While performing manual patch-clamp with hERG channel, we could see a rundown effect of ~4%/min (although it varies) even after adding Mg-ATP to the internal. My questions are: 1. Any other possible measure I should take? 2. How to factorise the run-down effect while calculating drug effect. 3. Astemizole effect does not stbilize even after 10 mins (we maintain a constant flow of compound dilution). You suggestions would be really helpful.
Can you provide some more information on your setup? Otherwise it is really hard to guess what's the problem.
E.g. What is your intra- and extracellular solution? What is your voltage-protocol?
we also add 0.3 mM Tris-GTP and 14 mM Creatine-phosphate in pipet solution to avoid running down quickly. hopes helpful.
Have you tried perforated patch? I am not familiar with hERG or what are your conditions but if you could do you experiments with perforated patch, it will probably solve your problem.
Hi all, many thanks for your help. I am including more details of my experiment which might help you to answer my query specifically. We use cultured hERG-CHO cells plated on PDL coated glass coverslip 48h before experiment; cell is not lifted after giga-seal.
Voltage protocol:
i) Holding potential: -80 mV
ii) Depolarization: +40 mV, 1 sec
iii) Repolarization: -40 mV, 600 msec
iv) Sweep duration: 5 sec
Internal solution:
i) KCl - 10 mM
ii) MgCl2 – 1 mM
ii) EGTA – 5 mM
iv) Mg-ATP – 5 mM
v) HEPES – 10 mM
vi) CaCl2 – 0.1 mM
vii) K-aspartate – 130 mM
pH: 7.2
Osmolarity: 310mOsm
External solution:
NaCl- 140mM
MgCl2- 1mM
CaCl2- 2mM
KCl- 4mM
Glucose- 10mM
HEPES- 10mM
pH: 7.4
Osmolarity: 320-325mOsm
Rajib, tail hERG currents in recombinant cells are notorious in showing significant amounts of run-down over time. I am not aware of any good recipe on how to prevent or reduce it.
Suggestion on trying perforating agent seems to be reasonable, but we ourselves never tried it with hERG. My colleague used the following protocol (for a different type of current): use beta-escin. It is water soluble, just add to internal solution. Dip the tip of electrode in regular internal solution and then backfill with internal solution containing Beta-escin. Get giga-seal and wait for perforation to occur. My colleague claims it worked well.
We never tried to fight hERG run-down, we just picked-up cells with a reasonable amount of run-down which still permitted pharmacological assessment of compounds in early cardio-tox screens. [Our internal solution did not contain ATP/GTP and we saw comparable (or lesser) levels of run-down described by you]. For pharmacological profiling, one needs to monitor the base line for time sufficient to extrapolate it. Extrapolation will provide one with the level of projected control at the times when compound is tested. If compound is washable to a reasonable extent, the wash-out level and initial control level would give a more reliable estimate of projected control than extrapolation.
To your stimulation protocol, I would add one more step: step Ia - depolarize to- 40 mV for 100 ms. This will help you to automatically subtract leak current from the peak tail current measured at step iii. Also, typically people use the repetition rate of 1/10s or 1/15s instead of 1/5s to remove "memory" from the previous cycle of stimulation
Hi,
as far as I can tell, if your run-down is consistent then I would strongly recommend Victor's suggestion: use beta-escin like he described! The success rate is much higher than with Amphotericin or Nystatin; you can even get similar series resistances like going whole-cell. Theory is, that it forms almost a net-like structure in the membrane patch in contrast to just small pores. Still it is somehow able to retain a lot of intracellular metabolites like ATP and stuff. It is also very cheap, so give it a try and let us know!
We have with a KF based internal solution without ATP approximately 20% run-down over 10 min. It is not optimal but sufficient to asses hERG blockade of known blockers. Usually we have a daily run of solvent controls in parallel.
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