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A kinetic analysis of protein transport through the anthrax toxin channel.

Basilio D, Kienker PK, Briggs SW, Finkelstein A - J. Gen. Physiol. (2011)

Bottom Line: As expected, the translocation rate is slower with more than one LF(N) bound.We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field.The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dab2043@-med.cornell.edu

ABSTRACT
Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

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The translocation kinetics of His6-LFN through the (PA63)7 channel determined from macroscopic experiments compared with that determined from single-channel experiments. The protocol before stepping the voltage from 20 to 50 mV in the macroscopic experiments (the red and black curves) was the same as that described in the legend to Fig. 2. The cumulative probability distribution of translocation times (blue curve) was obtained from a survival curve (derived from single-channel experiments), such as that shown in Fig. 6, except that in this case the voltage was stepped from 20 to 50 mV (instead of to 48 mV). In the macroscopic experiment depicted by the red curve, the concentration of His6-LFN was so small that it only produced an 18% fall in conductance. Thus, virtually all of the blocked channels had only one His6-LFN bound to them. Note that the kinetics are identical to those obtained from the single-channel experiment (blue curve). In the macroscopic experiment depicted by the black curve, the concentration of His6-LFN was much larger, producing a 94% fall in conductance. In this case, ∼70% of the blocked channels had two or three His6-LFNs bound to them (Fig. 4). Note that the rate of unblocking in this case was, as expected, slower than in the case when the blocked channels contained only one His6-LFN. The cartoon indicates the percentage of channels that have zero, one, two, and three His6-LFNs bound to them when there was an 18% fall in conductance produced by His6-LFN (red numbers) and when there was a 94% fall in conductance (black numbers).
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fig7: The translocation kinetics of His6-LFN through the (PA63)7 channel determined from macroscopic experiments compared with that determined from single-channel experiments. The protocol before stepping the voltage from 20 to 50 mV in the macroscopic experiments (the red and black curves) was the same as that described in the legend to Fig. 2. The cumulative probability distribution of translocation times (blue curve) was obtained from a survival curve (derived from single-channel experiments), such as that shown in Fig. 6, except that in this case the voltage was stepped from 20 to 50 mV (instead of to 48 mV). In the macroscopic experiment depicted by the red curve, the concentration of His6-LFN was so small that it only produced an 18% fall in conductance. Thus, virtually all of the blocked channels had only one His6-LFN bound to them. Note that the kinetics are identical to those obtained from the single-channel experiment (blue curve). In the macroscopic experiment depicted by the black curve, the concentration of His6-LFN was much larger, producing a 94% fall in conductance. In this case, ∼70% of the blocked channels had two or three His6-LFNs bound to them (Fig. 4). Note that the rate of unblocking in this case was, as expected, slower than in the case when the blocked channels contained only one His6-LFN. The cartoon indicates the percentage of channels that have zero, one, two, and three His6-LFNs bound to them when there was an 18% fall in conductance produced by His6-LFN (red numbers) and when there was a 94% fall in conductance (black numbers).

Mentions: PA63 prepore heptamer was added to the cis-compartment (to a final concentration of ∼10 pg/ml [∼20 fM]), which was held at a V of 20 mV with respect to the trans-compartment. After ∼20 min. a single channel appeared, and His6-LFN was added to the cis-compartment (final concentration of ∼30 pM). Typically, there was an ∼1-min waiting time to see a blocking event. After 5 s in the blocked state, the voltage was stepped from 20 to either 48 mV in one experiment or 50 mV in another experiment until the single-channel current reappeared (that is, His6-LFN had been translocated through the channel). The voltage was then stepped back to 20 mV, and this maneuver was repeated (Fig. 6, inset). Under this protocol, one can assume that generally only one molecule of His6-LFN was translocated, given that the mean time of blocking was five times shorter than the waiting time before the voltage step. The interval between the voltage step (from 20 to 48 or 50 mV) and the reopening of the channel is the lag time (Δt) used to build the survival curve presented in Fig. 6; this curve, by normalizing and inverting, is readily converted to a cumulative probability distribution of translocation times as depicted in Fig. 7.


A kinetic analysis of protein transport through the anthrax toxin channel.

Basilio D, Kienker PK, Briggs SW, Finkelstein A - J. Gen. Physiol. (2011)

The translocation kinetics of His6-LFN through the (PA63)7 channel determined from macroscopic experiments compared with that determined from single-channel experiments. The protocol before stepping the voltage from 20 to 50 mV in the macroscopic experiments (the red and black curves) was the same as that described in the legend to Fig. 2. The cumulative probability distribution of translocation times (blue curve) was obtained from a survival curve (derived from single-channel experiments), such as that shown in Fig. 6, except that in this case the voltage was stepped from 20 to 50 mV (instead of to 48 mV). In the macroscopic experiment depicted by the red curve, the concentration of His6-LFN was so small that it only produced an 18% fall in conductance. Thus, virtually all of the blocked channels had only one His6-LFN bound to them. Note that the kinetics are identical to those obtained from the single-channel experiment (blue curve). In the macroscopic experiment depicted by the black curve, the concentration of His6-LFN was much larger, producing a 94% fall in conductance. In this case, ∼70% of the blocked channels had two or three His6-LFNs bound to them (Fig. 4). Note that the rate of unblocking in this case was, as expected, slower than in the case when the blocked channels contained only one His6-LFN. The cartoon indicates the percentage of channels that have zero, one, two, and three His6-LFNs bound to them when there was an 18% fall in conductance produced by His6-LFN (red numbers) and when there was a 94% fall in conductance (black numbers).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105512&req=5

fig7: The translocation kinetics of His6-LFN through the (PA63)7 channel determined from macroscopic experiments compared with that determined from single-channel experiments. The protocol before stepping the voltage from 20 to 50 mV in the macroscopic experiments (the red and black curves) was the same as that described in the legend to Fig. 2. The cumulative probability distribution of translocation times (blue curve) was obtained from a survival curve (derived from single-channel experiments), such as that shown in Fig. 6, except that in this case the voltage was stepped from 20 to 50 mV (instead of to 48 mV). In the macroscopic experiment depicted by the red curve, the concentration of His6-LFN was so small that it only produced an 18% fall in conductance. Thus, virtually all of the blocked channels had only one His6-LFN bound to them. Note that the kinetics are identical to those obtained from the single-channel experiment (blue curve). In the macroscopic experiment depicted by the black curve, the concentration of His6-LFN was much larger, producing a 94% fall in conductance. In this case, ∼70% of the blocked channels had two or three His6-LFNs bound to them (Fig. 4). Note that the rate of unblocking in this case was, as expected, slower than in the case when the blocked channels contained only one His6-LFN. The cartoon indicates the percentage of channels that have zero, one, two, and three His6-LFNs bound to them when there was an 18% fall in conductance produced by His6-LFN (red numbers) and when there was a 94% fall in conductance (black numbers).
Mentions: PA63 prepore heptamer was added to the cis-compartment (to a final concentration of ∼10 pg/ml [∼20 fM]), which was held at a V of 20 mV with respect to the trans-compartment. After ∼20 min. a single channel appeared, and His6-LFN was added to the cis-compartment (final concentration of ∼30 pM). Typically, there was an ∼1-min waiting time to see a blocking event. After 5 s in the blocked state, the voltage was stepped from 20 to either 48 mV in one experiment or 50 mV in another experiment until the single-channel current reappeared (that is, His6-LFN had been translocated through the channel). The voltage was then stepped back to 20 mV, and this maneuver was repeated (Fig. 6, inset). Under this protocol, one can assume that generally only one molecule of His6-LFN was translocated, given that the mean time of blocking was five times shorter than the waiting time before the voltage step. The interval between the voltage step (from 20 to 48 or 50 mV) and the reopening of the channel is the lag time (Δt) used to build the survival curve presented in Fig. 6; this curve, by normalizing and inverting, is readily converted to a cumulative probability distribution of translocation times as depicted in Fig. 7.

Bottom Line: As expected, the translocation rate is slower with more than one LF(N) bound.We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field.The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dab2043@-med.cornell.edu

ABSTRACT
Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Show MeSH
Related in: MedlinePlus