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Evidence for a proton-protein symport mechanism in the anthrax toxin channel.

Basilio D, Juris SJ, Collier RJ, Finkelstein A - J. Gen. Physiol. (2009)

Bottom Line: Therefore, the translocated species is positively charged.Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited.We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dbseyler@aecom.yu.edu

ABSTRACT
The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

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The effect of mutating F427 to an alanine on the ion selectivity of the (PA63)7 channel. The experiments were started with the membrane (diphytanoyl-phosphatidylcholine) separating symmetric solutions of 100 mM KCl, 5 mM potassium succinate, and 1 mM EDTA, pH 5.5. After treating the membrane with either valinomycin (0.2 µg/ml), WT (PA63)7, or (PA63F427A)7, KCl was added in steps to the cis solution, and for each step the reversal potential (Erev) was measured. Plotted in the figure is Erev versus the activity ratio of KCl (acis/atrans). (Activity coefficients were obtained from Appendix 8.10, Table II in Robinson and Stokes [1965], where we took the KCl concentration to be equal to the K+ concentration. Approximately 10 mM of K+ was contributed by K-succinate and K-EDTA.) We see in the figure that, as expected, the valinomycin-treated membrane (open circles) displayed ideal K+ selectivity (continuous line). The selectivity of the WT (PA63)7–treated membrane (filled circles) deviated somewhat from ideal cation selectivity, and that of the (PA63F427A)7–treated membrane (filled triangles) deviated even more so; that is, it was more permeable to Cl− than was the WT channel. The dotted lines are drawn to connect the points. (Applying inappropriately, as usual, the Goldman-Hodgkin-Katz equation to the data, we calculate that PK/PCl is 21 and 7.6 for the WT (PA63)7 and (PA63F427A)7 channels, respectively.)
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fig7: The effect of mutating F427 to an alanine on the ion selectivity of the (PA63)7 channel. The experiments were started with the membrane (diphytanoyl-phosphatidylcholine) separating symmetric solutions of 100 mM KCl, 5 mM potassium succinate, and 1 mM EDTA, pH 5.5. After treating the membrane with either valinomycin (0.2 µg/ml), WT (PA63)7, or (PA63F427A)7, KCl was added in steps to the cis solution, and for each step the reversal potential (Erev) was measured. Plotted in the figure is Erev versus the activity ratio of KCl (acis/atrans). (Activity coefficients were obtained from Appendix 8.10, Table II in Robinson and Stokes [1965], where we took the KCl concentration to be equal to the K+ concentration. Approximately 10 mM of K+ was contributed by K-succinate and K-EDTA.) We see in the figure that, as expected, the valinomycin-treated membrane (open circles) displayed ideal K+ selectivity (continuous line). The selectivity of the WT (PA63)7–treated membrane (filled circles) deviated somewhat from ideal cation selectivity, and that of the (PA63F427A)7–treated membrane (filled triangles) deviated even more so; that is, it was more permeable to Cl− than was the WT channel. The dotted lines are drawn to connect the points. (Applying inappropriately, as usual, the Goldman-Hodgkin-Katz equation to the data, we calculate that PK/PCl is 21 and 7.6 for the WT (PA63)7 and (PA63F427A)7 channels, respectively.)

Mentions: Where within the channel is the negative charge exclusion of SO3− occurring? The (PA63)7 channel is a mushroom-like structure with a long stem and a cap that contains the binding site for LFN (Fig. 6 B) (Krantz et al., 2005; Katayama et al., 2008). Near the junction of the cap with the stem lies residue F427, which forms a ring of seven phenylalanines that plays an important role in protein translocation, and which we have dubbed the Φ-clamp (Krantz et al., 2005). If the phenylalanines were mutated to alanines, protein translocation was compromised (Krantz et al., 2005, 2006). When F427 was mutated to an alanine, the rate of voltage-driven translocation decreased by about a factor of three, but, strikingly, the rate of translocation of LFN with an attached SO3− enormously increased. (A much larger decrease occurs in the rate of translocation driven by a pH gradient [Krantz et al., 2005, 2006] and is probably a consequence of the creation of a leakage pathway for protons, thereby compromising the ΔpH across the Φ-clamp.) This effect of the alanine mutation is clearly seen in Fig. 6 A for LFNA59C; a similar effect was seen for the other residue tested, LFNN242C (not depicted). With either WT LFN or the neutral (CH2)2-CONH2 attached to the LFNA59C cysteine, the half-time of translocation (at +55 mV) increased from ∼5 to ∼15 s, whereas with SO3− attached to the cysteine, the half-time decreased from a value >>200 to ∼30 s. Thus, the mutation of F427 to an alanine has almost completely removed a barrier to SO3− translocation. The effect of the Φ-clamp on anion entry into the channel was also manifested in the channel becoming less selective for K+ over Cl−. We see in Fig. 7 that the (PA63F427A)7 channel, although still cation selective, is less so than is the WT channel.


Evidence for a proton-protein symport mechanism in the anthrax toxin channel.

Basilio D, Juris SJ, Collier RJ, Finkelstein A - J. Gen. Physiol. (2009)

The effect of mutating F427 to an alanine on the ion selectivity of the (PA63)7 channel. The experiments were started with the membrane (diphytanoyl-phosphatidylcholine) separating symmetric solutions of 100 mM KCl, 5 mM potassium succinate, and 1 mM EDTA, pH 5.5. After treating the membrane with either valinomycin (0.2 µg/ml), WT (PA63)7, or (PA63F427A)7, KCl was added in steps to the cis solution, and for each step the reversal potential (Erev) was measured. Plotted in the figure is Erev versus the activity ratio of KCl (acis/atrans). (Activity coefficients were obtained from Appendix 8.10, Table II in Robinson and Stokes [1965], where we took the KCl concentration to be equal to the K+ concentration. Approximately 10 mM of K+ was contributed by K-succinate and K-EDTA.) We see in the figure that, as expected, the valinomycin-treated membrane (open circles) displayed ideal K+ selectivity (continuous line). The selectivity of the WT (PA63)7–treated membrane (filled circles) deviated somewhat from ideal cation selectivity, and that of the (PA63F427A)7–treated membrane (filled triangles) deviated even more so; that is, it was more permeable to Cl− than was the WT channel. The dotted lines are drawn to connect the points. (Applying inappropriately, as usual, the Goldman-Hodgkin-Katz equation to the data, we calculate that PK/PCl is 21 and 7.6 for the WT (PA63)7 and (PA63F427A)7 channels, respectively.)
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654084&req=5

fig7: The effect of mutating F427 to an alanine on the ion selectivity of the (PA63)7 channel. The experiments were started with the membrane (diphytanoyl-phosphatidylcholine) separating symmetric solutions of 100 mM KCl, 5 mM potassium succinate, and 1 mM EDTA, pH 5.5. After treating the membrane with either valinomycin (0.2 µg/ml), WT (PA63)7, or (PA63F427A)7, KCl was added in steps to the cis solution, and for each step the reversal potential (Erev) was measured. Plotted in the figure is Erev versus the activity ratio of KCl (acis/atrans). (Activity coefficients were obtained from Appendix 8.10, Table II in Robinson and Stokes [1965], where we took the KCl concentration to be equal to the K+ concentration. Approximately 10 mM of K+ was contributed by K-succinate and K-EDTA.) We see in the figure that, as expected, the valinomycin-treated membrane (open circles) displayed ideal K+ selectivity (continuous line). The selectivity of the WT (PA63)7–treated membrane (filled circles) deviated somewhat from ideal cation selectivity, and that of the (PA63F427A)7–treated membrane (filled triangles) deviated even more so; that is, it was more permeable to Cl− than was the WT channel. The dotted lines are drawn to connect the points. (Applying inappropriately, as usual, the Goldman-Hodgkin-Katz equation to the data, we calculate that PK/PCl is 21 and 7.6 for the WT (PA63)7 and (PA63F427A)7 channels, respectively.)
Mentions: Where within the channel is the negative charge exclusion of SO3− occurring? The (PA63)7 channel is a mushroom-like structure with a long stem and a cap that contains the binding site for LFN (Fig. 6 B) (Krantz et al., 2005; Katayama et al., 2008). Near the junction of the cap with the stem lies residue F427, which forms a ring of seven phenylalanines that plays an important role in protein translocation, and which we have dubbed the Φ-clamp (Krantz et al., 2005). If the phenylalanines were mutated to alanines, protein translocation was compromised (Krantz et al., 2005, 2006). When F427 was mutated to an alanine, the rate of voltage-driven translocation decreased by about a factor of three, but, strikingly, the rate of translocation of LFN with an attached SO3− enormously increased. (A much larger decrease occurs in the rate of translocation driven by a pH gradient [Krantz et al., 2005, 2006] and is probably a consequence of the creation of a leakage pathway for protons, thereby compromising the ΔpH across the Φ-clamp.) This effect of the alanine mutation is clearly seen in Fig. 6 A for LFNA59C; a similar effect was seen for the other residue tested, LFNN242C (not depicted). With either WT LFN or the neutral (CH2)2-CONH2 attached to the LFNA59C cysteine, the half-time of translocation (at +55 mV) increased from ∼5 to ∼15 s, whereas with SO3− attached to the cysteine, the half-time decreased from a value >>200 to ∼30 s. Thus, the mutation of F427 to an alanine has almost completely removed a barrier to SO3− translocation. The effect of the Φ-clamp on anion entry into the channel was also manifested in the channel becoming less selective for K+ over Cl−. We see in Fig. 7 that the (PA63F427A)7 channel, although still cation selective, is less so than is the WT channel.

Bottom Line: Therefore, the translocated species is positively charged.Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited.We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dbseyler@aecom.yu.edu

ABSTRACT
The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

Show MeSH
Related in: MedlinePlus