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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 inactivation properties of NavAb WT and NavAb/N49K. (A) Time constant of the decay of current during depolarizations to the indicated potentials for NavAb WT (red circles) and NavAb/N49K (black triangles). (B) Voltage dependence of inactivation for NavAb WT (red circles) and NavAb/N49K (black triangles) by depolarizations to −10 mV after 100-ms long conditioning prepulses to the indicated potentials.
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fig3: Comparison of inactivation properties of NavAb WT and NavAb/N49K. (A) Time constant of the decay of current during depolarizations to the indicated potentials for NavAb WT (red circles) and NavAb/N49K (black triangles). (B) Voltage dependence of inactivation for NavAb WT (red circles) and NavAb/N49K (black triangles) by depolarizations to −10 mV after 100-ms long conditioning prepulses to the indicated potentials.

Mentions: Despite lacking the late use-dependent, slow-inactivation process, NavAb/N49K channels still inactivate during short depolarizing pulses, with a time constant that reaches a limiting value of ∼21 ms for potentials positive to +30 mV (Fig. 3 A). This is significantly slower than NavAb WT, which reaches a plateau of ∼11 ms at positive potentials (Fig. 3 A; Payandeh et al., 2012). The voltage dependence of inactivation of NavAb/N49K studied with 100-ms conditioning prepulses was shifted ∼60 mV toward more positive potentials. (Fig. 3 B; NavAb WT, V1/2 = −119.3 ± 0.8 mV; NavAb/N49K, V1/2 = −59.3 ± 0.7 mV). This positive shift in the voltage dependence of inactivation resembles the corresponding shift in the activation properties of mutant NavAb/N49K, but the magnitude of the shift is 16 mV less.


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 inactivation properties of NavAb WT and NavAb/N49K. (A) Time constant of the decay of current during depolarizations to the indicated potentials for NavAb WT (red circles) and NavAb/N49K (black triangles). (B) Voltage dependence of inactivation for NavAb WT (red circles) and NavAb/N49K (black triangles) by depolarizations to −10 mV after 100-ms long conditioning prepulses to the indicated potentials.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753604&req=5

fig3: Comparison of inactivation properties of NavAb WT and NavAb/N49K. (A) Time constant of the decay of current during depolarizations to the indicated potentials for NavAb WT (red circles) and NavAb/N49K (black triangles). (B) Voltage dependence of inactivation for NavAb WT (red circles) and NavAb/N49K (black triangles) by depolarizations to −10 mV after 100-ms long conditioning prepulses to the indicated potentials.
Mentions: Despite lacking the late use-dependent, slow-inactivation process, NavAb/N49K channels still inactivate during short depolarizing pulses, with a time constant that reaches a limiting value of ∼21 ms for potentials positive to +30 mV (Fig. 3 A). This is significantly slower than NavAb WT, which reaches a plateau of ∼11 ms at positive potentials (Fig. 3 A; Payandeh et al., 2012). The voltage dependence of inactivation of NavAb/N49K studied with 100-ms conditioning prepulses was shifted ∼60 mV toward more positive potentials. (Fig. 3 B; NavAb WT, V1/2 = −119.3 ± 0.8 mV; NavAb/N49K, V1/2 = −59.3 ± 0.7 mV). This positive shift in the voltage dependence of inactivation resembles the corresponding shift in the activation properties of mutant NavAb/N49K, but the magnitude of the shift is 16 mV less.

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