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Immobilizing the moving parts of voltage-gated ion channels.

Horn R, Ding S, Gruber HJ - J. Gen. Physiol. (2000)

Bottom Line: As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments.By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation.Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA. Richard.horn@mail.tju.edu

ABSTRACT
Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

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Relative reduction of peak gating current and ionic current for Shaker-IR A359C-BP Same as for Fig. 3, except using flash-lamp stimulation and measuring the ionic current at +70 mV. Solid curves are the best fit to an independent model (see ). The dashed line shows the predicted gating current reduction for a model including cooperativity (fcoop = 0.045). Data for n = 5 cells are shown.
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Figure 5: Relative reduction of peak gating current and ionic current for Shaker-IR A359C-BP Same as for Fig. 3, except using flash-lamp stimulation and measuring the ionic current at +70 mV. Solid curves are the best fit to an independent model (see ). The dashed line shows the predicted gating current reduction for a model including cooperativity (fcoop = 0.045). Data for n = 5 cells are shown.

Mentions: UV irradiation of A359C-BP at a hyperpolarized voltage reduced both ionic and gating currents (Fig. 3 A), and, as predicted above, the inward ionic current at 0 mV was reduced more than the gating current. Comparable exposure of unlabeled A359C channels to UV light had no effect (Fig. 3 B). The relative time courses of current reduction for normalized data are plotted in Fig. 5 for five cells. For these experiments, we measured the ionic current at +70 mV, avoiding any possibility of reducing the open probability by a step from +40 to 0 mV. The reduction of ionic current (Fig. 5▾) was fit by a single exponential function raised to a fourth power ( and ) with a time constant of 6.0 ± 0.8 (units of number of UV flashes). Only ∼27% of the S4 segments are immobilized at steady state (w = 0.267 ± 0.018), indicating that β ≃ 2.7 α. In other words, photoexcitation of the benzophenone is nearly three times more likely to destroy its function than to result in an immobilizing insertion.


Immobilizing the moving parts of voltage-gated ion channels.

Horn R, Ding S, Gruber HJ - J. Gen. Physiol. (2000)

Relative reduction of peak gating current and ionic current for Shaker-IR A359C-BP Same as for Fig. 3, except using flash-lamp stimulation and measuring the ionic current at +70 mV. Solid curves are the best fit to an independent model (see ). The dashed line shows the predicted gating current reduction for a model including cooperativity (fcoop = 0.045). Data for n = 5 cells are shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233689&req=5

Figure 5: Relative reduction of peak gating current and ionic current for Shaker-IR A359C-BP Same as for Fig. 3, except using flash-lamp stimulation and measuring the ionic current at +70 mV. Solid curves are the best fit to an independent model (see ). The dashed line shows the predicted gating current reduction for a model including cooperativity (fcoop = 0.045). Data for n = 5 cells are shown.
Mentions: UV irradiation of A359C-BP at a hyperpolarized voltage reduced both ionic and gating currents (Fig. 3 A), and, as predicted above, the inward ionic current at 0 mV was reduced more than the gating current. Comparable exposure of unlabeled A359C channels to UV light had no effect (Fig. 3 B). The relative time courses of current reduction for normalized data are plotted in Fig. 5 for five cells. For these experiments, we measured the ionic current at +70 mV, avoiding any possibility of reducing the open probability by a step from +40 to 0 mV. The reduction of ionic current (Fig. 5▾) was fit by a single exponential function raised to a fourth power ( and ) with a time constant of 6.0 ± 0.8 (units of number of UV flashes). Only ∼27% of the S4 segments are immobilized at steady state (w = 0.267 ± 0.018), indicating that β ≃ 2.7 α. In other words, photoexcitation of the benzophenone is nearly three times more likely to destroy its function than to result in an immobilizing insertion.

Bottom Line: As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments.By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation.Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA. Richard.horn@mail.tju.edu

ABSTRACT
Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

Show MeSH
Related in: MedlinePlus