Current flow through the channel formed in planar phospholipid hilayer membranes by the PA65 fragment of anthrax toxin is blocked, in a voltage-dependent manner, by tetraalkylammonium ions (at micromolar concentrations), which bind to a blocking site within the channel lumen. We have presented evidence that diffusion plays a significant role in the kinetics of blocking by tetrabutylammonium ion (Bu4N+) from the cis (toxin-containing) side of the membrane (Blaustein, R. O., E.J.A. Lea, and A. Finkelstein. 1990. f Gen. Physiol. 96:921-942); in this paper we examine the implications and consequences of diffusion control for binding kinetics. As expected for a diffusion-affected reaction, both the entry rate constant (ka1cis) of Bu4N+ from the cis solution to the blocking site and the exit rate constant (k-1cis) of Bu4N+ from the blocking site to the c/s solution are reduced if the viscosity of that medium is increased by the addition of dextran. In conformity with both thermodynamics and kinetic arguments, however, the voltage-dependent equilibrium binding constant, Keq (=k-1cis/k1cis), is not altered by the dextrarl-ir-duced viscosity increase of the c/a solution. The entry rate constants (k1cis) for tetrapentylammonium (Pe4N+), tetrahexylalmnonium (Hx4N+), and tetraheptylammonium (Hp4N+) are also diffusion controlled, and all of them, including that for Bu4 N+, attain a voltage-independent plateau value at large positive cis voltages consistent with diffusion limitation. Although the plateau value of k1cis for Hx4N+ is only a factor of 3 less than that for Bu4N+, the plateau value for Hp4N+ is a factor of 35 less. This precipitous fall in value indicates, from diffusionlimitation theory, that the diameter of the channel entrance facing the cis solution is not much larger than the diameter of Hp4N+, i.e., ~12Å.
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