Voltage-dependent Block of Anthrax Toxin Channels in Planar Phospholipid Bilayer Membranes by Symmetric Tetraalkylammonium Ions

Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PAn5 fragment o f anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N + induces a flickery block of the PA65 channel when present on either side o f the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (>2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants rb and ~'u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction o f time the channel spends unblocked [Tu/(Tu + ~'b)] as a function o f voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements o f blocking, thus demonstrating that these single channels mirror the behavior seen with many (> 10,000) channels in the membrane. In going from large negative to large positive voltages ( 1 0 0 to + 160 mV) on the c/s (PA~n-containing) side of the membrane, one sees the mean blocked time (Tb) increase to a maximum at + 60 mV and then steadily decline for voltages greater than + 6 0 mV, thereby clearly demonstrating that Bu4N + is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N +. An interesting finding that emerges from analysis o f the voltage dependence o f mean blocked and unblocked times is that the blocking rate, with Bu4N + present on the c/s side o f the membrane, plateaus at large positive c/s voltages to a voltage-independent value consistent with the rate o f Bu4N + entry into the blocking site being diffusion limited. Address reprint requests to Dr. Alan Finkelstein, Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. J. GEN. PHYSIOL. O The Rockefeller University Press 9 0022-1295/90/11/0921/22 $2.00 Volume 96 November 1990 921-942 921 on Jne 5, 2017 D ow nladed fom Published November 1, 1990