I'm doing cell-attached recordings with external ACSF in the pipette and hoping to see some currents in voltage clamp mode. The aim is to see whether adding glycine/GABA to the pipette would depolarize or hyperpolarize the cell at different age. The question is, how does holding potential work in cell-attached mode (vs. whole-cell intracellular mode)? In whole-cell mode, the pipette is a continuation of the cytoplasm but in cell-attached mode, the pipette solution is extracellular. Does that mean to depolarize the cell I would hold it at a more negative potential in cell-attached mode (opposite as whole-cell)? Also if I hold it at 0mV, does that mean I'm just leaving the cell at its own RMP? Another related question, do the currents observed in cell-attached mode in opposite direction as well? e.g. inward = hyperpolarizing, outward = depolarizing?
If you are in cell-attached configuration, you cannot clamp the membrane potential of the entire cell. You may control the potential within the pipette up to the membrane patch; but on the other sider of the membrane patch, the cell controls the potential. The potential across the patch is the potential inside the cell minus the potential inside the pipette. If you put GABA or glycine in the pipette, you will cause opening of the respective channels, which would normally exert a hyperpolarizing potential on the cell. Instead of seeing square steps of single-channel current (if you are lucky enough to trap only a few channels within the patch), you will see currents that appear to droop, because as current flows, the membrane potential will move towards the equilibrium potential of the conducting channel. This leads the single channel currents to "droop" in amplitude. An excellent paper showing this effect with ACh channels is the one from Fenwick, Marty and Neher in Journal of Physiology from 1982 (http://jp.physoc.org/content/331/1/577.full.pdf). In that paper, the opening of AChRs leads to inward current that depolarizes the cell, and ultimately leads to action potentials. I think that paper will answer your question clearly by example.
I neglected to say that if you are in cell-attached configuration, and you DO want to clamp the cell membrane potential, there is a way to do it. This idea came from Dick Tsien's lab in the 1980's. After you get a gigaseal to achieve the cell-attached configuration, raise extracellular potassium concentration to about 140 mM. This will raise the K equilibrium potential to about 0 mV. Since the resting membrane potential is essentially set by the K channels, that will result in a clamp of the membrane potential at about zero. Transiently, you might see some inward currents. You could, of course, block those with the typical cocktail of blockers of sodium and calcium channels -- TTX and a few micromolar cadmium.
Being an alumnus of Dick Tsien's lab in the 1980's, I second Robert Chow's suggestion. Since you can only control the potential of one side of the patch of membrane, you put the cell in isotonic potassium to 'zero out' the membrane potential (generally, we did so in zero-calcium extracellular solution to avoid massive calcium influx at 0 vM Vm,. This is more effective at preventing calcium influx than using blockers, because there are so many different calcium conductances to block). Since you know that the overall membrane potential is 0mV, you can also know the potential of your membrane patch (since you also know the applied holding potential). Keep in mind however, that the holding potential in this configuration is "opposite". By this, I mean that if you depolarize the electrode, you are hyperpolarizing the patch (i.e. the outside is more positive than the inside).
Hello Miaomio
You may have a look at this nice article. Everything is explained.
Regards
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