Limits...
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.

Show MeSH

Related in: MedlinePlus

The S-shaped kinetics of LFN translocation through the (PA63)7 channel. After the (PA63)7-induced conductance had reached a more or less steady state, LFN (with the N-terminal His6 tag removed) was added (at the arrow) to the cis-side to a concentration of 6 nM, resulting in a rapid fall in conductance. LFN (along with (PA63)7) was then perfused out of the cis-compartment (during the ∼4-min break in the record); the conductance increased only slightly over this time. When the voltage was stepped from 20 to 45 mV, there was an S-shaped rise of conductance to a value comparable with that before the addition of LFN, and it remained at that value when the voltage was stepped back from 45 to 20 mV. The rate of conductance rise directly reflects the rate of translocation of LFN; i.e., the rate of its traversing the channel and exiting into the trans-solution. This figure was adapted from Fig. 2 of Finkelstein (2009).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3105512&req=5

fig2: The S-shaped kinetics of LFN translocation through the (PA63)7 channel. After the (PA63)7-induced conductance had reached a more or less steady state, LFN (with the N-terminal His6 tag removed) was added (at the arrow) to the cis-side to a concentration of 6 nM, resulting in a rapid fall in conductance. LFN (along with (PA63)7) was then perfused out of the cis-compartment (during the ∼4-min break in the record); the conductance increased only slightly over this time. When the voltage was stepped from 20 to 45 mV, there was an S-shaped rise of conductance to a value comparable with that before the addition of LFN, and it remained at that value when the voltage was stepped back from 45 to 20 mV. The rate of conductance rise directly reflects the rate of translocation of LFN; i.e., the rate of its traversing the channel and exiting into the trans-solution. This figure was adapted from Fig. 2 of Finkelstein (2009).

Mentions: A characteristic of the translocation of LFN through the (PA63)7 channel is its nonexponential S-shaped or sigmoidal kinetics (Fig. 2). That is, after the voltage step, there is a lag time with minimal conductance increase followed by a more rapid increase. In general, this sort of S-shaped kinetics is expected for a process with multiple sequential steps, even if the individual steps have exponential kinetics. Under the conditions of the experiments reported in the literature (e.g., Zhang et al., 2004b; Krantz et al., 2005, 2006) as well as those for the experiment depicted in Fig. 2, it is likely that most of the (PA63)7 channels bound two or three LFNs (see Theory section). If this is so, it is possible that the S-shaped kinetics of translocation are solely caused by this and are not an intrinsic characteristic of the kinetics of translocation. Before one can make a quantitative kinetic model of translocation, it is essential to determine the translocation kinetics of a single LFN molecule. In this paper, we address this issue both at the macroscopic and single-channel level. We report that with only one LFN bound to the channel, both the macroscopic and single-channel experiments gave S-shaped kinetics, that they were in quantitative agreement with each other, and, as might be expected, their kinetics of translocation were faster than those obtained in experiments with multiple LFN occupancy. In addition, we present a simple drift-diffusion model of translocation in which LFN is represented as a charged rod that moves under the combined influence of random thermal motion and an applied electrical potential difference. This model adequately accounts for the S-shaped kinetics and their voltage dependence.


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

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

The S-shaped kinetics of LFN translocation through the (PA63)7 channel. After the (PA63)7-induced conductance had reached a more or less steady state, LFN (with the N-terminal His6 tag removed) was added (at the arrow) to the cis-side to a concentration of 6 nM, resulting in a rapid fall in conductance. LFN (along with (PA63)7) was then perfused out of the cis-compartment (during the ∼4-min break in the record); the conductance increased only slightly over this time. When the voltage was stepped from 20 to 45 mV, there was an S-shaped rise of conductance to a value comparable with that before the addition of LFN, and it remained at that value when the voltage was stepped back from 45 to 20 mV. The rate of conductance rise directly reflects the rate of translocation of LFN; i.e., the rate of its traversing the channel and exiting into the trans-solution. This figure was adapted from Fig. 2 of Finkelstein (2009).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105512&req=5

fig2: The S-shaped kinetics of LFN translocation through the (PA63)7 channel. After the (PA63)7-induced conductance had reached a more or less steady state, LFN (with the N-terminal His6 tag removed) was added (at the arrow) to the cis-side to a concentration of 6 nM, resulting in a rapid fall in conductance. LFN (along with (PA63)7) was then perfused out of the cis-compartment (during the ∼4-min break in the record); the conductance increased only slightly over this time. When the voltage was stepped from 20 to 45 mV, there was an S-shaped rise of conductance to a value comparable with that before the addition of LFN, and it remained at that value when the voltage was stepped back from 45 to 20 mV. The rate of conductance rise directly reflects the rate of translocation of LFN; i.e., the rate of its traversing the channel and exiting into the trans-solution. This figure was adapted from Fig. 2 of Finkelstein (2009).
Mentions: A characteristic of the translocation of LFN through the (PA63)7 channel is its nonexponential S-shaped or sigmoidal kinetics (Fig. 2). That is, after the voltage step, there is a lag time with minimal conductance increase followed by a more rapid increase. In general, this sort of S-shaped kinetics is expected for a process with multiple sequential steps, even if the individual steps have exponential kinetics. Under the conditions of the experiments reported in the literature (e.g., Zhang et al., 2004b; Krantz et al., 2005, 2006) as well as those for the experiment depicted in Fig. 2, it is likely that most of the (PA63)7 channels bound two or three LFNs (see Theory section). If this is so, it is possible that the S-shaped kinetics of translocation are solely caused by this and are not an intrinsic characteristic of the kinetics of translocation. Before one can make a quantitative kinetic model of translocation, it is essential to determine the translocation kinetics of a single LFN molecule. In this paper, we address this issue both at the macroscopic and single-channel level. We report that with only one LFN bound to the channel, both the macroscopic and single-channel experiments gave S-shaped kinetics, that they were in quantitative agreement with each other, and, as might be expected, their kinetics of translocation were faster than those obtained in experiments with multiple LFN occupancy. In addition, we present a simple drift-diffusion model of translocation in which LFN is represented as a charged rod that moves under the combined influence of random thermal motion and an applied electrical potential difference. This model adequately accounts for the S-shaped kinetics and their voltage dependence.

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