3.1.1 Ion Channel Conduction

The conduction of ions across biological membranes is essential for life and a phenomenon that has preoccupied biologists, physiologists, physicists, and chemists for over a hundred years.  Ions cross biological membranes through so-called ion channels. These pore-like structures are generally formed by protein molecules that span the membrane; ion flow through the channels depends on concentration gradients and electrical potential differences across the channels.  Because the study of this process in vivo is rather difficult, many experiments are carried out using artificial membranes. These laboratory membranes are made from self-assembling phospholipid bilayers, similar to the ones that form the walls of living cells.  Different types of channels can be introduced in such membranes and their permeability to different ions can be studied. Typically, electrical conduction is used to characterize the flow of ions across the membrane.

 

In this experiment, we use a lipid bilayer synthetic membrane (prepared by the student for each experiment) and  gramicidin A channels. Gramicidin A has a simple, known structure, can be produced in large quantities and can be chemically modified. Moreover, there are reasonable models of the channel so that it is possible to relate ion transport properties to the known structure. Finally, gramicidin has the kind of ion selectivity that is often encountered in biological membranes. In the laboratory, an artificial membrane is constructed between two electrolyte solutions of identical equal concentration and gramiciden channels are inserted into the membrane. An externally applied electric potential difference causes a flow of ions through the channels. The purpose of the experiment is to observe single channel conduction by observing changes in current flow across the membrane over time.  The opening and closing of the channels results in discrete incremental changes in current.  From the data it is possible to determine the size and electrical properties of the channels as well as the kinetics of channel formation.

 

Since ion-current through a single channel is rather small, extraneous noise must be controlled very carefully. A better low-noise amplifier, increased signal filtering and a reduction of low frequency drift are improvements that can be made to the experiment. One extension of the experiment is a statistical analysis of the lifetime of such channels. The channels are formed by two gramicidin A molecules linked through only three hydrogen bonds that may easily break at room temperature and interrupt the flow of ions, an effect that can be observed in real-time. Channel conductance as a function of potential difference and concentration gradient are also aspects of the experiment that warrant examination.

 

References:

  1. Anderson, O. S. & Koeppe. R. E. 2nd (1992). “Molecular determinants of channel function”, Physiol. Rev. 72, S89-158.

  2. Busath, D. & Szabo, G. (1988). “Permeation characteristics of gramicidin conformers”, Biophys. J. 53 (5), 697-707.

  3. Colombini, M. (1987) “Characterization of channels isolated from plant mitochondria”, Methods Enzymol. 148, 465-75.

  4. Finkelstein, A. & Anderson, O.S. (1981), “The gramicidin A channel: a review of its permeability characteristics with special reference to the single file aspect of transport”, J. Membr. Bio. 59 (3), 155-71.

  5. Hille, B. (2001), “Ion channels of excitable membranes”, 3rd ed., Sunderland, Mass.: Sinauer.

  6. Ives-David, J. G. and George J.J. (1961), “Reference electrodes: theory and practice”, Academic Press.

  7. Montal, M. & Mueller, P. (1972), “Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties”, Proc. Natl. Acad. Sci. USA, 69 (12), 3561-6.

  8. Mueller, P., Rudin, D.O. et al. (1962), “Reconstitution of cell membrane structure in vitro and its transformation into an excitable system”, Nature, 194, 979-80.

  9. Quilter, R.E. & Surwillo, W.W. (1966), “A simple method for preparing silver-silver chloride electrodes for recording of skin-potential”, Am. J. Psychol. 79 (2), 309-13.

 

Technical Notes

 Experimental protocol for preparation of lipid bilayer membranes with gramicidin A channels

 Handling and Storage of Lipids

Material Safety Data Sheet for Lipids

Gramicidin Spec Sheet

List of chemicals

 

 

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