Hi
I'm trying to calculate the Monovalent cation permeability of TRPC6 channels. I did the experiments and got the results.
However, the problem is the values I get for NMDG. NMDG gets about 0.1 permiablity relative to Cs (Cesium and Sodium has similar permiabilities). I expected very low permeability for NMDG than the value I got.
I think reversal potential is the reason for this value. I get around +8mV for reversal of Sodium and about -50mV for the reversal potential for NMDG.
I used the calculation PX/PNa = exp(VrevF/RT), for the calculation of permeability. Since I used 3M KCl agar reference electrode, the junction potential all the external solutions (including the NMDG solution) is around 15mV. (Vm=Vp-15.0mV).
I guess the current is due to a leak current or an offset in the acquisition. system. I've seen in published papers that the reversal potential for NMDG is around -100mV.
The permeability value I get for the NMDG is causing problems when I try to calculate pore size of the ion channel.
Is there a way for me to refine my results? Can I improve my calculations?
I've attached the IV curves I got for NMDG and Sodium from two cells. I've attached one continuous trace too. The NMDG Bath solution has only 140mM NMDG, 10mM HEPES and 0.5mM EGTA (pH balanced with HCl). The ion channel I'm testing is a non selective cation channel (TRPC6)
Thank you :)
Any possibility of monovalent contamination in your external?
Have you tried Tris+ instead of NMDG+?
What do the currents look like in control cells that lack your channel?
Hi Tharaka,
I'm not sure what solutions you use, but your sodium reversal potential sounds like it's off. Use the Nernst equation and your total inside/outside Na concentration to determine its theoretical value (ENa). It should be significantly more positive if you're using near-physiological pipette/bath solutions. How did you determine ENa? Maybe I'm misunderstanding what you did...
You'll want a very tight seal to conduct these experiments.
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