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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 NavAb/N49K and NavAb WT channels. (A) NavAb/N49K currents in response to depolarizations to potentials ranging from −55 to +60 mV in 5-mV increments. (B) Peak current–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). (C) Conductance–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). The lines through the curves are fits of Boltzmann relationships with values of Va = −21.9 mV and k = 8.3 mV for NavAb N49K, and Va = −97.8 mV and k = 11.0 mV for NavAb WT.
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fig1: Comparison of NavAb/N49K and NavAb WT channels. (A) NavAb/N49K currents in response to depolarizations to potentials ranging from −55 to +60 mV in 5-mV increments. (B) Peak current–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). (C) Conductance–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). The lines through the curves are fits of Boltzmann relationships with values of Va = −21.9 mV and k = 8.3 mV for NavAb N49K, and Va = −97.8 mV and k = 11.0 mV for NavAb WT.

Mentions: NavAb WT channels expressed in High Five insect cells activate at very negative potentials, with observable sodium currents at approximately −130 mV and peak current at approximately −75 mV (Fig. 1 B, red). The negative voltage dependence and strong use-dependent inactivation (Payandeh et al., 2012, and see below) make electrophysiological measurements of the biophysical properties of the channel challenging. We previously found that substituting the outer conserved negatively charged Asp in the S2 segment of the voltage sensor of NaChBac with Lys resulted in a +75-mV shift in the voltage dependence of activation (Zhao et al., 2004b). The amino acid in the analogous position of NavAb is Asn49. As in NaChBac, mutating NavAb Asn49 to a Lys, N49K, resulted in a +76-mV shift in the current–voltage relationship (Fig. 1, A and B, black) and the voltage dependence of activation (Fig. 1 C; NavAb WT, half-activation voltage, Va = −97.8 ± 1.2 mV; NavAb/N49K, Va = −21.9 ± 0.3 mV). Insertion of Lys at this key position in NavAb strongly favors the deactivated state of the voltage sensor relative to the activated state.


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 NavAb/N49K and NavAb WT channels. (A) NavAb/N49K currents in response to depolarizations to potentials ranging from −55 to +60 mV in 5-mV increments. (B) Peak current–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). (C) Conductance–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). The lines through the curves are fits of Boltzmann relationships with values of Va = −21.9 mV and k = 8.3 mV for NavAb N49K, and Va = −97.8 mV and k = 11.0 mV for NavAb WT.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3753604&req=5

fig1: Comparison of NavAb/N49K and NavAb WT channels. (A) NavAb/N49K currents in response to depolarizations to potentials ranging from −55 to +60 mV in 5-mV increments. (B) Peak current–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). (C) Conductance–voltage relationships for NavAb WT (red circles) and NavAb/N49K (black triangles). The lines through the curves are fits of Boltzmann relationships with values of Va = −21.9 mV and k = 8.3 mV for NavAb N49K, and Va = −97.8 mV and k = 11.0 mV for NavAb WT.
Mentions: NavAb WT channels expressed in High Five insect cells activate at very negative potentials, with observable sodium currents at approximately −130 mV and peak current at approximately −75 mV (Fig. 1 B, red). The negative voltage dependence and strong use-dependent inactivation (Payandeh et al., 2012, and see below) make electrophysiological measurements of the biophysical properties of the channel challenging. We previously found that substituting the outer conserved negatively charged Asp in the S2 segment of the voltage sensor of NaChBac with Lys resulted in a +75-mV shift in the voltage dependence of activation (Zhao et al., 2004b). The amino acid in the analogous position of NavAb is Asn49. As in NaChBac, mutating NavAb Asn49 to a Lys, N49K, resulted in a +76-mV shift in the current–voltage relationship (Fig. 1, A and B, black) and the voltage dependence of activation (Fig. 1 C; NavAb WT, half-activation voltage, Va = −97.8 ± 1.2 mV; NavAb/N49K, Va = −21.9 ± 0.3 mV). Insertion of Lys at this key position in NavAb strongly favors the deactivated state of the voltage sensor relative to the activated state.

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