The theme of this work is that proteins, especially those designed to transport ions across channels, are sensitive to osmotic stress from materials unable to enter their aqueous cavities. We have used the ability to exert osmotic stress to measure the amount of water that goes in or comes out when a protein, particularly a trans-membrane ionic channel opens or closes. Among our accomplishments during the past year, we have determined that some 20 to 40 water molecules enter a typical potassium channel of the squid giant axon when it opens under electrical stimulation. These measurements, or whole axon preparations, suggest that rather significant charges in protein structure must occur during channel opening. Such large changes are quite different from what is usually imagined to be channel """"""""gating"""""""". We have developed a new method for separating out vesicles containing single channels and for incorporating these into artificial systems. This development is enabling more controlled protein reconstitution into artificial bilayer membranes for single-channel study. Major effort has been successfully expended in designing computer hardware and software to allow high-speed data acquisition while recording channel electrical activity, concurrently analyzing results and putting the variation of experimental conditions under computer control. This system, a prototype for personel computer laboratory use, also makes possible better correction with powerful mainframe equipment for more sophisticated data analyses if need be. Finally we have begun to look at the sensitivity of enzyme activity to osmotic stress to see how such stress, naturally occurring in living cells, is a part of intracellular activity.
