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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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Single-channel recordings of Y401C, E758C, and  reduced Y401C/E758C mutant  channels following fenvalerate  application at −80 mV. (A) Currents recorded in Y401C, E758C,  and Y401C/E758C channels in  the presence of 10 μM fenvalerate which is added to maintain  the channels in the open state  for tens to hundreds of milliseconds. (B) Currents recorded in  Y401C and reduced Y401C/ E758C mutants channels with 5  μM extracellular Cd2+ and in  E758C channels with 400 μM  Cd2+. Cd2+ caused full closures  of the Y401C and Y401C/E758C  channels while blocking E758C  channels to a sub-conductance  level (closed level indicated by  the broken line). In the presence  of 5 μM Cd2+ the double-mutant  channel Y401C/E758C displayed  bursts of short-lived blocking  events separated by long-lived  blocking events not seen in either single-mutant. These long-lived blockages likely represent  the “trapping” of the Cd2+ ion as a result of simultaneous interactions with the two free sulfhydryl groups. (C) The mean block-time histograms are shown for the channels illustrated in A for Y401C, E758C, and Y401C/E758C channels. The blocked-time histograms could be  adequately fit using a mono-exponential equation for Y401C and E758C channels, while a bi-exponential function was required for  Y401C/E758C channels. Note: the time axes are different in the different panels. See text for further details. (D) The mean unblocked-time  (i.e., open-time) histogram is shown for the same channels illustrated in B. For Y401C, E758C, and Y401C/E758C channels the unblocked-time histogram could be adequately fit using a mono-exponential function.
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Figure 5: Single-channel recordings of Y401C, E758C, and reduced Y401C/E758C mutant channels following fenvalerate application at −80 mV. (A) Currents recorded in Y401C, E758C, and Y401C/E758C channels in the presence of 10 μM fenvalerate which is added to maintain the channels in the open state for tens to hundreds of milliseconds. (B) Currents recorded in Y401C and reduced Y401C/ E758C mutants channels with 5 μM extracellular Cd2+ and in E758C channels with 400 μM Cd2+. Cd2+ caused full closures of the Y401C and Y401C/E758C channels while blocking E758C channels to a sub-conductance level (closed level indicated by the broken line). In the presence of 5 μM Cd2+ the double-mutant channel Y401C/E758C displayed bursts of short-lived blocking events separated by long-lived blocking events not seen in either single-mutant. These long-lived blockages likely represent the “trapping” of the Cd2+ ion as a result of simultaneous interactions with the two free sulfhydryl groups. (C) The mean block-time histograms are shown for the channels illustrated in A for Y401C, E758C, and Y401C/E758C channels. The blocked-time histograms could be adequately fit using a mono-exponential equation for Y401C and E758C channels, while a bi-exponential function was required for Y401C/E758C channels. Note: the time axes are different in the different panels. See text for further details. (D) The mean unblocked-time (i.e., open-time) histogram is shown for the same channels illustrated in B. For Y401C, E758C, and Y401C/E758C channels the unblocked-time histogram could be adequately fit using a mono-exponential function.

Mentions: Single-channel recordings were used to establish the mechanism underlying the enhanced Cd2+ sensitivity of reduced Y401C/E758C mutants. Fig. 5 shows typical single-channel recordings at −80 mV for Y401C, E758C, and reduced Y401C/E758C channels recorded from inside-out cell attached patches in the absence (A) and presence (B) of Cd2+. All recordings were made in the presence of 10 μM fenvalerate, which maintains Na+ channels in the open state for tens to hundreds of milliseconds (Backx et al., 1992). For Y401C channels, Fig. 5 B shows representative single-channel sweeps measured in the presence of 5 μM Cd2+ in the pipette. Notice the discrete flicker blockade of the unitary current in Y401C channels (i.e., represented by O) often lasting several milliseconds which was not observed in the absence of Cd2+. Cd2+ totally occludes the passage of Na+ ions (i.e., represented by C) consistent with Cd2+ binding within the permeation pathway. The corresponding blocking-time histogram, illustrated in Fig. 5 C, could be adequately fit by a mono-exponential function, as expected if a single Cd2+ binding site exists within the pore. The estimated average block-time of Cd2+ ions within the pore (i.e., equal to the estimated time constant for the mono-exponential fit of the blocked-time histogram) was 1.43 ms for this Y401C (average 1.36 ± 0.12 ms, n = 3).


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

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

Single-channel recordings of Y401C, E758C, and  reduced Y401C/E758C mutant  channels following fenvalerate  application at −80 mV. (A) Currents recorded in Y401C, E758C,  and Y401C/E758C channels in  the presence of 10 μM fenvalerate which is added to maintain  the channels in the open state  for tens to hundreds of milliseconds. (B) Currents recorded in  Y401C and reduced Y401C/ E758C mutants channels with 5  μM extracellular Cd2+ and in  E758C channels with 400 μM  Cd2+. Cd2+ caused full closures  of the Y401C and Y401C/E758C  channels while blocking E758C  channels to a sub-conductance  level (closed level indicated by  the broken line). In the presence  of 5 μM Cd2+ the double-mutant  channel Y401C/E758C displayed  bursts of short-lived blocking  events separated by long-lived  blocking events not seen in either single-mutant. These long-lived blockages likely represent  the “trapping” of the Cd2+ ion as a result of simultaneous interactions with the two free sulfhydryl groups. (C) The mean block-time histograms are shown for the channels illustrated in A for Y401C, E758C, and Y401C/E758C channels. The blocked-time histograms could be  adequately fit using a mono-exponential equation for Y401C and E758C channels, while a bi-exponential function was required for  Y401C/E758C channels. Note: the time axes are different in the different panels. See text for further details. (D) The mean unblocked-time  (i.e., open-time) histogram is shown for the same channels illustrated in B. For Y401C, E758C, and Y401C/E758C channels the unblocked-time histogram could be adequately fit using a mono-exponential function.
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Show All Figures
getmorefigures.php?uid=PMC2229360&req=5

Figure 5: Single-channel recordings of Y401C, E758C, and reduced Y401C/E758C mutant channels following fenvalerate application at −80 mV. (A) Currents recorded in Y401C, E758C, and Y401C/E758C channels in the presence of 10 μM fenvalerate which is added to maintain the channels in the open state for tens to hundreds of milliseconds. (B) Currents recorded in Y401C and reduced Y401C/ E758C mutants channels with 5 μM extracellular Cd2+ and in E758C channels with 400 μM Cd2+. Cd2+ caused full closures of the Y401C and Y401C/E758C channels while blocking E758C channels to a sub-conductance level (closed level indicated by the broken line). In the presence of 5 μM Cd2+ the double-mutant channel Y401C/E758C displayed bursts of short-lived blocking events separated by long-lived blocking events not seen in either single-mutant. These long-lived blockages likely represent the “trapping” of the Cd2+ ion as a result of simultaneous interactions with the two free sulfhydryl groups. (C) The mean block-time histograms are shown for the channels illustrated in A for Y401C, E758C, and Y401C/E758C channels. The blocked-time histograms could be adequately fit using a mono-exponential equation for Y401C and E758C channels, while a bi-exponential function was required for Y401C/E758C channels. Note: the time axes are different in the different panels. See text for further details. (D) The mean unblocked-time (i.e., open-time) histogram is shown for the same channels illustrated in B. For Y401C, E758C, and Y401C/E758C channels the unblocked-time histogram could be adequately fit using a mono-exponential function.
Mentions: Single-channel recordings were used to establish the mechanism underlying the enhanced Cd2+ sensitivity of reduced Y401C/E758C mutants. Fig. 5 shows typical single-channel recordings at −80 mV for Y401C, E758C, and reduced Y401C/E758C channels recorded from inside-out cell attached patches in the absence (A) and presence (B) of Cd2+. All recordings were made in the presence of 10 μM fenvalerate, which maintains Na+ channels in the open state for tens to hundreds of milliseconds (Backx et al., 1992). For Y401C channels, Fig. 5 B shows representative single-channel sweeps measured in the presence of 5 μM Cd2+ in the pipette. Notice the discrete flicker blockade of the unitary current in Y401C channels (i.e., represented by O) often lasting several milliseconds which was not observed in the absence of Cd2+. Cd2+ totally occludes the passage of Na+ ions (i.e., represented by C) consistent with Cd2+ binding within the permeation pathway. The corresponding blocking-time histogram, illustrated in Fig. 5 C, could be adequately fit by a mono-exponential function, as expected if a single Cd2+ binding site exists within the pore. The estimated average block-time of Cd2+ ions within the pore (i.e., equal to the estimated time constant for the mono-exponential fit of the blocked-time histogram) was 1.43 ms for this Y401C (average 1.36 ± 0.12 ms, n = 3).

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