Information is transmitted along nerve cells by the controlled opening and closing of protein channels in the nerve membrane. We have applied our technique of osmotic stress to the squid giant axon to measure the change in volume of the potassium channels. Some 20 molecules of water appear to enter the average channel when it opens. We postulate a number of closed states under voltage control and a final opening state that is voltage-independent but osmotically sensitive. Towards a more detailed study of protein channels in planar bilayers of defined lipid environment, we have been developing a PC computer software/hardware combination to accurately measure equilibrium statistics of channel conformation. We are currently interfacing the computer to the experimental setups, to allow closed-loop voltage control as well. We have used our new techniques for membrane channel purification and incorporation into planar lipid membranes to study the gap junction of the rat liver (connexin32). We isolated vesicles permeable to sucrose which contained junctional material. When fused to bilayers, these vesicles induced channel activity which had three major conductance levels (20, 50 and 150 pS), were asymmetrically voltage-dependent, and somewhat anion selective. Western blotting techniques quantitatively confirmed the presence of connexin32 in the shifted vesicles in amounts more than sufficient to mediate the shift.
