I have recently started using patch clamp to measure BKca channel current in neurons (whole brain culture) and in mesenteric vascular smooth muscle cells.
But after getiing a gigaohm patch in voltage clamp method, when i switch to recording mode, I don't see any channel current. Instead I am seeing big spikes of about 2000pa height in the recording.
I am quite new to this, so if someone could kindly share their opinion on this, i would really appreciate it.
I have attached the recording here.
Dear Sayeman Isalam Nilogy: Are the spikes you saw during the recordings downward or upward deflection? Current amplitude measured at “2000 pA = 2 nA” appears to be very large and it should be voltage-gated sodium currents or emergency of action currents! Note that single channel currents, even BKca channels, are hard to reach such high current amplitude. One may distinguish cell-attached from whole-cell configuration during the recordings.
Looking at your traces. 1) Firstly your BK single channel currents are likely to be of the order of 10 pA so the traces you show are far too low resolution to see that. 2) The holding current is positive by +500 pA which means that you definitely don't have gigohm seals. These big 'spikes' are completely saturating (note there is no longer any 'noise' at the peak of each of these steps. Id start by checking the whole system without real cells.
First step Id suggest is to check your setup with a model cell (Are you in clamp mode or 'search mode? (what amplifier are you using)? Do you have a model cel? Use the model cell to check that there are no problems with the set up. Bias currents poorly adjusted baseline etc. (always do this if you 'inherit' a set up or when you are setting up for the first time). This will also allow you to increase the gain of the amplifier so you can resolve 10 pA steps accurately (there should be no 50 or 60 cycle noise visible)
Second step if all above is good. Check that with a pipette in the bath and in clamp mode that the current through the tip is Zero when the holding potential is zero. Your pipette resistance for BKs to be used in cell attached should be a bit higher than 5 meg in my experience. Im presuming your pipette solution is the same as the external solution.
Bring the pipette gently to the surface of the cell and gently suck onto the membrnae without rupturing it. Hold until a gigaohme seal forms. you need at least 4-5 GH and almost no leak current. Do not change the HP until the seal looks good. Now step the Holding potential to -65 and observe currents (hopefully) with a gain that gives a full scale of 100 pA or better less.
By the way, if the cell was held at -65 mV, the BKca channel activity would be expected to be low.
Yes Sheng-Nan Wu you are right. But I think if he is in cell -attached mode (not clear from the question) he will have to assume the RMP to eb around -65 mV. If however he polarises the pipette potential to -56 without rupturing the patch the transmebrane potential will be around 0 mV in which case he should see channel activity.
For measuring BKCa channel activity, apart from the single-channel conductance, one need to know membrane polarization and channel rectification property.
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude