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A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb.

Gamal El-Din TM, Martinez GQ, Payandeh J, Scheuer T, Catterall WA - J. Gen. Physiol. (2013)

Bottom Line: Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation.Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation.Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V1/2 of approximately -98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.

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Comparison of recovery from inactivation for NavAb WT and NavAb/N49K. (A) Recovery from inactivation for NavAb WT studied with a 100-ms pre-depolarization (red protocol and circles) and for NavAb/N49K studied with a 4-s pre-depolarization (black protocol and triangles). Pre-depolarizations to induce inactivation were followed after a variable recovery interval by a test depolarization to −10 mV. Peak current measured during the test depolarization was normalized to the peak current during the pre-depolarization and plotted as a function of the recovery interval. (B) Recovery from inactivation for NavAb/N49K studied as described above but using a 10-s pre-depolarization to induce inactivation. (C) Eight superimposed depolarizations to 0 mV for NavAb/N49K applied at 0.2 Hz from an HP of −120 mV (left-hand traces) were followed by a 2-min maintained depolarization to 0 mV. The membrane potential was then returned to −120 mV, and the 0.2-Hz train of depolarizations to 0 mV was resumed (right-hand traces). The first depolarization, applied 10 ms after returning to −120 mV, resulted in the trace with no inward current. Current traces elicited by nine additional subsequent depolarizations are shown superimposed.
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fig4: Comparison of recovery from inactivation for NavAb WT and NavAb/N49K. (A) Recovery from inactivation for NavAb WT studied with a 100-ms pre-depolarization (red protocol and circles) and for NavAb/N49K studied with a 4-s pre-depolarization (black protocol and triangles). Pre-depolarizations to induce inactivation were followed after a variable recovery interval by a test depolarization to −10 mV. Peak current measured during the test depolarization was normalized to the peak current during the pre-depolarization and plotted as a function of the recovery interval. (B) Recovery from inactivation for NavAb/N49K studied as described above but using a 10-s pre-depolarization to induce inactivation. (C) Eight superimposed depolarizations to 0 mV for NavAb/N49K applied at 0.2 Hz from an HP of −120 mV (left-hand traces) were followed by a 2-min maintained depolarization to 0 mV. The membrane potential was then returned to −120 mV, and the 0.2-Hz train of depolarizations to 0 mV was resumed (right-hand traces). The first depolarization, applied 10 ms after returning to −120 mV, resulted in the trace with no inward current. Current traces elicited by nine additional subsequent depolarizations are shown superimposed.

Mentions: NavAb WT recovers from inactivation with multiple exponential components (Payandeh et al., 2012). Recovery after a 100-ms depolarizing prepulse followed a double-exponential time course, with mean time constants of 101 ms representing 77% of the recovering current and 3.2 s representing 23% of the recovering current; 8% of the sodium current failed to recover in the time allotted (20 s). These values yielded a weighted time constant of 819 ms (Fig. 4 A, red). Recovery from inactivation of NavAb/N49K at −120 mV after a 2.5-s depolarization was characterized by a double-exponential time course, with a fast time constant of ∼73 ms and a slow one of ∼727 ms. Recovery from inactivation after a 4-s prepulse (Fig. 4 A, black) was also biexponential, with a fast time constant of 47.6 ms representing 68% of the current and a slow time constant of 320 ms representing 32% of the current. Extending the prepulse to 10 s resulted in 62% of the current recovering with a time constant of 62 ms and 38% recovering with a time constant of 561 ms; 12% of the current failed to recover in the time allotted (Fig. 4 B). These values represent a weighted time constant of 250 ms. Even after this 100-fold longer prepulse, NavAb/N49K recovers from inactivation approximately three times faster than NavAb/WT. In addition, the slowest component of recovery is still 5.7-fold faster for NavAb/N49K. The slowly recovering component of inactivation is far less stable in NavAb/N49K than in NavAb/WT. In addition, this slowly recovering component of inactivation in NavAb/WT is induced within 100 ms but requires far longer depolarizations to be observed in NavAb/N49K.


A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb.

Gamal El-Din TM, Martinez GQ, Payandeh J, Scheuer T, Catterall WA - J. Gen. Physiol. (2013)

Comparison of recovery from inactivation for NavAb WT and NavAb/N49K. (A) Recovery from inactivation for NavAb WT studied with a 100-ms pre-depolarization (red protocol and circles) and for NavAb/N49K studied with a 4-s pre-depolarization (black protocol and triangles). Pre-depolarizations to induce inactivation were followed after a variable recovery interval by a test depolarization to −10 mV. Peak current measured during the test depolarization was normalized to the peak current during the pre-depolarization and plotted as a function of the recovery interval. (B) Recovery from inactivation for NavAb/N49K studied as described above but using a 10-s pre-depolarization to induce inactivation. (C) Eight superimposed depolarizations to 0 mV for NavAb/N49K applied at 0.2 Hz from an HP of −120 mV (left-hand traces) were followed by a 2-min maintained depolarization to 0 mV. The membrane potential was then returned to −120 mV, and the 0.2-Hz train of depolarizations to 0 mV was resumed (right-hand traces). The first depolarization, applied 10 ms after returning to −120 mV, resulted in the trace with no inward current. Current traces elicited by nine additional subsequent depolarizations are shown superimposed.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig4: Comparison of recovery from inactivation for NavAb WT and NavAb/N49K. (A) Recovery from inactivation for NavAb WT studied with a 100-ms pre-depolarization (red protocol and circles) and for NavAb/N49K studied with a 4-s pre-depolarization (black protocol and triangles). Pre-depolarizations to induce inactivation were followed after a variable recovery interval by a test depolarization to −10 mV. Peak current measured during the test depolarization was normalized to the peak current during the pre-depolarization and plotted as a function of the recovery interval. (B) Recovery from inactivation for NavAb/N49K studied as described above but using a 10-s pre-depolarization to induce inactivation. (C) Eight superimposed depolarizations to 0 mV for NavAb/N49K applied at 0.2 Hz from an HP of −120 mV (left-hand traces) were followed by a 2-min maintained depolarization to 0 mV. The membrane potential was then returned to −120 mV, and the 0.2-Hz train of depolarizations to 0 mV was resumed (right-hand traces). The first depolarization, applied 10 ms after returning to −120 mV, resulted in the trace with no inward current. Current traces elicited by nine additional subsequent depolarizations are shown superimposed.
Mentions: NavAb WT recovers from inactivation with multiple exponential components (Payandeh et al., 2012). Recovery after a 100-ms depolarizing prepulse followed a double-exponential time course, with mean time constants of 101 ms representing 77% of the recovering current and 3.2 s representing 23% of the recovering current; 8% of the sodium current failed to recover in the time allotted (20 s). These values yielded a weighted time constant of 819 ms (Fig. 4 A, red). Recovery from inactivation of NavAb/N49K at −120 mV after a 2.5-s depolarization was characterized by a double-exponential time course, with a fast time constant of ∼73 ms and a slow one of ∼727 ms. Recovery from inactivation after a 4-s prepulse (Fig. 4 A, black) was also biexponential, with a fast time constant of 47.6 ms representing 68% of the current and a slow time constant of 320 ms representing 32% of the current. Extending the prepulse to 10 s resulted in 62% of the current recovering with a time constant of 62 ms and 38% recovering with a time constant of 561 ms; 12% of the current failed to recover in the time allotted (Fig. 4 B). These values represent a weighted time constant of 250 ms. Even after this 100-fold longer prepulse, NavAb/N49K recovers from inactivation approximately three times faster than NavAb/WT. In addition, the slowest component of recovery is still 5.7-fold faster for NavAb/N49K. The slowly recovering component of inactivation is far less stable in NavAb/N49K than in NavAb/WT. In addition, this slowly recovering component of inactivation in NavAb/WT is induced within 100 ms but requires far longer depolarizations to be observed in NavAb/N49K.

Bottom Line: Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation.Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation.Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V1/2 of approximately -98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.

Show MeSH
Related in: MedlinePlus