Hi :)
Im trying to start obtain on Single channel data on Potassium ion channels. So far I have worked with whole cell condition and I have no experience with single channel experiments.
There is a part of single channel recording protocols which I do not understand.
Usually when I record whole cell current from Slo1 (BK) Potassium channels I give step pulses from -100 to +200mV and I can see the channels activate and produce robust outward currents beyond 0mv (positive command potentials). This I can understand as the channels are activating when the voltage across the cell membrane is positive which is beyond the activation Pentecostal BK1 channels.
However, in inside out single channel recordings, Since the inside of membrane is now outside the patch pipette, I assumed I should give a negative holding/command potential to activate channel. (the reason is unlike the whole cell configuration, now the Potassium current has to come in to the pipette and the voltage drop across the membrane has to be positive.)
However, the researches are still using positive holding potentials to record single channel potassium currents. (Image attached from a published paper)
Can I know the reason for still using positive potentials to record the single channel current?
The figure I attached are from an already published paper (http://www.jbc.org/cgi/doi/10.1074/jbc.M117.776013) and I'm not in anyway questioning the results in the paper. I'm just using the figures to elaborate my question.
Thank you all :)
Dear Tharaka Darshana Wijerathne : The voltage level designed in most of patch amplifier is “upside down”. As the paper showed the single-channel activity in positive potential, the experiments should be tested in the “negative” potential. Indeed, that really confused some of researchers.
Than you so much Professor Sheng-Nan Wu. I tested the protocol as you said (cell attached single channel mode) and it worked.
In my opinion, the published data is absolutely correct. The attached figure can help you understand why they were able to obtain single outward conductance at +60 mV.
Hi Tharaka,
not to confuse you further (hopefully). You are right when you say that in cell attached the voltage is reversed so, for instance, if you want to see a voltage-dependent current that in whole cell is elicited by a positive voltage, THEN in cell attached you must REVERSE the sign of the voltage by amplifier, irrespective of the solutions used. If the channel is strictly voltage -dependent, in cell-attached configuration you wont be able to see it if you use the voltage employed in the whole cell. So electrophysiologists have two options when designing a protocol by instrument: 1) you either design a protocol with ALL opposite signs to your correspondent whole-cell and then when you analyze your recordings you also have to invert your current traces (with Clampfit there is a special command in the analysis window) or 2) you copy and paste the protocol used with whole cell but in your amplifier you must invert the SIGN of BOTH your Voltage command and Current Input by putting a - (minus) in front of them. This way you will see what you are supposed to see real time because everything is reversed by instrument. In the paper you have shown, they either assume electrophysiologists would know or they must have specified it in the methods section what they did.
Best
Francesca
Hi Xuexin
Thank you so much for the elaborated reply.
What i don't understand is, how does a BK channel send current in to a cell as you have shown in the top right figure. When we make a patch as you have shown and when we provide +60mV command voltage, the BK channel has to send current from outside to inside (Potassium from pipette to bath). That current flow is in the opposite direction of the regular BK channel current flow (from inside to out side). Furthermore, the BK channels are activated at positive membrane potentials. So how does the BK channels activate as illustrated in the top right figure? The real voltage difference that the channel(s) feeling at that configuration is -60 (because the inside of the pipette is +60mV and outside of the pipette is 0mV thus making the net voltage across the ion channel's voltage sensor being -60mV). I may be missing some basic concepts here. I'll be thankful if you explain where I have gone wrong.
I know the data in the original paper is not wrong, I'm just trying to understand where I have gone wrong in the process of understanding.
Hi Francesca Fieni
Thank you so much for taking your time to explain the issue in detail to me. Yes, I understand what you said and the basis of the data in the original paper.
Thank you again
Indeed, some of previous papers have already confused the experimental results regarding the activity of K(+) channels with either "inward" or "outward" rectification.
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