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Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel.

Li RA, Vélez P, Chiamvimonvat N, Tomaselli GF, Marbán E - J. Gen. Physiol. (2000)

Bottom Line: Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway.The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV.Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The deep regions of the Na(+) channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

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A schematic representation of the proposed “double-dipp” orientation of the domain II P-S6 linker. This segment of the channel may reverse direction, dipping back into the pore. It could do so by forming a partial ring that extends horizontally at a tilted angle. Figures are not drawn to scale. Electrical distances may not directly correlate with actual physical distances (see text).
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Figure 7: A schematic representation of the proposed “double-dipp” orientation of the domain II P-S6 linker. This segment of the channel may reverse direction, dipping back into the pore. It could do so by forming a partial ring that extends horizontally at a tilted angle. Figures are not drawn to scale. Electrical distances may not directly correlate with actual physical distances (see text).

Mentions: The electrical distances of domain II pore residues reveal a striking pattern: they ascend (I757 and E758), and then descend (D762 and E765) back into the pore. Assuming that no significant structural or conformational changes of the pore are induced by the mutations and upon Cd2+ binding to the substituted cysteine, one possibility for this observation is that this region of the pore (i.e., the DII linker) may reverse direction and dip back into the membrane. Fig. 7 demonstrates a schematic representation of such possible orientations of the domain II P-S6 linker. This could occur by forming a partial ring that extends horizontally at a tilted angle. One should, however, recognize that electrical distances do not directly translate into physical distances, particularly in regions where the transmembrane electric field gradient is not linear. Nevertheless, our data raise new possibilities about the local topology of the domain II P segment since many of the previous pore mutations studied in this domain either did not express or were inaccessible, making its topology relatively uncertain (Yamagishi et al. 1997).


Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel.

Li RA, Vélez P, Chiamvimonvat N, Tomaselli GF, Marbán E - J. Gen. Physiol. (2000)

A schematic representation of the proposed “double-dipp” orientation of the domain II P-S6 linker. This segment of the channel may reverse direction, dipping back into the pore. It could do so by forming a partial ring that extends horizontally at a tilted angle. Figures are not drawn to scale. Electrical distances may not directly correlate with actual physical distances (see text).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887778&req=5

Figure 7: A schematic representation of the proposed “double-dipp” orientation of the domain II P-S6 linker. This segment of the channel may reverse direction, dipping back into the pore. It could do so by forming a partial ring that extends horizontally at a tilted angle. Figures are not drawn to scale. Electrical distances may not directly correlate with actual physical distances (see text).
Mentions: The electrical distances of domain II pore residues reveal a striking pattern: they ascend (I757 and E758), and then descend (D762 and E765) back into the pore. Assuming that no significant structural or conformational changes of the pore are induced by the mutations and upon Cd2+ binding to the substituted cysteine, one possibility for this observation is that this region of the pore (i.e., the DII linker) may reverse direction and dip back into the membrane. Fig. 7 demonstrates a schematic representation of such possible orientations of the domain II P-S6 linker. This could occur by forming a partial ring that extends horizontally at a tilted angle. One should, however, recognize that electrical distances do not directly translate into physical distances, particularly in regions where the transmembrane electric field gradient is not linear. Nevertheless, our data raise new possibilities about the local topology of the domain II P segment since many of the previous pore mutations studied in this domain either did not express or were inaccessible, making its topology relatively uncertain (Yamagishi et al. 1997).

Bottom Line: Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway.The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV.Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The deep regions of the Na(+) channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

Show MeSH