Hi everyone!

I have trying to figure out how to determine if the number of functional Cav2.3 channels is different under my test conditions.

These channels are stably expressed in HEK293 cells.

One of our ideas was to perform non-stationary fluctuation analysis of our voltage-step evoked currents. I was surprised to see that basically there is virtually no examples of this kind of analysis of calcium channels in the literature. Is this so difficult to do?

The main questions I have are:

1) As expected, my calcium currents have run down, which varies among the different cells and impedes a proper analysis of noise variance, becuase the variance of the run down effect is way larger.

2) I attached an example of 21 consecutive sweeps (test.abf) that I would use for this kind of analysis. Each sweep was evoked every 4 seconds using a 500 ms voltage step from -80 to 10 mV. Is this a suitable protocol?

3) Normally I apply P/5 online protocols for leak substration (the one served by Clampex). Can I used these corrected traces for the noise analysis? (I guess not).

4) If you see my traces, in which part of the curve would you analyse the noise? e.g. from the beggining to end of the step? from the peak to the end of the step?

5) In pClamp 10, there are two tools to perform variance analysis: "Nonstationary Fluctuation" and "V-M Analysis". Can I use any of this? I tried the Nonstationary Fluctuation tool with the sweeps I attached here (Figure 2), but as you can see, the results don´t look logical (Figure 2) (from peak to end of step).

I know, these are lots of questions. I would appreciate some input.

Thanks a lot for reading!

Diego Fernández