Try 1mM 8-Bromo-cAMP or dibuteryl-cAMP (more potent). You can add them
via the bath as they will easily permeate the membrane. PKA activity
should increase within a few minutes. Walsh peptide is the standard PKA
inhibitor. This can be added via the pipet. Look at a paper by Pusch
and Neher for estimating the timecourse of diffusion into the cell. I
can dig out the reference if you need it.
On Sat, 1 Jul 2000, Zakaria
> Hi everybody
>>> I am trying to augment cAMP dependent protein kinase (PKA) activity in a
> neuron, in order to reveal it's impact on certain ionotrophic events in
> an acutely isolated dentate granule cell, during voltage clamp
> recording. For this purpose I think to include PKA catalytic subunit
> into my recording pipette. however, I don't know what should be right
> (closed to physiologically relevant) concentration of the protein. media
> etc. Also, I need to inhibit PKA. Which inhibitors and what
> concentrations are recommended?
> I will highly appreciate if someone will share his/her experience and
> suggestions on this matter.
Gary G Wilson
Center for Molecular Recognition