Hi
I'm testing an effect of a drug on voltage gated potassium channel Slo3. I can see a clear change in current response to Step Pulses (shown in attached image). The voltage step protocol that I used for the channel activation is shown in middle.
I'm trying to calculate weather the time taken for current to saturate at each step pulse changes with drug application. Therefore, I believe I have to calculate the time Constant (tau value) at each step pulse. So I want to plot a graph with Step pulse Voltage (mV) as X axis and Tau value (ms) as Y axis.
I may be able to calculate all the tau values manually, but it will be a very time consuming process.
I have two questions
I have a background in biology therefore and my Mathematics knowledge is bit blunt.
Any advice would be highly appreciated
S.O.S
Thank You :)
Hi,
You can calculate the tau values by using clampfit software. It is specialised for electrophysiological recordings. In that software you have to put cursers at the beginning and end of the region of the trace for which you want to calculate the tau. It will give you the values of tau for all the voltage steps in a tabular form.
Dear Swagata Ghatak
Thank you so much. It worked really well.
The calculations can be done via Clampex & Originlab (I found out these later and I'm editing the original post)
1. From Originlab
Download the Simple pCLAMP Analyzer extension from Originlab to your existing origin software. It will do the trick
http://www.originlab.com/fileExchange/details.aspx?fid=315
File Exchange > DataAnalysis > Simple pCLAMP Analyzer
Unfortunately, this app works only for Origin 2016 Sr1
2. Using Clampex
I use Clampfit daily to view my PClamp data. Never thought about using it to analyse Data as I try to use Origin software for all my analysis.
Thank you again :)
For anyone who want more information. I'll put what I found, below.
You must first set up the fitting function, then perform the fit.
For more details on the significance of the fitting options, consult the Clampfit Curve Fitting chapter in the pCLAMP manual (Chapter 11 in the pCLAMP 9 and 10 manuals).
Press OK to complete the fit.
The fit results will appear in the Clampfit Results window under the Fit Params tab.
A1 and tau1 represent the amplitude and time constant of the first exponential component.
A2 and tau2 represent the amplitude and time constant of the second exponential component.
C represents the y-axis offset
Source http://mdc.custhelp.com/app/answers/detail/a_id/17217/~/obtaining-the-time-constants-tau-from-fits
http://mdc.custhelp.com/app/answers/detail/a_id/17217/~/obtaining-the-time-constants-tau-from-fits
http://www.originlab.com/fileExchange/details.aspx?fid=315
You have to be very careful with the data that Clampfit calculate. Specially in the Tau value. Sometimes it doesn't fit well.
It's evident that the Tau changes after the addition of the compound. You also need to test if the addition of the drug doesn't have an indirect effect. As Slo3 channels are activated by intracelullar Ca2+ and pH, you should test if this compound is not increasing, at least, these two parameters.
Best,
Oscar
Thank you Oscar
Yes indeed, the Tau value behave strangely sometimes with Clampfit. I noticed the Tau value can change quite strongly depending on the slightest changes of cursor positions. I would prefer to use Origin. However, my knowledge on Statistics and Origin is not enough to pull it off. And this is my first time working on Voltage gated channels. So most of these kinetics work is bit new to me.
If you follow a different method to calculate the Time Constant, please let me know.
Thank you for your advise on pH and Calcium. I tested the effect of drug at different pH and different internal free calcium levels.
Thank you again for your valuable insight :)
I calculate it with Clampfit or FitMaster, however in both cases it is the same, if you change the cursor position, the value changes. What I'd recommend you is to calculate the Tau of your control (Tau-fast and Tau-slow) and compare it with previous results.
http://molehr.oxfordjournals.org/content/20/5/392.long
In my knowledge (or I don't remember), it has not been published the Tau value of Slo3 channel in a heterologous system. I have preliminary results about that.
Best,
Oscar
Dear Oskar
Thank you for all the help. You made things lot clear to me. I'll od as you said
Good Luck with your work
TC :)
Have you tried repeating the protocol 3-5 times, averaging the sweeps and then fitting to the average trace? It might help with the tau variability.
Dear Marcin
Thank you for the valuable advise. Did you mean repeating the step protocol for the same cell few times and then averaging or Averaging the step protocols for few cells?
Sorry for not clarifying, I meant repeating the step protocols for the same cell. You would have to make sure that each series of voltage steps is independent of the previous series, i.e. you need to introduce a long enough delay between the sets so that the electrochemical gradients across the membrane can regenerate.
Altrrnatively you can simply try smoothing the signal slightly to reduce fit variability. Just remember to consequently apply the same parameters and cursor placement between cells so you don't introduce non-normal variability.
Thank you Marcin Siwiec
It's clear to me now. Yes I understand It would be helpful. I have noticed when calculating the GV curves (using the IV curves plotted form the step pulses), sometimes the slope of the curve become highly variable at higher voltages as the max conductance has already been reached. If I smooth the plots by normalizing with few readings, I would be able to get a curve with less variation. Same would apply for Time Constant calculations too.
Thank You gain
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