Hi Bill,
Correcting for liquid junction potentials when doing perforated
patches can be a rather tricky situation. First, you have the liquid
junction potential between the patch pipette and the batch solution,
which was 'zeroed' when the patch pipette was first placed in the
bath. Second, there is the junction potential across the cell
membrane permeabilized with gramacidin. This junction potential will
be determined by the activities of the permeable ions on either side
of the membrane and the permeability of the membrane with the
gramacidin. Thus, you will need to find out the permeability of
gramacidin - I guess that this information is in the literature, but
I confess that I haven't looked. You will also need to make the
assumption that the gramacidin conductance in the membrane is far
greater than any other conductance that is present in the membrane
under the pipette tip, and so dominates the junction potential; this
is a fairly reasonable assumption. Depending on the composition of
your pipette solution and the permeability of gramacidin, it may be
necessary to also take account of the Donnan potential across the
membrane under the pipette.
For whole-cell recordings when the cell contents is dialysed with the
pipette solution, the correction for the liquid junction potential is simply:
Vm = Vp - LJP
where the LJP is what was cancelled when the pipette offset was
'zeroed'. The LJP is best calculated using the Junction Potential
Calculator in Clampex. Vm is the membrane potential and Vp is the
command potential at the pipette.
With the perforated patch, the correction is:
Vm = (Vp + Vpf) - LJP
where the Vpf is the potential you will need to calculate for the
perforated patch potential due to gramacidin in the patch.
If you look at the Junction Potential Calculator, there is some
(small) online help with calculating perforated patch LJPs.
If you want to discuss this further, happy to help out off-line.
Regards,
Trevor
>Message: 1
>Date: Sat, 4 Oct 2008 14:00:28 -0700 (PDT)
>From: Bill <connelly.bill from gmail.com>
>Subject: [Neuroscience] Gramicidin Perforated Patches and liquid
> function potentials
>To: neur-sci from net.bio.net>Message-ID:
> <a1e0d20f-2b76-4384-9808-0681c6da2fae from o40g2000prn.googlegroups.com>
>Content-Type: text/plain; charset=ISO-8859-1
>>Hi,
>>I may have already posted this, but it isn't showing up on google
>groups, so I'm thinking there may have been a problem.
>>To get to the point I finally have got gramicidin perforated patching
>working regularly (~30mOhms in 15 minutes) however all my cells appear
>to be "resting" ~20mV more hyperpolarized than they do when I do whole-
>cell recording using either a K-gluconate or KCl based pipette
>solution. Does this make sense to anyone?
>>I presume this is something to do with the fact that during whole-cell
>recordings the cell interior eventually becomes the same as your
>pipette solution and hence there is no liquid junction potential (so
>the added voltage which was 'bucked' out of the system when the
>electrode was zeroed and the LJP was in force, and is added to your
>recorded membrane potential)? However in perforated patch mode, you
>have a difference situation, where different ions have different
>mobilities, not only based on their mobilities in solution, but their
>conductivity through the gramicidin pores? Or am I over analysing
>stuff here?
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