Limits...
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.

Show MeSH

Related in: MedlinePlus

Photocross-linking D4/S4 mutants. Black and blue traces represent currents before and after, respectively, exposure to continuous-power UV light at the holding potential of −150 mV. All cysteine mutants were labeled with BPMTS. (A) D4:R1C-BP currents at 0 mV. Traces show progressive effects of 4.2-s exposures to UV light. This residue was labeled externally. (B) D4:R3C labeled with extracellular BPMTS. Currents at −30 mV using 4-s exposures to UV light. (C) D4:R3C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −30 mV using 4-s exposures to UV light. (D) D4:R4C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −45 mV using 4-s exposures to UV light. The current reductions seen in C and D were uniform over all voltages, as seen for D2:R1C-BP in Fig. 7 C.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233689&req=5

Figure 8: Photocross-linking D4/S4 mutants. Black and blue traces represent currents before and after, respectively, exposure to continuous-power UV light at the holding potential of −150 mV. All cysteine mutants were labeled with BPMTS. (A) D4:R1C-BP currents at 0 mV. Traces show progressive effects of 4.2-s exposures to UV light. This residue was labeled externally. (B) D4:R3C labeled with extracellular BPMTS. Currents at −30 mV using 4-s exposures to UV light. (C) D4:R3C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −30 mV using 4-s exposures to UV light. (D) D4:R4C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −45 mV using 4-s exposures to UV light. The current reductions seen in C and D were uniform over all voltages, as seen for D2:R1C-BP in Fig. 7 C.

Mentions: Fig. 8A and Fig. B, shows that UV irradiation of extracellularly labeled D4/S4 mutants has a qualitatively different effect from that observed for Shaker and for the D2/S4 mutant. Instead of decreasing the peak current while having only minor effects on kinetics, irradiation of either D4:R1C-BPout or externally labeled D4:R3C (D4:R3C-BPout) causes an increase in current amplitude and a marked slowing of inactivation kinetics. Similar effects were seen whether the cells were irradiated at hyperpolarized or depolarized voltages (data not shown). These results show that immobilizing D4/S4 decreases the effectiveness of inactivation. The increase in peak current could be due to a decrease in the rate of inactivation of both closed and open channels after a depolarization (Gonoi and Hille 1987).


Immobilizing the moving parts of voltage-gated ion channels.

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

Photocross-linking D4/S4 mutants. Black and blue traces represent currents before and after, respectively, exposure to continuous-power UV light at the holding potential of −150 mV. All cysteine mutants were labeled with BPMTS. (A) D4:R1C-BP currents at 0 mV. Traces show progressive effects of 4.2-s exposures to UV light. This residue was labeled externally. (B) D4:R3C labeled with extracellular BPMTS. Currents at −30 mV using 4-s exposures to UV light. (C) D4:R3C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −30 mV using 4-s exposures to UV light. (D) D4:R4C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −45 mV using 4-s exposures to UV light. The current reductions seen in C and D were uniform over all voltages, as seen for D2:R1C-BP in Fig. 7 C.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Photocross-linking D4/S4 mutants. Black and blue traces represent currents before and after, respectively, exposure to continuous-power UV light at the holding potential of −150 mV. All cysteine mutants were labeled with BPMTS. (A) D4:R1C-BP currents at 0 mV. Traces show progressive effects of 4.2-s exposures to UV light. This residue was labeled externally. (B) D4:R3C labeled with extracellular BPMTS. Currents at −30 mV using 4-s exposures to UV light. (C) D4:R3C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −30 mV using 4-s exposures to UV light. (D) D4:R4C labeled with intracellular BPMTS (125 μM in the pipette solution). Currents at −45 mV using 4-s exposures to UV light. The current reductions seen in C and D were uniform over all voltages, as seen for D2:R1C-BP in Fig. 7 C.
Mentions: Fig. 8A and Fig. B, shows that UV irradiation of extracellularly labeled D4/S4 mutants has a qualitatively different effect from that observed for Shaker and for the D2/S4 mutant. Instead of decreasing the peak current while having only minor effects on kinetics, irradiation of either D4:R1C-BPout or externally labeled D4:R3C (D4:R3C-BPout) causes an increase in current amplitude and a marked slowing of inactivation kinetics. Similar effects were seen whether the cells were irradiated at hyperpolarized or depolarized voltages (data not shown). These results show that immobilizing D4/S4 decreases the effectiveness of inactivation. The increase in peak current could be due to a decrease in the rate of inactivation of both closed and open channels after a depolarization (Gonoi and Hille 1987).

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