What models are you looking in? Expression systems (e.g. HEK293 cells)? Primary cells (e.g. astrocytes, neurones)? Your protocols and set up will depend on the cell type you are investigating. If you could provide some further details then we could aim to assist you.

Hi, I am using C6 glioma cells. Looking forward to your help. They are transiently transfected ones. basically GFP-fusion proteins.

Thanks

Depending on your electrophysiological set up you can perfuse glutamate onto your cells and look for Na+ dependent currents (in voltage clamp mode). If you have microinjection capabilities then you can puff on glutamate. You can characterise these currents via biophysical properties, Na+ dependence and their pharmacology using commercially available inhibitors.

I have not used the cell line you are using before so I am unsure on the amplitude of your currents (pA range?). You can modify your intracellular solution to increase permeability through the pore and thus increase the magnitude of you associated currents.

See Dallas et al. 2007 for further details of solutions.

http://www.ncbi.nlm.nih.gov/pubmed/17428968

Hope this helps.

Hi Mark,

I am manually perfusing glutamate and seeing the effect of it on Na dependent currents Yeah. We don't have microinjection capability. But I am now trying to sort GFP positive cells to have more of transfected population.

pA range is 500 and beyond. But what I am confused about the pulsing protocol. How to be sure that they are sodium or anion currents. Yes I do use a blocker and even see the wash out effect when glutamate is not there. But since my study is about comparing Wt vs Mutant expressing cells, I need to be careful. Right now I am holding cells at -80. pulsing interval of 5 ms. increment of 10mV from -120 till +60mV.

Am I missing some point here? I have constant Na conc now in solution. My aim is to look for glutamate dependent currents than Na dependence.

Thanks

You can be sure that you are measuring Na or anion currents by replacing the extracellular Na in your perfusate. The Na depedence comes for the transporter electrogenic properties, therefore is a requirement. Your inhibition data seems confusing, what blocker did you try and are you seeing effects of the blocker alone?

Do your cells express any glutamate receptors?

If you are looking to see changes in EAAT activity (with your mutation), you can do this by comparing the kinetics and amplitude of your glutamate induced currents in voltage clamp mode (holding the cell at -70mV or equivalent). This response should be a) Na dependent and b) sensitive to EAAT blockers.

Hope this makes sense.

Hi,

I use DL TBOA. Yes I do see a blocker specific effect in the amplitude. I have done choline based experiment to check for the Na dependence.

Actually I add blocker and wait for few min and then take readings. Wash out the blocker and add glutamate to see if it still shows similar amplitude or has actually reduced. I see a drop in amp. But for some mutants, the amp has increased. I am confused about the result here.

What I do not see in my trace is a typical inward current. The current keeps increasing from -100mV to a +60mV unlike a proper depolarization profile. Am I doing something wrong here.

Also I have not checked for the expression of receptors but according to literature, there are mGlu receptors and NMDA too. Yes the only specific way seems removal of Na as a check that antagonists. Correct me if I am wrong.

Thanks

Kalpita

Is there a linear relationship in your current voltage profile? It seems as if you are recording leak currents from your cells. Are you leak subtracting your dataset- this can be done online using a P/4 protocol? In order to generate a current voltage profile of the EAATs you should be changing the holding potential of your cell and then bath applying your glutamate and presenting the magnitude of the response against your holding potential. You should see a classical inward rectifying current profile. Have you tried this? You could also look to subtract your control traces from those generated in the presence of DL TBOA.

Hi Mark,

I actually fit into non-linear regression but I do see most times linear profile except for few mutants. I have just acquired reading yet and simultaneously take leak current readings in the P/4 protocol. I have not subtracted them yet. I am very new to the technique. But intuitively and through literature I am trying to work it out. I have some affinity data for my mutants through kinetics experiments and I am trying to see what happens in a patch clamp setup to this comparison.

I use Nanion port a patch for my work. I am using there default protocols and some I have generated based on literature. I want to know if there are any open softwares which i can use to fit my data. Nanion does give fit master. But apart from that, the leak subtraction and regression fits etc, is it available online?

Also, I see the magnitude post glu application at -80mV only as holding. I have not changed the holding. Does it differ from the sweep traces? which I take form a negative to a positive potential ?

Thank again and again for the inputs.

Fit master should have these functions built in as options in terms of leak subtraction and regression fits. Are you seeing any rectification in your currents? It seems that you are not set up to look at the transporter currents rather you are recording global currents.

I would set up your system to run continuous sweeps (do not alter the voltage), while holding at -80mV. Then bath apply glutamate, do you see anything?

Hi,

No I havenot done that. As in only hold at -80 and taking multiple sweeps.

Also, if I see changes in conductivity in a global profile across various constructs with a blocker in place, is it still not meaningful.

I am having some issues with my amplifier, so cant run experiments. But I already have leak subtraction enables while taking a trace. Also I take without the leak subtraction.

I am trying to understand the rationale behind not changing a voltage from holding, is it just to control any external effect apart from glutamate if I was looking at transporter current?

Your inputs are appreciated.