Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions: Single-channel analysis

Robert O. Blaustein, E. J A Lea, Alan Finkelstein

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Abstract

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 cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.

Original languageEnglish (US)
Pages (from-to)921-942
Number of pages22
JournalJournal of General Physiology
Volume96
Issue number5
StatePublished - Nov 1990

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Ion Channels
Phospholipids
Membranes
Ions
anthrax toxin

ASJC Scopus subject areas

  • Physiology

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Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions : Single-channel analysis. / Blaustein, Robert O.; Lea, E. J A; Finkelstein, Alan.

In: Journal of General Physiology, Vol. 96, No. 5, 11.1990, p. 921-942.

Research output: Contribution to journalArticle

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abstract = "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 cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.",
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