How do I maintain desired potassium concentration in patch pipet solution when adjusting pH with KOH?

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zang
zang's picture
How do I maintain desired potassium concentration in patch pipet solution when adjusting pH with KOH?

When I prepare 100 mL of the pipet solution (K-gluconate or KCl : 130 mM), I have to add some mL of 1N KOH to adjust pH (7.2).
I think this will increase the K concentration of pipet solution. Comparing with carefully measured amount of chemicals for the solution preparation, I am not sure if I can ignore this additional KOH amount. As my feeling, most of papers seem to ignore the amount of KOH when mentioning the intra-pipet K concentration.
What should I consider about K concentration when I check the K reversal potential?
Thanks.

JennaMo
JennaMo's picture
If you are recording K

If you are recording K currents you might not want to change the K concentration by pHing with KOH as it will change the reversal potential of your current, like you said. You could use NaOH instead. If you need to use KOH you can calculate how much it will change the concentration of K in your solution and calculate the new reversal potnetial.
Hope that helps a bit!

The FFM
The FFM's picture
Sorry JennaMo but using NaOH

Sorry JennaMo but using NaOH is NOT a good idea and here is why:

Think about it in terms of what is within the bounds of the measurable error of your experiment.

The reason that labs do not report changes in overall potassium concentration when pH ing with KOH is that the impact on EK (reversal potential for K+) is going to be negiligible in most cases.

Consider your solution.  It contains 130mM KCl.  If you have to add 0.5 ml of 1M KOH to your 100 ml intracellular solution to adjust the pH to 7.2  then your final intracellular K+ concentration is going to be 135 mM.

Potassium is the predominant ion inside the cell and it is only about 2 mM outside the cell. Therefore a 5 mM shift in [K+]in is only going to produce about a 1mV change in the EK. 

EK= (RT/F) ln ([K+]out/[K+]in)

So at room temp consider the two possible intracellular potassium concentrations and their effect on EK

(RT/F) ln ([2 mM]out/[130 mM]in) = -106 mV

(RT/F) ln ([2 mM]out/[135 mM]in) = -107 mV

Plus or  minus 1 mV is almost certainly going to be within the bounds of the error of your measurements and for that reason most labs do not sweat reporting the effects of adjusting the intracellular solution pH with KOH

NOW  consider JennaMo's suggestion 

Na+ concentration inside the cell is ten times lower than outside the cell.

So if you were to adjust the pH of your same solution using 0.5 ml NaOH, then there is going to be a much bigger effect. e.g.

ENa = (RT/F) ln ([98 mM]out/[9.8 mM]in) =  +58 mV

But if you add 5mM of NaOH then ENa is going to shift 10 mV more negative 

ENa = (RT/F) ln ([98 mM]out/[14.8 mM]in) =  +48 mV

This may significantly impact the measured properties of the channel from which you are recording.

acsm
acsm's picture
Replace 10mM KCl from your

Replace 10mM KCl from your pipette solution for KOH. The solution will be alkaline and you will need to add HCl to pH your solution.
KOH + HCl ==> KCl + H2O

You will keep your K+ concentration and get to pH your solution.
You can do the same with K-gluconate, unless you are replacing the whole chloride from your intracellular solution, so, try another acid.

JennaMo
JennaMo's picture
Yes, I suppose it depends on

Yes, I suppose it depends on what you are measuring. I was coming at it from thinking about recording pure currents- like isolating a native K current or recording from a HEK cell over-expressing a K channel. For my experiments Na concentration is not important to the biophysical properties of the K channels which I am studying. Of course if you are measuring action potentials or synaptic currents, or even recording Na-dependent K currents, changing Na concentration is going to matter.