Struggling to get consistent reversal potentials with gluconate

1 post / 0 new
aoirsae's picture
Struggling to get consistent reversal potentials with gluconate

 Hi all,
For the past few weeks I have been struggling to record consistent reversal potentials using axopatch 200b / Clampex software and HEK cells expressing P2X receptors.  I can't figure out if my Erevs are being influenced by me or whether I should expect a... dynamic range from my recordings.  

Generally speaking, I run a protocol clamped at 0 mV in reciprocal NaCl-based external / internal solutions to tweak my pipette offset and baseline any current.  I then perfuse a gluconate solution, wait several seconds before swapping into gluconate + ATP, and ramp from -40 to +40 mV over 4 seconds.  When I do this my Erev's vary anywhere from -15 mV to +15 mV, even though I expect my reversal potential to always be at 0 mV (... at least for a wild-type channel...).  I have noticed that my baseline / pipette offset jumps up or down slightly (~5 pA) when I swap into my gluconate buffer compared to the NaCl-based solutions, so I've begun to adjust my pipette offset based on that.  However, this still gives me the same amount of variance in my recordings.  I have also started to outside-out patch to try and reduce cell to cell variability,but I still get bouncey Erevs.  Sometimes after running my ramp protocol I notice that my baseline is not at zero anymore, in which case I tweak my pipette offset again and re-ramp - which occasionally helps but doesn't always fix my variance.  And, to be honest, I'm scared of playing with the offset knob too much in fear of influencing my recordings.

I plan on trying shorter (1 sec) ramps from -10 to +10 mV to try reducing ion build-up in my pipette.  But, beyond that, I am lost as to why I can't get consistent reversal potentials from patch to patch.  I'm curious if anyone has thoughts or advice?  Any help would be much appreciated!