Limits...
Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.

Kienker PK, Wu Z, Finkelstein A - J. Gen. Physiol. (2015)

Bottom Line: Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface.Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side.We also determined that this constriction is located near the middle of the TH8 helix.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 paul.kienker@einstein.yu.edu.

Show MeSH

Related in: MedlinePlus

Estimation of MTS-ET reaction rates at 0 mV. (A) S305C. (B) S312C. Each graph is a semilog plot of the second-order rate constant, k, for cis (●) or trans (Δ) MTS-ET reaction versus voltage. Each data point represents one experiment. Linear fits of log(k) versus voltage are shown. Interpolating to 0 mV gives kcis/ktrans ≈ 2.4 for S305C and 0.7 for S312C. Solutions were as in Fig. 2, except that for A, the buffers were 30 mM MES, pH 5.3 (cis), versus 50 mM HEPES, pH 7.2 (trans).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4306713&req=5

fig6: Estimation of MTS-ET reaction rates at 0 mV. (A) S305C. (B) S312C. Each graph is a semilog plot of the second-order rate constant, k, for cis (●) or trans (Δ) MTS-ET reaction versus voltage. Each data point represents one experiment. Linear fits of log(k) versus voltage are shown. Interpolating to 0 mV gives kcis/ktrans ≈ 2.4 for S305C and 0.7 for S312C. Solutions were as in Fig. 2, except that for A, the buffers were 30 mM MES, pH 5.3 (cis), versus 50 mM HEPES, pH 7.2 (trans).

Mentions: Protection of S312C channels from reaction with MTS-ET by His6-tag blockade. Records from three experiments are superimposed in each panel; the top traces are normalized current, and the bottom traces are voltage, with all plotted against time (relative to the moment of MTS-ET addition). The protection experiments (blue and green traces) used S312C with an amino-terminal His6-tag, whereas the unblocked control experiments (red traces) used S312C from which the His6-tag had been removed by thrombin treatment. About 4–10 min before the start of each record, 8–9 ng S312C was added to the cis compartment, and the channel activity was allowed to stabilize. At the arrow, while holding at −30 mV, 2.6 mg MTS-ET was added to the cis (A) or trans (B) compartment. In the unblocked control experiments (red traces), reaction with MTS-ET at −30 mV caused a decrease in conductance that was largely complete within 1–2 min. In the His6-tag protection experiments (blue and green traces), the potential was held at −30 mV for 1–4 min of exposure to MTS-ET. Upon switching to 60 mV, there was a rapid increase in current, representing unblocking of the channels by the His6-tag, followed by a slower decay of the current, representing reaction with MTS-ET. (No such decay was seen without MTS addition.) The relatively large peak currents after unblocking show that His6-tag blockade protected Cys-312 from reaction. Note that the reaction rate of cis MTS-ET with S312C channels is voltage dependent (see Fig. 6 B); the relevant rate here is that at −30 mV (seen in the unblocked control experiment), rather than the faster rate seen at 60 mV. Solutions were as in Fig. 2. Some of the voltage traces are shown as dashed lines so that all the superimposed traces can be seen. The time scale bar applies to both panels.


Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.

Kienker PK, Wu Z, Finkelstein A - J. Gen. Physiol. (2015)

Estimation of MTS-ET reaction rates at 0 mV. (A) S305C. (B) S312C. Each graph is a semilog plot of the second-order rate constant, k, for cis (●) or trans (Δ) MTS-ET reaction versus voltage. Each data point represents one experiment. Linear fits of log(k) versus voltage are shown. Interpolating to 0 mV gives kcis/ktrans ≈ 2.4 for S305C and 0.7 for S312C. Solutions were as in Fig. 2, except that for A, the buffers were 30 mM MES, pH 5.3 (cis), versus 50 mM HEPES, pH 7.2 (trans).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4306713&req=5

fig6: Estimation of MTS-ET reaction rates at 0 mV. (A) S305C. (B) S312C. Each graph is a semilog plot of the second-order rate constant, k, for cis (●) or trans (Δ) MTS-ET reaction versus voltage. Each data point represents one experiment. Linear fits of log(k) versus voltage are shown. Interpolating to 0 mV gives kcis/ktrans ≈ 2.4 for S305C and 0.7 for S312C. Solutions were as in Fig. 2, except that for A, the buffers were 30 mM MES, pH 5.3 (cis), versus 50 mM HEPES, pH 7.2 (trans).
Mentions: Protection of S312C channels from reaction with MTS-ET by His6-tag blockade. Records from three experiments are superimposed in each panel; the top traces are normalized current, and the bottom traces are voltage, with all plotted against time (relative to the moment of MTS-ET addition). The protection experiments (blue and green traces) used S312C with an amino-terminal His6-tag, whereas the unblocked control experiments (red traces) used S312C from which the His6-tag had been removed by thrombin treatment. About 4–10 min before the start of each record, 8–9 ng S312C was added to the cis compartment, and the channel activity was allowed to stabilize. At the arrow, while holding at −30 mV, 2.6 mg MTS-ET was added to the cis (A) or trans (B) compartment. In the unblocked control experiments (red traces), reaction with MTS-ET at −30 mV caused a decrease in conductance that was largely complete within 1–2 min. In the His6-tag protection experiments (blue and green traces), the potential was held at −30 mV for 1–4 min of exposure to MTS-ET. Upon switching to 60 mV, there was a rapid increase in current, representing unblocking of the channels by the His6-tag, followed by a slower decay of the current, representing reaction with MTS-ET. (No such decay was seen without MTS addition.) The relatively large peak currents after unblocking show that His6-tag blockade protected Cys-312 from reaction. Note that the reaction rate of cis MTS-ET with S312C channels is voltage dependent (see Fig. 6 B); the relevant rate here is that at −30 mV (seen in the unblocked control experiment), rather than the faster rate seen at 60 mV. Solutions were as in Fig. 2. Some of the voltage traces are shown as dashed lines so that all the superimposed traces can be seen. The time scale bar applies to both panels.

Bottom Line: Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface.Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side.We also determined that this constriction is located near the middle of the TH8 helix.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 paul.kienker@einstein.yu.edu.

Show MeSH
Related in: MedlinePlus