...about capacitance

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Hss
Hss's picture
...about capacitance

Hi

...of what depends the capacitance of one cell during one recording? I´m doing wholle cell patch for recorgind Ca++ currents in HEK cells, and in some cases the capacitance is about 15pF and in some cases is about 35pF (in the same culture), and the size of the cells in both cases is similar, and the pipette resistence is similar too, so I don´t understand why so much difference in the capacitance.

Thanks.

Fraser Moss
Fraser Moss's picture
The physical dimensions of

The physical dimensions of the cell membrane are the most important factor in determining the whole cell capacitance (Cm): the more membrane surface area, the more charge can build up and therefore the capacitance is directly proportional to the cell surface area. Another factor that can contribute to the membrane capacitance is membrane thickness, but in the case of phospholipid bilayers of HEK cells these are pretty uniform and so are not normally considered an experimental variable. The final variable that can affect the capacitance is the composition of the intracellular and extracellular environments, but assuming your solutions are remaining constant through your experiments this too is not a large variable.

The above facts about Cm measurement assume that Cm is constant for a given cell, and that membrane resistance (Rm) is fairly constant over membrane potential (Vm) changes, at least in the voltage range of application of the protocol used to measure whole cell currents. However, Rm can change around the resting potential (Vr) and there is a second assumption that these changes are negligible with respect to Cm estimate. HEK cells express endogenous rectifying potassium channels to varying degrees depending on the source of the original HEK line that your lab obtained. Furthermore, varying expression levels of the heterologously expressed ion channels may also affect the Vr. It is possible that Rm voltage-changes around Vr are not negligible when standard voltage clamp protocols are used to calculate Cm.

Therefore, cells of apparently the same size can have different measured Cm due to the variable expression of voltage dependent channels in the membrane which can set significantly different Vr in each cell. Thus during a given voltage step at which the cell parameters are being measured the holding voltage (Vh) deflection from Vr could be many mV different between each cells and cause a significant difference in measured Cm. Typically, the more hyperpolarized the Vh from Vr the smaller is the measured Cm.

That said the magnitude of the differences in Cm between your cells is huge and I suggest that you should check the cells’ health. i.e. do they have a negative Vr (measured when I=0) when you go whole cell (a healthy nontransfected HEK has a Vr around -15mV)? Dead or leaky cells with 0mV or even positive Vr may give much smaller measured Cm than you would expect if you are measuring Cm at -60mV or -80mV.

Hss
Hss's picture
Thanks, there are good points

Thanks, there are good points; now I can test if the cells are healthy enought and considerer differents parameters for changes in the capacitance.