4.2.3  Patch clamping

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Patch clamp analysis involves sealing very fine glass capillaries, with tip diameters of about 1 µm, to the surface of protoplast or vacuole membranes. Using a brief (a few milliseconds) pulse of 1 V, it is possible to rupture the small area of membrane sealed across the orifice at a capillary tip, leaving a protoplast or vacuole like a balloon stuck on the end of a straw (Figure 4.13a). Solution within the capillary diffuses into the membrane-bound space, replacing the cytoplasm or vacuolar contents. Data from this preparation provide infor-mation on the whole membrane surface and it is called the ‘whole-cell’ configuration. Alternatively, a small patch of mem-brane sealed across the tip can be retained after tearing it away from the protoplast, which is discarded. The inside surface of the plasma membrane then faces the solution in the experimental chamber which can be changed at will. This configuration is called an ‘inside-out’ patch. (It is also possible to get ‘outside-out’ patches — Figure 4.14.)

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Figure 4.13 Patch clamp recordings of orthophosphate channels in vacuole membranes. (a) A photomicrograph of a vacuole (c. 40 µm diameter) isolated from beetroot, sealed to the end of a glass microcapillary (Figure 4.14). The capillary is filled with a salt solution which diffuses into the vacuole. (b) Currents that flowed across the vacuole membrane when pulses of voltage ranging from -111 to +129 mV were applied. Data from these and other voltages suggest that the currents were due to flow of both K+ and orthophosphate ions. (c) Currents flowing through individual ion channels in an ‘outside—out’ patch (Figure 4.14). The individual steps in current represent the opening or closing of a single channel measured at 29, 39, 49 and 59 mV. There are at least four channel molecules in this patch of membrane. (d) Increasing applied voltage from 29 to 59 mV raises electrical current through a single channel (O) and probability of channel opening (•). Because current extrapolates to zero at a negative voltage close to the Nernst potential for orthophosphate, it is likely that orthophosphate carries current through these channels. Decreasing probability of opening as potentials decrease towards zero suggests that these channels only function at positive potentials.

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Figure 4.14 Patch clamping is used to measure tiny electrical currents across membranes. A patch of membrane is sealed against the rim of the tip of a glass microcapillary such that all charge passes through the membrane. Keeping voltage (V) constant (clamped) across the patch will reveal whether ion channels are present. Opening and closing of channels is indicated when square steps are seen in current (I) across the patch after amplification (AMP). Useful configurations include: whole cell (e.g. current flow across an entire tonoplast); inside out (capillary solution in contact with outer face of membrane); and outside out (capillary solution in contact with inner face of membrane)

Ions are charged so their fluxes can then be studied by the flow of electric current that accompanies ion movement. Voltages are applied to simulate the potentials developed by electrogenic pumps and other processes. Because the area of a membrane patch is so small, a millionth of a square millimetre, it usually contains only very few, may be one to ten, ion channels. Consequently the opening and closing of even a single ion channel molecule significantly alters the flow of current across the patch. These single channel events can be seen on-line as they happen. Ogden and Stanfield (1994) give a good description of these techniques.

Controlling the composition of solutions on either side of the membrane is both a great strength and a weakness of patch clamp experiments. By setting the conditions on both sides of the membrane we can obtain incisive information about the function of a number of components involved in solute movement. However, we can only mimic the chemical composition of the cytoplasm, with its thousands of con-stituents, and therefore suffer the limitations of normal in vitro experimentation. The cytoplasm contains many molecules which affect ion transport; until these are identified, they will probably not be used in bathing media. So while we might have definitive information for the conditions of our experi-ments, we must still make estimates and extrapolations to speculate about what happens in cells that are not disturbed by the experimental procedures we use. This does not negate the great contribution that patch clamp methods are making to our knowledge, instead it emphasises that there is still a lot of work to be done.

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