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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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(A) Na+ current tracings of Y401C, E758C, and  Y401C/E758C mutant channels in response to depolarization to  −10 mV from a holding potential of −120 mV before (solid traces)  and after (broken traces) the addition of 100 μM Cd2+. Na+ currents  for Y401C/E758C channels are shown before and after the addition of DTT. The currents have been scaled for ease of comparison. The current amplitudes in the absence of Cd2+ were (μA): 3.5  (Y401C), 2.7 (E758C), 2.2 (Y401C/E758C −DTT), and 4.6 (Y401C/ E758C + DTT). (B) Dose-response curves of the normalized peak  Na+ current as a function of the [Cd2+]. The channels become  about 1,200-fold more sensitive to Cd2+ (KD changes from 1,353 ±  382 μM to 1.1 ± 0.2 μM) following the addition of 2 mM DTT.
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Figure 3: (A) Na+ current tracings of Y401C, E758C, and Y401C/E758C mutant channels in response to depolarization to −10 mV from a holding potential of −120 mV before (solid traces) and after (broken traces) the addition of 100 μM Cd2+. Na+ currents for Y401C/E758C channels are shown before and after the addition of DTT. The currents have been scaled for ease of comparison. The current amplitudes in the absence of Cd2+ were (μA): 3.5 (Y401C), 2.7 (E758C), 2.2 (Y401C/E758C −DTT), and 4.6 (Y401C/ E758C + DTT). (B) Dose-response curves of the normalized peak Na+ current as a function of the [Cd2+]. The channels become about 1,200-fold more sensitive to Cd2+ (KD changes from 1,353 ± 382 μM to 1.1 ± 0.2 μM) following the addition of 2 mM DTT.

Mentions: Remarkably, all double-cysteine mutant channels created (except W402C/I757C and those constructed with G1238C) formed functional channels. Fig. 3, A and B, presents typical results for Y401C/E758C channels; the measured dissociation constant for Cd2+ block (KD,ob) for Y401C/E758C channels was 1,353 ± 382 μM (mean ± SD, n = 7) compared to 12 μM predicted for independent binding (i.e., KD(Y401C) = 13.7 ± 3.0 μM (n = 6) and KD(E758C) = 454 ± 47 μM (n = 7)). Following reduction with 10 mM DTT, applied for a period of 8–10 min, the KD for Cd2+ block decrease about 1,200-fold to 1.1 ± 0.2 μM (n = 4). In this mutant, DTT increased the whole-cell current about 2.5-fold; DTT washin also caused comparatively large increases in whole-cell current and/or conductance in other cross-linked mutant channels studied. The increase in current invariably occurred in less than 30 s after DTT application, indicating rapid separation of the cross-linked cysteines. Furthermore, reduction with DTT enhanced the sensitivity of cross-linked channels to Cd2+ blockade (see below). Subsequent to DTT-reduction, Cd2+-sensitive Y401C/E758C and other cross-linked double-cysteine channels could be made Cd2+-insensitive again by applying 1 mM MTSEA (data not shown).


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

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

(A) Na+ current tracings of Y401C, E758C, and  Y401C/E758C mutant channels in response to depolarization to  −10 mV from a holding potential of −120 mV before (solid traces)  and after (broken traces) the addition of 100 μM Cd2+. Na+ currents  for Y401C/E758C channels are shown before and after the addition of DTT. The currents have been scaled for ease of comparison. The current amplitudes in the absence of Cd2+ were (μA): 3.5  (Y401C), 2.7 (E758C), 2.2 (Y401C/E758C −DTT), and 4.6 (Y401C/ E758C + DTT). (B) Dose-response curves of the normalized peak  Na+ current as a function of the [Cd2+]. The channels become  about 1,200-fold more sensitive to Cd2+ (KD changes from 1,353 ±  382 μM to 1.1 ± 0.2 μM) following the addition of 2 mM DTT.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2229360&req=5

Figure 3: (A) Na+ current tracings of Y401C, E758C, and Y401C/E758C mutant channels in response to depolarization to −10 mV from a holding potential of −120 mV before (solid traces) and after (broken traces) the addition of 100 μM Cd2+. Na+ currents for Y401C/E758C channels are shown before and after the addition of DTT. The currents have been scaled for ease of comparison. The current amplitudes in the absence of Cd2+ were (μA): 3.5 (Y401C), 2.7 (E758C), 2.2 (Y401C/E758C −DTT), and 4.6 (Y401C/ E758C + DTT). (B) Dose-response curves of the normalized peak Na+ current as a function of the [Cd2+]. The channels become about 1,200-fold more sensitive to Cd2+ (KD changes from 1,353 ± 382 μM to 1.1 ± 0.2 μM) following the addition of 2 mM DTT.
Mentions: Remarkably, all double-cysteine mutant channels created (except W402C/I757C and those constructed with G1238C) formed functional channels. Fig. 3, A and B, presents typical results for Y401C/E758C channels; the measured dissociation constant for Cd2+ block (KD,ob) for Y401C/E758C channels was 1,353 ± 382 μM (mean ± SD, n = 7) compared to 12 μM predicted for independent binding (i.e., KD(Y401C) = 13.7 ± 3.0 μM (n = 6) and KD(E758C) = 454 ± 47 μM (n = 7)). Following reduction with 10 mM DTT, applied for a period of 8–10 min, the KD for Cd2+ block decrease about 1,200-fold to 1.1 ± 0.2 μM (n = 4). In this mutant, DTT increased the whole-cell current about 2.5-fold; DTT washin also caused comparatively large increases in whole-cell current and/or conductance in other cross-linked mutant channels studied. The increase in current invariably occurred in less than 30 s after DTT application, indicating rapid separation of the cross-linked cysteines. Furthermore, reduction with DTT enhanced the sensitivity of cross-linked channels to Cd2+ blockade (see below). Subsequent to DTT-reduction, Cd2+-sensitive Y401C/E758C and other cross-linked double-cysteine channels could be made Cd2+-insensitive again by applying 1 mM MTSEA (data not shown).

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