PLS HELP ASAP: Assaying transient VSCGs w/ voltage sensitive dyes

7 posts / 0 new
Last post
Vicky
Vicky's picture
PLS HELP ASAP: Assaying transient VSCGs w/ voltage sensitive dyes

Hello All,

This is my first posting & I hope some of you out there can help.

I would like to perform some studies of the NaV family using the voltage-sensitive dyes. Unfortunately I do not have easy access to a rig; I do have access to an FDSS (sim. to the TETRA).

I have seen several papers using these VS dyes to measure response w/ recombinant NaVs (293 or CHO) in the presence of veratridine with and without ATX2 & demonstrating response (Benjamin 2005, Felix 2004, posters at conferences (Astra Zeneca 2005, Ionix 2004)

1) Isn't the channel in an inactivated state, considering the RMP of the host cell is higher than the activation threshold (CHO=0- -10 & HEK= -20 - -40mV)?

2) To my knowledge veratridine binds to the open state, so I do not understand how veratridine can work if the channel is indeed inactivated. Can someone explain this?

3) Is it that these groups are using a stable, or one chosen w/ a lower RMP? Considering my results w/ ligand gated channels, etc. the expression levels should not be an issue.

4) Beta Subunits: Is it better or worse to express them w/ a flourometric readout?

I greatly appreciate your help, and hopefully can return the kindness in the future.

Vicky :-)

Bluejay
Bluejay's picture
I don't have a lot of answers

I don't have a lot of answers but have perhaps some helpful comments on Membrane Potential Dyes (MPD). A) Using the dye in CHO hERG cells, we saw activity with standard blockers without activating the channel, although the channel does open at -20mV or so. B) Be prepared for numerous false positive and false negative responses and poor correlation with patch clamp data, based on our experience and the literature on hERG. MPDs are still good for a general screen and are easy to use and give a strong response. C) Is it possible the presence of the NAV channels are changing the resting potential of the cell?

Vicky
Vicky's picture
Thank you BlueJay for your

Thank you BlueJay for your reply. As these are sodium channels the activation threshold is -100mV or so. I have read that for one recombinant NaV in HEK the RMP was -45mV, and at -70mV the channels were almost 70% inactivated at -70mV. So I would think even if the presence of NaV somehow shifted the HEK RMP more negative, at -45mV, the channel's state equilibrium would be shifted to a very inactivated state. That is why I do not understand how veratridine has the opportunity to shift the channel's equilibrium towards the open state under these conditions.

As for native NaV channels, to my knowledge they are not present in HEK or at least at a detectable level. Actually there is a sodium channel in CHO-K1, although I have not seen anything written about the original CHO cell line.

hERG is indeed an easier channel to tackle as a recombinant as its activation threshold is well within the RMP limits of most host cells used for stables. That is why MPDs are an excellent readout for hERGs.

Thank you once more for your kind reply :-)

Fraser Moss
Fraser Moss's picture
You have to remember that you

You have to remember that you are working with a population of channels in equilibrium, and that although at the RMPs you describe the tendency is for a single channel to be in the inactivated state, you have to remember that the population you are recording a mixture of channels in different equilibrium states.

i.e. at any given time in your cells there will be a proportion of your cells that leave the inactivated state and return to closed and/open states.

Your statement that the channels are 70% inactivated at -70mV describes that 70% of the population are inactivated, however, over time this does not have to be the same 70% of channels and they are constantly moving between different equilibrium states. The membrane potential just sets which state is the most favorable.

So the longer you incubate with veratridine, the more channels will have at some point passed thorough an open state allowing drug to bind.

Vicky
Vicky's picture
Thank you very much for your

Thank you very much for your reply frasermoss. As my background is more in cell physiology techniques than in ephys, could you kindly answer 2 questions if possible?

1) I was aware that there is always an equilibrium dynamic, but if the RMP is now -40 to -50 mV in this recombinant environment, wouldn't the probability of the population being in an open state be exceedingly low?

2) Since I am performing a cell based assay w/ a voltage sensitive dye readout, as the MP of the cell population is somewhere around -45mV (if I am assuming correctly), would you expect this eventual population shift towards an open state to occur within the course of a few minutes?

Again thank you for your time.
V :-)

Fraser Moss
Fraser Moss's picture
I'll just state clearly here

I'll just state clearly here that I don't not work with MP dyes or Na channels but am answering from my general electrophys background.

at -45mV, the probability of being in an inactivated state will be high, but the channels will still spontaneously move to an open state from time to time allowing the drug to bind which then prevents the channel re-inactivating. I don't actually work with this system so I cannot make a conclusive statement about the time courses but the application/activation times by veratridine must be in the literature.

Some useful background reading if you have not already read them are:
(PMID = pubmed ID)

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes.
PMID: 15285907

Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.
PMID: 16550174

Veratridine modifies open sodium channels.
PMID: 2454286

Kinetics of veratridine action on Na channels of skeletal muscle
PMID: 2419478

Voltage-dependent gating of veratridine-modified Na channels.
PMID: 2419480

Effects of veratridine on sodium currents and fluxes.
PMID: 9600010

Vicky
Vicky's picture
Thank you for your reply.

Thank you for your reply. Vicky