I am trying to perform whole-cell patch clamp on HeLa cells over expressed with a calcium/proton exchanger. I am failing to understand why there is no shift in the reversal potential even if I change the concentration of either Ca/H+ on either side (pipette/bath). I have tried this experiment for a particular combination of external and internal buffer in the HeLa cells knock-out of the exchanger under study. This was done just to compare the contribution of endogenous effect of the exchanger (if at all present).
I am using NMDG-MSA buffer for my experiments as I don't want interference from any other cation or even chloride current.
However, the problem starts here:
Even if I vary any one concentration of the ions involved (Ca/H+) in the transport process across the pipette and the bath, there should be a shift in the reversal potential which I could calculate theoretically. The reversal potential doesn't change significantly even if I keep a very steep ionic gradient.
I have been trying to figure this out for a long time now! Unfortunately, nothing is working for me. Could you guys show some light?
Thanks lot in advance!