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Evolutionarily conserved intracellular gate of voltage-dependent sodium channels.

Oelstrom K, Goldschen-Ohm MP, Holmgren M, Chanda B - Nat Commun (2014)

Bottom Line: The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved.These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels.Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53706, USA [2] Molecular Pharmacology Graduate Program, University of Wisconsin, Madison, Wisconsin 53706, USA.

ABSTRACT
Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

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Related in: MedlinePlus

State-dependent modification of L1580C.Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 200 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW-L1580C channels. Test pulse current traces for closed-state modification (a, bottom) and open-state modification (b, bottom) are plotted before and after each subsequent exposure to MTSET. Peak sodium current does not change when MTSET is applied to closed channels; however, sodium current decreases after each 100 ms exposure of MTSET to open channels.
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f2: State-dependent modification of L1580C.Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 200 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW-L1580C channels. Test pulse current traces for closed-state modification (a, bottom) and open-state modification (b, bottom) are plotted before and after each subsequent exposure to MTSET. Peak sodium current does not change when MTSET is applied to closed channels; however, sodium current decreases after each 100 ms exposure of MTSET to open channels.

Mentions: If an intracellular gate exists within voltage-gated sodium channels, then we hypothesize that residues above the gate will be inaccessible to internally applied membrane impermeable MTSET while the gate is closed, but will become accessible when the gate is open. To test this prediction, we measured the accessibility of the introduced cysteine L1580C in Nav1.4-WSW channels to intracellularly applied MTSET in excised inside-out patches from Xenopus oocytes (Fig. 2). On the basis of homology with bacterial sodium channel structures, L1580 is likely to line the pore above the putative gate at the S6 helix bundle crossing. To determine the state-dependent accessibility of L1580C, we estimated the rate of MTSET modification under conditions where we expect the channels to be predominantly either closed (−130 mV) or open (0 mV). For both cases, the extent of modification was assayed intermittently by examining the peak current response to a test pulse to −30 mV. Figure 2 shows that L1580C undergoes no apparent modification while the channel is closed, whereas MTSET application while the channel is held open results in a time-dependent decrease in current amplitude, indicating that this position is accessible in the open state. A cytoplasmic gate below L1580 could explain why this site is protected from MTSET while the channels are closed, but accessible while the channel is open.


Evolutionarily conserved intracellular gate of voltage-dependent sodium channels.

Oelstrom K, Goldschen-Ohm MP, Holmgren M, Chanda B - Nat Commun (2014)

State-dependent modification of L1580C.Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 200 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW-L1580C channels. Test pulse current traces for closed-state modification (a, bottom) and open-state modification (b, bottom) are plotted before and after each subsequent exposure to MTSET. Peak sodium current does not change when MTSET is applied to closed channels; however, sodium current decreases after each 100 ms exposure of MTSET to open channels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3959192&req=5

f2: State-dependent modification of L1580C.Closed-state (a, top) and open-state (b, top) modification protocols for monitoring modification by 200 μM MTSET applied to inside-out patches excised from Xenopus oocytes containing Nav1.4-WSW-L1580C channels. Test pulse current traces for closed-state modification (a, bottom) and open-state modification (b, bottom) are plotted before and after each subsequent exposure to MTSET. Peak sodium current does not change when MTSET is applied to closed channels; however, sodium current decreases after each 100 ms exposure of MTSET to open channels.
Mentions: If an intracellular gate exists within voltage-gated sodium channels, then we hypothesize that residues above the gate will be inaccessible to internally applied membrane impermeable MTSET while the gate is closed, but will become accessible when the gate is open. To test this prediction, we measured the accessibility of the introduced cysteine L1580C in Nav1.4-WSW channels to intracellularly applied MTSET in excised inside-out patches from Xenopus oocytes (Fig. 2). On the basis of homology with bacterial sodium channel structures, L1580 is likely to line the pore above the putative gate at the S6 helix bundle crossing. To determine the state-dependent accessibility of L1580C, we estimated the rate of MTSET modification under conditions where we expect the channels to be predominantly either closed (−130 mV) or open (0 mV). For both cases, the extent of modification was assayed intermittently by examining the peak current response to a test pulse to −30 mV. Figure 2 shows that L1580C undergoes no apparent modification while the channel is closed, whereas MTSET application while the channel is held open results in a time-dependent decrease in current amplitude, indicating that this position is accessible in the open state. A cytoplasmic gate below L1580 could explain why this site is protected from MTSET while the channels are closed, but accessible while the channel is open.

Bottom Line: The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved.These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels.Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53706, USA [2] Molecular Pharmacology Graduate Program, University of Wisconsin, Madison, Wisconsin 53706, USA.

ABSTRACT
Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Show MeSH
Related in: MedlinePlus