Hello everybody,

I have the next problem. I measure hERG channels expressed in CHO cells. While there is no problem for inactivation of for activation tail current, I still have strange curves for steady-state activation currents. It's not bell-shaped as supposed to be. Only slightly. How it could be?

My protocol: -80 -> test pulses (2sec) -> -50mV (2 sec) -> -80 mV (4sec)

It sounds like you are not applying any form of leak subtraction, so the leak currents are dominating over the inactivated hERG currents at the more depolarized potentials. Are you maintaining a Giga seal during reacording or a at least a high hundreds of megaohm seal resistance?

To find out the seal resistance during a recording, and subtract the leak apply a short pre-pulse to a voltage that does not activate hERG (e.g. -80mV holding step to -70mV for 100-200 ms or so and back again ) before your actual test protocol.

When analyzing your traces use the change in current during this step to assess the resistance and apply this value to the leak subtraction routine built into your analysis software (if you are using pClamp Clampfit this will be in the menus: "Analyze">"Adjust">"Leak Subtraction")

P/N leak subtraction does not work well for hERG

Yes, I don't apply any subtraction. I should try to do this. The resistance seems ok.
It looks the effect depends on the amplitude of hERG current. So, quite possible that it's leak or endogenous currents. Thanks, I'll try again.
By the way, does trypsinisation influence hERG currents? How long do you wait after it (after putting the cells onto dishes)? I just realized that during the day the hERG amplitude was increased (it was around 24h after trypsinization). Maybe it's just occasion, because of small amount of cells (n=4)? Or it was observed by you too?

I've try to use the leak subtraction. It helps a little bit. But still, the curves are not so nice :(

Trypsinization has a big effect as it cleaves proteins on the surface of the cell including your hERG channels.

For transfected cells from which I want to record I use non-enzymatic cell dissociation solutions to replate the cells about 12-16h before I rewcord from them.


you can buy "cellstripper" solution (25-056-CI) from mediatech (www.cellgro.com)

Can you please upload a couple of pictures of your traces before and after leak subtraction and include the information on the holding current to maintain resting potential, the access resistance in whole cell mode, and membrane capacitance of each cell?

Use the "Add attachment" feature to upload your trace images as jpegs ior a similar image format.

I'm not sure I will be able to give you a definitive answer but it's easier to look at traces than imagine themk from a description

Thanks

Hello, here are the pdf file with 3 figs:
1. WT hERG in CHO (not corrected) - just best what I have. Others looks worse.

2. WT hERG in CHO (corrected). Others currents have higher right shoulder.

3. hERG with mutation in CHO (no correction). The mutation was based on another plasmid. WT hERG was obtained from a company.

Actually, I want to do next WT based on the plasmid used for mutation. But still - the WT supplied looks strange.