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P-loop flexibility in Na+ channel pores revealed by single- and double-cysteine replacements.

Tsushima RG, Li RA, Backx PH - J. Gen. Physiol. (1997)

Bottom Line: Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents.These results allow identification of residue pairs capable of approaching one another to within less than 3.5 A.These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Toronto, Ontario, Canada.

ABSTRACT
Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na+ channels (SkM1) caused an increased sensitivity to current blockade by Cd2+ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd2+ block relative to the individual single mutants, suggesting coordinated Cd2+ binding and cross-linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 A. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd2+ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.

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Effect of cross-linking on conductance and selectivity properties of double-cysteine  mutants. (A) Raw current traces  following depolarization to −10  mV from a holding potential of  −120 mV for E403C/D1532C  channels expressed in oocytes  before (□) and after (▪) reduction with DTT. Note the nearly  twofold increase in current after  reduction at this voltage. (B) The  corresponding current-voltage  relationships for E403C/D1532C.  In this particular mutant, there is  little change in the channel's  conductance (161 μS before  and 156 μS after DTT), estimated from the slope of the current-voltage curve at voltages  above 0 mV, but the reversal potential is shifted by 19 mV to the  right following reduction with  DTT. (C) Raw traces for E403C/ A1529C before (○) and after  (•) the application of DTT. The  current increased more than sixfold following reduction with  DTT. (D) The current-voltage relationship for E403C/A1529C mutants shows a fivefold increase in slope at voltages above 0 mV (22 μS before and 97 μS after DTT),  whereas the reversal potential is only slightly shifted rightward (7 mV) by reduction with DTT.
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Figure 6: Effect of cross-linking on conductance and selectivity properties of double-cysteine mutants. (A) Raw current traces following depolarization to −10 mV from a holding potential of −120 mV for E403C/D1532C channels expressed in oocytes before (□) and after (▪) reduction with DTT. Note the nearly twofold increase in current after reduction at this voltage. (B) The corresponding current-voltage relationships for E403C/D1532C. In this particular mutant, there is little change in the channel's conductance (161 μS before and 156 μS after DTT), estimated from the slope of the current-voltage curve at voltages above 0 mV, but the reversal potential is shifted by 19 mV to the right following reduction with DTT. (C) Raw traces for E403C/ A1529C before (○) and after (•) the application of DTT. The current increased more than sixfold following reduction with DTT. (D) The current-voltage relationship for E403C/A1529C mutants shows a fivefold increase in slope at voltages above 0 mV (22 μS before and 97 μS after DTT), whereas the reversal potential is only slightly shifted rightward (7 mV) by reduction with DTT.

Mentions: While the data in Fig. 4 suggests P-loop flexibility, the functional importance of pore motion on channel behavior, as previously postulated (Läuger, 1987; Eisenman and Horn, 1983; Eisenman, 1984), remains speculative. The presence of cross-linkages for a number of double-cysteine mutants provides a unique opportunity to further investigate the significance of pore motion. Indeed, we expect cross-linked channels to have reduced pore flexibility and motion compared to the same channels following reduction with DTT. As examples, Fig. 6, A and C, shows raw current traces following depolarization to −10 mV from a holding potential of −120 mV for E403C/D1532C and E403C/A1529C before (□, ○) and after (▪, •) disruption of the disulfide linkage with DTT. DTT application caused about a twofold and sixfold increase in peak current for E403C/D1532C and E403C/A1529C, respectively, indicating that these double-mutant channels are less capable of conducting current in the oxidized, cross-linked state versus the reduced state. The increase in whole-cell current following DTT exposure is not solely due to subtle changes in channel gating as illustrated in Fig. 6, B and D, which shows the current-voltage relationships for the corresponding mutants before (□, ○) and after (▪, •) the application of DTT. Not only is the peak of the current-voltage relationship significantly affected by DTT but the reversal potentials were also shifted: from 8 to 27 mV for E403C/D1532C and from 34 to 41 mV for E403C/A1529C after DTT application. Average shifts in reversal potential for 4 double-cysteine mutants and WT channels studied are summarized in Table III. Significant rightward shifts in reversal potential were observed in all cross-linked double-mutant channels, in which it was studied, following reduction with DTT. Furthermore, after reduction, the measured reversal potential closely matched the potential observed in the least selective of the corresponding single-cysteine mutants. Changes in selectivity following reduction could not be studied in cross-linked double-mutant channels involving cysteine replacements at position W1531 since W1531C channels are nonselective. Furthermore, 1.8-fold to 8-fold increases in whole-cell current were observed for cross-linked double-mutant channels (Fig. 4) following DTT exposure (data not shown) establishing that ionic conductance is strongly influenced by disulfide reduction. These results suggest that channel selectivity and permeation are impaired when channel motion is reduced by cross-linking. Alternatively, cross-linking of cysteine pairs could cause sufficient distortion of the P-loop structure to interfere with ion selectivity and permeation.


P-loop flexibility in Na+ channel pores revealed by single- and double-cysteine replacements.

Tsushima RG, Li RA, Backx PH - J. Gen. Physiol. (1997)

Effect of cross-linking on conductance and selectivity properties of double-cysteine  mutants. (A) Raw current traces  following depolarization to −10  mV from a holding potential of  −120 mV for E403C/D1532C  channels expressed in oocytes  before (□) and after (▪) reduction with DTT. Note the nearly  twofold increase in current after  reduction at this voltage. (B) The  corresponding current-voltage  relationships for E403C/D1532C.  In this particular mutant, there is  little change in the channel's  conductance (161 μS before  and 156 μS after DTT), estimated from the slope of the current-voltage curve at voltages  above 0 mV, but the reversal potential is shifted by 19 mV to the  right following reduction with  DTT. (C) Raw traces for E403C/ A1529C before (○) and after  (•) the application of DTT. The  current increased more than sixfold following reduction with  DTT. (D) The current-voltage relationship for E403C/A1529C mutants shows a fivefold increase in slope at voltages above 0 mV (22 μS before and 97 μS after DTT),  whereas the reversal potential is only slightly shifted rightward (7 mV) by reduction with DTT.
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Related In: Results  -  Collection

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Figure 6: Effect of cross-linking on conductance and selectivity properties of double-cysteine mutants. (A) Raw current traces following depolarization to −10 mV from a holding potential of −120 mV for E403C/D1532C channels expressed in oocytes before (□) and after (▪) reduction with DTT. Note the nearly twofold increase in current after reduction at this voltage. (B) The corresponding current-voltage relationships for E403C/D1532C. In this particular mutant, there is little change in the channel's conductance (161 μS before and 156 μS after DTT), estimated from the slope of the current-voltage curve at voltages above 0 mV, but the reversal potential is shifted by 19 mV to the right following reduction with DTT. (C) Raw traces for E403C/ A1529C before (○) and after (•) the application of DTT. The current increased more than sixfold following reduction with DTT. (D) The current-voltage relationship for E403C/A1529C mutants shows a fivefold increase in slope at voltages above 0 mV (22 μS before and 97 μS after DTT), whereas the reversal potential is only slightly shifted rightward (7 mV) by reduction with DTT.
Mentions: While the data in Fig. 4 suggests P-loop flexibility, the functional importance of pore motion on channel behavior, as previously postulated (Läuger, 1987; Eisenman and Horn, 1983; Eisenman, 1984), remains speculative. The presence of cross-linkages for a number of double-cysteine mutants provides a unique opportunity to further investigate the significance of pore motion. Indeed, we expect cross-linked channels to have reduced pore flexibility and motion compared to the same channels following reduction with DTT. As examples, Fig. 6, A and C, shows raw current traces following depolarization to −10 mV from a holding potential of −120 mV for E403C/D1532C and E403C/A1529C before (□, ○) and after (▪, •) disruption of the disulfide linkage with DTT. DTT application caused about a twofold and sixfold increase in peak current for E403C/D1532C and E403C/A1529C, respectively, indicating that these double-mutant channels are less capable of conducting current in the oxidized, cross-linked state versus the reduced state. The increase in whole-cell current following DTT exposure is not solely due to subtle changes in channel gating as illustrated in Fig. 6, B and D, which shows the current-voltage relationships for the corresponding mutants before (□, ○) and after (▪, •) the application of DTT. Not only is the peak of the current-voltage relationship significantly affected by DTT but the reversal potentials were also shifted: from 8 to 27 mV for E403C/D1532C and from 34 to 41 mV for E403C/A1529C after DTT application. Average shifts in reversal potential for 4 double-cysteine mutants and WT channels studied are summarized in Table III. Significant rightward shifts in reversal potential were observed in all cross-linked double-mutant channels, in which it was studied, following reduction with DTT. Furthermore, after reduction, the measured reversal potential closely matched the potential observed in the least selective of the corresponding single-cysteine mutants. Changes in selectivity following reduction could not be studied in cross-linked double-mutant channels involving cysteine replacements at position W1531 since W1531C channels are nonselective. Furthermore, 1.8-fold to 8-fold increases in whole-cell current were observed for cross-linked double-mutant channels (Fig. 4) following DTT exposure (data not shown) establishing that ionic conductance is strongly influenced by disulfide reduction. These results suggest that channel selectivity and permeation are impaired when channel motion is reduced by cross-linking. Alternatively, cross-linking of cysteine pairs could cause sufficient distortion of the P-loop structure to interfere with ion selectivity and permeation.

Bottom Line: Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents.These results allow identification of residue pairs capable of approaching one another to within less than 3.5 A.These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Toronto, Ontario, Canada.

ABSTRACT
Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na+ channels (SkM1) caused an increased sensitivity to current blockade by Cd2+ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd2+ block relative to the individual single mutants, suggesting coordinated Cd2+ binding and cross-linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 A. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd2+ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.

Show MeSH
Related in: MedlinePlus