I'm patch-clamp recording from neurons that strongly rectify in response to depolarizing current injections. I have tried to eliminate this by using a cesium-based internal. Recent recordings have shown that cesium has had no effect on the rectification.
What possible reasons for this are there? Any suggestions on how to circumvent this problem?
Cesium blockade is due to cesium trying to enter the K+ channel, but unable to go past, because of its size. Therefore, if cesium was present only in the internal solution, it will enter the channel only from inside under conditions where an outward K+ current is expected. When conditions favour inward rectification, K+ in external solution can still find entry. The problem can be dealt with by adding some cesium (say 5 mM) to the external solution as well (or probably by keeping the external solution potassium-free).
Current through inwardly-rectifying K+ channels is highly sensitive to barium and cesium (but much less to TEA). Typically, a 1-3 mM BaCl2 solution is sufficient to block all Kir current and generate control currents, which are subsequently subtracted from the actual Kir recording, if that is what you're interested in. The best concentration for BaCl2 varies from Kir channel to Kir channel and has to be established via a dose-response curve. The dihydrate salt of BaCl2 can simply be added to the regular bath solution.
Note: Barium block of Kir channels is voltage-dependent. It works best at negative potentials, e.g., -100 mV. The remaining step potentials have to be extrapolated from this value. Barium block is also time-dependent, in other words, steady-state must be achieved to ensure complete efficiency of the blocker. Barium block is reversible, i.e., after washout the current should reappear. This can be difficult at times, which is why only one cell of a barium-exposed bath should be analyzed. You can use CsCl in a similar fashion; it is faster, but less effective.
One more thing regarding barium: Make sure not to have any sulfate in your solution, as this would cause BaSO4 to precipitate (i.e., no Ba2+ available for blockade). For complete Ba2+ blockade of your Kir currents, you'll have to go fairly high in concentration. This is based on experience with heterologous expression of Kir4.1 and Kir4.1/5.1 a couple years ago. Block was evident at 300 uM BaCl2, but not complete until almost 10fold that concentration, which is, likely, experimentally prohibitive in your situation, since high Ba2+ is also known to block Kv channels.
Hi ! I'm a PhD Student (first year) in a lab working as an electrophysiologist in the cerebellum. I would like to block especially Ki4.1 on one cell patched in situ. I have one question: in a patch-clamp situation, how could I be sure to "block" Kir4.1 with cesium inside the electrode? Do we know anything more in good blockers of these kinds of channels? Thank you :)
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