Hello there,
We use cell-attached patch clamp for BK channel activity in Vascular smooth muscle cells. Unfortunately 99% of the time I don't see any channel activity even at high command voltage (+70mV). Maybe 1 or 2 cells will have good channel opening out of 100. Not even the Kv channels will open.
I keep the pH at 7.35-7.4 for bath solution and 7.2-7.3 for pipette solution. Solutions are made every week. The cells look very clear and healthy. My pipette tips have resistance around 6 mOhm when it's in the bath solution.
We use either a physiological bath solution or a high potassium, low calcium solution without any Sodium.
Can someone who have some experience in this kindly share your opinion? Is it normal?
Bk channels display an outward rectifying property. One should hold at +70 mV.
Hi Dr. Wu, So this was the cell attached patch clamp so -70mV that i mentioned, is actually +70mV I should have specified. Not even any Kv channel will open
Dr. Lis, I have tried 3mgaohm pipettes but no luck.
During getting a seal, can suctioning a bit too hard destroy the channel? The cells do look good after i get a seal.
Thank you both for your answers.
Its frustrating to have mainly empty patches. Our lab uses stably transfected cells and we find it useful to do whole cell recordings to select cell lines that have currents between 150-300 pA prior to doing cell attached patches.
Dear Linda M Nowak: Do you have idea of how we can purchase or acquire such stably transfected cells?
We make them in our lab. I would give them to you, but we study glutamate receptors, not K+ channels.
Hi Sayeman, try to switch in excised patch-clamp mode with patch pipette electrode near 7 Mohms ...maybe the noise is masking your signals.
Good luck
Marcello
1 - BK conductance? about 100 to 200 pS? is that correct? that means that even if you have a 5 pA noise, you will still see your BK activity when it opens. I don't think you have that much noise! do you?
2 - Linda works on Glutamate channels (stably transfected cells). I can tell you from experience, it's heaven for an electrophysiologist. In fact, the only problem with that system is that you get too many channels when you patch. So, you pull high resistance pipettes (10 MOhms or more) to try get as fewer channels as you can (The dream is to get one single channel of course).
3 -I don't know why you can't get a higher success getting you BK channel in Cell-Attached. How do you get your cells? Enzymatic digestion? If it is, I can tell you from experience as well, that working with Detrusor muscle cells (smooth muscle cells from mice bladder) and trying to find HCN channel (in Whole-cell mode) is really difficult. Maybe smooth muscle cells are sensitive to the enzymatic cocktail we use(???). By the time, we patch them, the channel density tend to go down. In my case, I did see HCN a couple of times but only when I had cyclic nucleotides (cGMP or cAMP) in the pipette. So, maybe you need to activate your channel. BK channels are activated by high Calcium, so why don't you try to patch your cells, go inside out and wash in high Ca solution to try activate you BK channel even if you don't see it in Cell-attached. I can't think of anything else for now.
Hi Fouad, thank you for your detailed thoughtful answer.
We use Papain+DTT and then Collagenase+Elastase+trypsin inhibitor to isolate the VSMCs from mesenteric arteries.
Indeed the channel opening is higher when we try a higher calcium solution but even then the number of cells that have proper channel opening is very low. For most of them it seems the patch is completely devoid of any channels, no matter what holding voltage/ amount of calcium we use.
Why don't you try recording BK in whole cell first to see if every cell in your prep even has BK currents. And see how large they are. If the majority of cells don't show substantial macro BK current then one would not expect to see single channel activity either.
One may test the activity of large-conductance calcium-activated potassium channels in endocrine cells like pituitary tumor cells. ( https://www.x-mol.com/paper/1293623328478208000)
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