using Cs there is no K+ current and this help to have a precise evaluation of the Na+ peak current. In general Cs is used any time it is convenient to ablish the K+ currents. Of course this does not hold for some receptors where a current of Cs similar to that produced by K+ mimics the K+ components because these receptors are permeables to Cs as to K...
A further note: make sure that the pipette solution contains ALWAYS a certain amount of Cl-, even if the main anion is SO3 or gluconate. The Ag/AgCl wire inside the patch pipette is stable if bathed by a solution of Cl-. Usually Cl- from other compounds occurring in the pipette solution, such as CaCl2 or MgCl2 should do it.
Albertino
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