Lecture 13 and 14: Techniques

Voltage clamp

Many of the membrane transporters exhibit very distinct p.d.- dependence. Consequently, the control of the trans-membrane p.d. allows us to distinguish the different transporters and sometime to determine their function. The device, which controls the membrane p.d., is called "voltage clamp" and was introduced by Hodgkin and Huxley in 1950's to unravel the origin and propagation of the nerve action potential.

Making electrical contact with living cells

In the electric circuit the conduction is electronic, but in the cell and across the membrane,

The conduction is ionic, mainly by K⁺ and Cl^-. To transduce electronic current to ionic current, silver electrodes coated with silver chloride are employed. To make a contact with cells, which are small and easily damaged, glass micro-pipettes are pulled, while the glass is heated, into very thin (<0.5 m) points. The micro-pipettes are filled with 0.5 to 3 M KCl and impaled into cells, while silver/silver chloride electrodes are in contact with the filling solution. Such electrodes have a very high resistance of ~10 Mohms and the voltmeter measuring the membrane p.d. has to have a very high input impedance. The trans-membrane p.d. is measured by impaling one electrode into the cell (vacuole or cytoplasm) and placing the other electrode for reference in the outside medium. The trans-membrane p.d. is input into a comparator and is compared to a command voltage generated by a computer. A current is then passed across the membrane until the membrane p.d. matches the command voltage. This can be achieved by an insertion of another electrode (usually a wire to lower the resistance) or by placing the cell between two compartments.

Patch Clamp

The advances in electronics enabled biophysicists to pass a current through a patch of membrane small enough to contain only a single channel. This amazing experimental method was also made possible by the property of the membrane to seal onto the glass pipette (see attached Fig.1). There are several configurations of applying patch clamp (see attached Fig.2). The solution in the patch pipette can be controlled as well as the solution in the bath. Therefore, the specificity of the channel can be measured by controlling the p.d. across the patch and comparing the current reversal p.d. with the Nernst potential for ions in the media on both sides of the membrane patch. The currents through the channels appear as pulses (see attached Fig.3). In the initial experiments the pulses were seen as rectangular, but in later experiments sublevels were identified (substates). In plant cells, the application of patch clamp is difficult due to cell wall. The cells have to be prepared by digesting the cell wall (enzymes from various herbivore stomachs) and then patching before it re-forms.

Microelectrode ion flux estimation (MIFE)

The idea for this technique came from Bill Lucas (Lucas and Kochian, 1986, in Advanced Agricultural Instrumentation: Design and Use (W. G. Gensler, editor), pp 402 – 425). It has been implemented by I. Newman and S. Shabala at University of Tasmania. A microelectrode containing a specific liquid ion exchanger (LIX) is placed at a distance x from living tissue. The electrochemical potential at this point can be determined in terms of concentration of the ion and electrical potential V. The electrode is then moved slowly (as not to disturb the solution) to x + dx and new value of electrochemical potential is obtained. The gradient can thus be calculated and the ion flux determined.

Set of data from H⁺ sensitive electrode from Shabala et al. (1997, Plant Physiol 113: 111 – 118). At 60 s the manipulator moved the microelectrode to the position near the corn root tissue surface (20 mm). The electrochemical potential decreased by ~ 1 mV. At 65 s the microelectrode was moved to more distant position (60 mm) and the electrochemical potential increased again. The cycle was repeated with the 10 s period and average difference in the electrochemical potential was calculated. The protons move down the electrochemical gradient and consequently, there is a net influx of protons into the root.

N. A. Walker, Visiting Professor in Biophysics group, is building a version of MIFE, TRIFID (Transient Ion Flux Identification) to improve time resolution in flux measurement upon changes of medium in the experimental chamber. Linkages of ion flux at membrane level (symports and antiports) are usually hard to measure in the steady state, but will be discernible, if the substrate ion concentration is changed in the medium.

AC Impedance Spectrometry

This technique was developed H. Coster, J. R. Smith and T. Chilcott here in the biophysics group. A small AC current is applied to a system (cell membranes, black lipid membranes, electrode-electrolyte interface, unstirred layers, suspensions of cells). Usually four terminal configuration is used: two electrodes to pass current across the region of interest and two electrodes to measure the voltage drop generated. The phase shift between the current and voltage is measured and the complex impedance,  , is calculated. (G = conductance, C = capacitance). A heterogeneous system, which can be represented by a number of different planar regions sandwiched together, produces a “dispersion curve” as G and C are measured at a range of frequencies.