HEK293 cells can form large clusters connected by gap junctions - a nightmare for voltage clamp experiments. I've had success with over-expression of ligand-gated channels in lower density cultures where these electrically-coupled clusters are less abundant; you can also just scrap the cells of the dish mechanically and replate -- patching the isolated cells. L-cell fibroblasts are a good alternative, but the expression levels are generally lower in my hands. Note that different cell lines can alter channel physiology/pharmacology due to interactions with intracellular cytoskeletal elements and signalling pathways.

Without knowing your target protein, I would suggest to go to voltage clamp, hold the cell at -120mV and record the current flowing. after having a stable baseline, perfuse the ligand, until you reach a stabe current level again. Then if it is possible, record wash-out of your ligand, to be sure that You are not fooled by some changes in the clamp conditions ( eg changes in series resistence etc. )

I forgot to mention to use inta and extracellular solutions close to physiological ion concentrations ( 140 mM Na+ extracellular, 10 mM Na* intracellular)

Thanks Brian, we have to start the experiment and hence we are looking for inputs , although I have the study design, I am not an expert in handling the instrument. Well we do detach cells , wash with PBS and try capturing at a single cell dilution and the instrument is automated suction type, so till the cell is captured and a good seal is formed we wait. We surely avoid clusters but HEK cells and later on a neuronal cell line will be used for the work. So aim is to capture the currents generated by ligand binding. Na+ entry inside and a K+ going out is what happens here.

Brian, I am also into a protein which in an endogenous level expresses less on the membrane. So over expression will be done and hopefully something is seen.

I have one naive question, what do you call an excitable cell, ant cell which can generate current? or only action potential? In that case HEK would not have been excitable, its akidney cell line but there are different things which can generate current. the ion channels, the ligand gating etc right?

Thanks Peter, We will be using that conc. for our solutions. The perfusion has to be seen. when we start capturing we will see how it goes according to your suggestion.

It's better to use low serum or serum-free solutions for the first 12-18 h after plaiting- it'll reduce density. If you are using cells in the suspension, you can substitute Ca for Ba to prevent aggregation

Thanks Maria, now its adherent cells but I may use suspension later.

First, what Na2+ current are you trying to measure? Or more specifically what channel are you overexpressing? Moreover, what is your hypothesis since you will be doing the experiment in mammalian cells as well? Are you looking at cardiac or smooth muscle? If cardiac, are you looking at SA nodal cells, ventricular, or atrial cells? In mammalian cells, you must realize first is that Ca2+ current is going to contaminate your Na2+ current recordings so you will need to a Ca2+ ionic replacement such as cesium and make sure your bath solution is at the proper osmotic balance ~300 mOsm. Also, you must use a calcium buffer, either BAPTA or EGTA, depending on what channel you are recording? BAPTA is a fast buffer and EGTA is a slower buffer of calcium.

Hi, William thanks for the input. Well the case is looking at a membrane transporter which will be overexpressed, a neuronal transporter. The stoichiometry being that the intake of the ligand will also take 2 Na+ in and throw one K+ out. We are interested in looking at Na+ induced transport current and anion current in the whole cell mode.

There will be WT and mutant comparisons and will be done in a neuronal or glial cell line. Yes the Ca+ part will be taken care of but we first would see whether a patch clamp unlike voltage clamp will be able to detect these currents. To be hopeful it should as there are lot of literature around with HEK cells itself.

Understood. So for a Na/K transporter, as with other membrane transporters maybe a ramp protocol can be utilized as which what is used for to measure Na/Ca exchanger current (NCX). Induction could be accomplished by altering bath K+ concentrations and/or altering K+ concentrations in the pipette solution since K+ extrusion will be the rate-limiting step. Another approach would be Li replacement and ouabain replacement compared to large injections of Na. This would give you a baseline by blocking the transporter and then a large injection of Na for induction of current. This could be accompllshed in wild-type and mutant tranfections of the transporter. This should get you on the right track if you google or pubmed search lithium replacement, ouabain replacement, and Na/K exchanger in neurons or HEKs. I came up with multiple hits that have various stratgies and protocols that should you should be able to find a protocol that would provide you a basis for experiment along with bath and pipette solutions. I hope this helps.

Actually i honestly wanted some experiences regarding protocols as I have loads of references for how I go about the protocol. point here is that the system is an automated planar patch clamp and what you could modify is internal and external solutions, perfusion rates and I would look at the experiments at various temperatures. I am completely novice in electrophysiology but what i have been reading is that mostly people work with voltage clamp and we only have to manage with cell lines and not hippocampal neurons or anything because the purpose is to transfect patient specific mutations and capture minute differences of Km and current. The transport mechanism being of the order of 100ms, things have to be right.

the ligand being glutamate, Na+/glu in an exchange mode will be seen and ions will be manipulated to check the specificity of currents generated. Blockers will be used.

I have a question, what is actually homoexchange mode in ligand gated activity?

Holding -90mV ( 50mS)

Step from -60mV to 90mV by 10mV (300mS)

Back to -90mV (50mS)

Total 16 sweeps

Thanks Zhang, can you give a brief picture of whats the significance of the steps you mentioned.I am entering the world of electrophysiology..

Thanks

Xuexin Zhang is right. But I would increase the length of the first step up to 300 ms to get rid of Na channel incativation

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Thanks Zhang and Maria for the inputs, I would see with one of my colleagues how things come up. If any query would get back to you guys. Hope its not a problem.

well one query, if one has to capture mEPSCs, does it make your life different? As in a ligand gated Na+ dependent transport leads to uncoupled anion and transport currents and sometimes mini currents has to be looked at.

Briefly: 1) to block Ca2+ channels, you will need to use ACSF with 0.1 mM Ca2+ and 5 mM Mg2+.

2) to block K+ channels, you will need to use Cs+-based intracellular solution. Additionally, you might add 1-10 mM TEA to ASCF, if necessary.

3) Stimulation protocol: Vhold, -80 mV. Conditioning pre-hyperpolarization -120 mV for 50 ms. Then testing voltages starting from -60 with increments of 5-10 mV.

You can start with all this and then you will see.

Thanks Igor for the input. Will try and see as soon as possible. since i will be looking at currents irrespective of any channel but due to ion exchange I might need to block these and see the specificity of the current after overexpression of my protein which binds glutamate as a ligand.