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The effect of enzymatic proteolysis on structural and functional properties of the isolated lobster nerve membrane was investigated. The membranes were treated with different amounts of either trypsin or unspecific protease. Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in proteoliposomes prepared with nerve membrane and soybean lipids. The changes in the membrane proteins were followed by electrophoresis in polyacrylamide gradient gels. From the densitometric scan of the gels the relative area for each protein was obtained, and the ratio of enzyme-treated to control areas was evaluated. Under a similar degree of proteolysis catalyzed either by trypsin or by unspecific protease, the sensitive sodium influx is not affected by trypsin, whereas it is about 60% diminished by the unspecific protease. In this condition the zones corresponding to molecular weights of 240,000 and 166,000 daltons appear modified in the electrophoretic gels by both enzymes. The 117,000-dalton range is modified only by the unspecific protease. Increasing trypsin concentration diminishes sodium influx about 60%; and the 240,000-, 166,000-, and 117,000-dalton zones appear modified. A further increase of the protease concentration totally abolishes the sensitive sodium influx and modifies practically all of the membrane proteins. The present results indicate the rather high sensitivity of the membrane sodium channel activity to proteolytic action, and show that the membrane sites that respond to veratridine appear to be highly affected by proteolysis. In contrast, the tetrodotoxin receptor retains its binding capacity even after treatment of the membrane with protease concentrations 1,000 times higher than those affecting the sensitive sodium influx [Benzer and Raftery, 1972; Villegas et al, 1973].

The nonlinear cable equation was solved numerically by means of an implicit procedure. The correlation between end-plate length and fiber diameter was determined in frog (Rana pipiens) sartorius muscles stained with gold chloride (Löwit, 1875). The diameter of the fibers stained by the Löwit method was 80 (74-85) micron (median and its 95% confidence interval for 52 fibers), the length of the end plates in the same fibers was 382 (353-417) micron. The fibers simulated were 80 micron in diameter. To solve the equation the muscle fibers were represented by 500 segments 20 micron long, and the equation was solved in steps of 10 microseconds; a double exponential function was incorporated to the first seven segments to represent the neuromuscular junction. The potential of the first segment of the cable was set to the clamping level and the membrane potential of the remaining segments calculated. The current needed to hold the first segment was estimated by adding the current flowing through the first segment to the current flowing from it to the second segment. Our results indicate that the lack of space clamp in the point voltage-clamp studies of the frog neuromuscular junction introduces serious errors in the estimates of the end-plate conductance value, the kinetics of the conductance changes, and the reversal potential of the end-plate currents. The possibility of an efficient voltage-clamp technique is also explored. Our calculations suggest that the study of end-plate current and conductance is possible with little error if the end-plate potential is controlled at both ends of the synaptic area simultaneously.