Paolo Tammaro, Kenju Shimomura, and Peter Proks
10.4. Notes
correspond to hyperpolarization of Vm. In cell-attached configura-tion, inward membrane currents appear as positive signals at the current monitor outputs. Further, in the inside-out configuration, where Vm = −Vp, the sign of the control potential and the recorded current are inverted. In order to follow the standard electrophysi-ological convention (i.e., outward currents are positive and positive voltages are depolarizing), the acquisition software used must also be properly programmed, in order to invert digital stimulus and sampled values. Conversely, in the outside-out configuration, Vm = Vp, and currents have the conventional polarity.
The basic technique for micropatch recording is similar to that which we have described for macropatch. The electrodes are pulled to a much smaller diameter than that typically used for macropatch pipettes. Formation of gigaseal with small pipettes is also very similar to that described for macropatches. Following initial contact with the membrane, suction is applied to form a tight gigaseal. Outside-out patches can be obtained by rupture of the patch after seal formation and slow removal of the pipette from the oocytes. Formation of outside-out patches with a mac-ropipette is also possible, but the rate of success is considerably lower than with micropipettes. The intensity of the suction used in order to favor seal formation with micropipettes can be 2–5-fold greater than that used to assist macropatch formation.
The large size of the oocyte is particularly suitable for patch-cramming, i.e., insertion of the patch-containing pipette into the oocyte (see Fig. 10.2). After excision of an inside-out patch, the pipette is pushed deep into the oocyte. We found that patch sur-vival with this technique is quite high (80–90%); thus the patch can be repeatedly exposed to cytoplasmic and bath environments.
For example, this approach has been used to demonstrate that cytoplasmic constituents (e.g., protein kinases) play a role in reg-ulating the activity of certain ion channels (22–25).
1. It should be noted that oocytes from other amphibians (such as oocytes from the cane toad Bufo marinus (26, 27) ) have been successfully used as heterologous protein expression systems and may represent an alternative to Xenopus oocytes.
This may be particular relevant in Australia, Asia and the Americas where B. marinus is widely available.
2. In the early days, receptors and ion channels were expressed in Xenopus oocytes by injection of total poly(A) mRNA 10.3.4.5. Other
Patch-Clamp Configurations Micropatches
Patch-Cramming
extracted from tissue samples (e.g., see refs. 28, 29). This method has the obvious disadvantage that all possible mRNA are translated into proteins. With the advent of gene cloning, the use of complementary RNA (cRNA) became predominant.
3. With respect to the expression of ion channels that are com-posed of two or more subunits, the amount of cRNA for each subunit that needs to be injected depends, in principle, on the stoichiometry of the subunits comprising the channel and the length of the cRNA encoding each subunit. However, dif-ferent efficiency of RNA translation, or difdif-ferent posttransla-tional processing could modify this relationship. For example, the pancreatic beta cell KATP channel is formed by four Kir6.2 subunits, forming the channel pore, and an equal number of auxiliary subunits (SUR1). SUR1 is a protein of 1,581 amino acids whereas Kir6.2 is about five times shorter (390 amino acids). We found that the best KATP channel expression is achieved by injecting that five times more of the cRNA cod-ing for SUR1 than codcod-ing for Kir6.2 RNA. Presumably, this results in the translation of approximately the same number of Kir6.2 and SUR1 subunits.
4. Examples include the IKs channel formed from the heterol-ogously expressed IsK (minK) subunit and the endogenous KvLQT1 subunit (30). Oocyte-specific interactions between Fig. 10.2. Macroscopic KATP current recorded at −60 mV from a Xenopus oocyte coexpressing Kir6.2 and SUR1.
The patch was excised in nucleotide-free solution at the time indicated by the arrow and reinserted into the oocytes (crammed) as indicated. The dashed line indicates the zero current level. Note that the current is virtually completely blocked in cell-attached or cramming mode, presumably because the high intracellular ATP level within the oocytes results in the complete blockage of the KATP channel.
voltage-gated sodium channels and the cytoskeleton have also been reported (31).
5. The expression levels are sometimes higher around the site of injection. For this reason, it can be useful to use a similar injection site in all oocytes, such as the centre of the animal hemisphere.
6. If blockers of endogenous channels are used, it is always important to check that they do not affect the activity of heterologously expressed ion channels. For example, of high concentration gadolinium might have electrical screening effects on the plasma membrane.
We wish to thank Drs. Oscar Moran and Tim Craig for their criti-cal reading of the manuscript and helpful comments. P.T. holds a Junior Research fellowship at Wolfson College, Oxford.
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