Bryan Krantz Laboratory

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We have three Axon Axoclamp 200B voltage-clamp amplifiers (Fig. 2), which are now made by Molecular Devices. They interface with a custom bilayer perfusion cell (Fig. 3), which is based on the basic cell made by Warner Instruments.

Bilayer cell. Top view of a bilayer cell, showing the cis and trans chambers, which are connected to the head stage by the Ag/AgCl electrodes. The bilayer cup in the rear has the small ~50- to 200-μm hole, wherein the lipid bilayer membrane is formed.

Our bilayer cell is made out of PEEK, and the bilayer cup is made of white Delrin or polysulfone plastic. A Bilayer perfusion cell schematic PDF is available. Schematics are also available so that the membrane can visualized via either a Polished polysulfone bilayer window PDF or standard microscope slide with a Bilayer cell glass window support PDF.

These instruments are capable of low noise recordings in single-channel mode as well as less sensitive multi-channel recordings. The sturdy metal Faraday cage (Fig. 4) blocks both sound vibrations and electrical noise (e.g., cell phones and AC power-line hum) from the experiment. Schematics for mounting the head stage PDF and Schematics for the bilayer Faraday cage PDF are available.

Amplifier head stage interfaced to a bilayer cell. Inside an aluminum Faraday cage is the bilayer cell connected to the voltage-clamp amplifer. The Faraday cage screens electromagnetic interference and sound vibrations from the sensitive head stage.

The capacitance test is the practical means of determining the size and quality of an artificial bilayer membrane created during a typical planar lipid bilayer experiment.

Recall that a capacitor stores charges of opposite sign within conductive plates on either side of a dielectric medium (also known as an insulator). This situation is exactly represented by a phospholipid bilayer membrane (of low dielectric constant), whereupon two oppositely charged layers can build-up on either side. Thus a capacitor is analogous to a jar that can hold a set volume of water. [In fact, the first capacitor was a glass jar with metal plates on either side of the glass, and the first unit of capacitance was called a 'jar' (~ 1 nF).] As suggested in the above analogy, a pair of parallel disk conductors (of radius, r ) have a capacity that is directly proportional to the area of the disks and ε—the permittivity of the dielectric medium—and is inversely proportional to the thickness of the dielectric medium separating the disks, d: C = ε πr 2/d . This relationship is nice for bilayer membranes, which are formed in round holes, ranging in diameter from 50 to 200 μm. A useful number to consider is that phospholipid membrane has a capacitance per unit area of 1 μF/cm2. Thus, a 200 μm hole has a theoretical maxium of ~300 pF in capacitance! In reality, it is hard to achieve this ideal number (see below).

A photograph of the oscilloscope output during a capacitance test, using a model cell, which is not a membrane but rather an actual electronic circuit of a series capacitor (100 pF) and resistor. Model cells are often used to calibrate the voltage-clamp amplifier. Remember that the outputs are scaled in various ways. So, the applied triangle function appears to be ±10 mV/ms, but it is really ±1 mV/ms, as it is scaled 10× before being read by the oscilloscope. The current output square wave is ±100 pA, since the gain on the amplifier is 1 pA/mV. Recalling the capacitance is directly equal to the current when dV / dt is 1 V/s, the correct capacitance, 100 pF, is observed. We are using a Fluke 271 function generator [50% symmetric triangle wave, 50 Hz, 500 mV p-p (VhiZ)].

A convenient way to go about measuring membrane capacitance is to apply a symmetric triangle wave function as the membrane potential, where the rate of change in voltage is either ±1 V/s. Given the relationship between charge, q, in Coulombs (C) and voltage, V, for capacitance, C, is: C = q / V; you then can take the time-dependent derivative of this expression, i = dq /dt = dV/dt × C, where i is the instantaneous current [in units of Amps (C/s)]. Therefore, the current is directly equal to the capacitance (as dV/dt = ±1). Thus the current flows in either direction (depending on the sign of the voltage change with time) and appears in the oscilloscope as square-wave output. One-half of the peak-to-peak output is equal to the capacitance [of course after you include all the gain settings and output scaling factors in your particular setup (see Fig 5)].

Usually, the capacitance test is used to monitor membrane formation. Lipid solution is applied to the aperture with a paint brush or other means, and the initially observed capacitance is often lower than expected ideally. Bilayer then begins to spontaneously nucleate, and the capacitance gradually begins to climb. A 'good membrane' nearly approaches the limit expected above; e.g., a 200 μm hole yields ~200 pF in practice. A 'bad membrane' has a much lower than expected final capacitance. What is physically happening? When viewed optically, a thick torus of amorphous lipid surrounds the bilayer formed in the center. For a so-called 'good' membrane, this torus is much smaller than in a 'bad', poorly formed membrane.