Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PA65 fragment of 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 of 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 τb and τu that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [τu/(τu + τb)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of 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 (-100 to + 160 mV) on the cis (PA65-containing) side of the membrane, one sees the mean blocked time (τb) increase to a maximum at + 60 mV and then steadily decline for voltages greater than +60 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 of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N+ present on the cis side of the membrane, plateaus at large positive c/s voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.
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