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Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Brock MW, Mathes C, Gilly WF - J. Gen. Physiol. (2001)

Bottom Line: SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect.Finally, SNDTT is remarkably selective for Kv1 channels.When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

View Article: PubMed Central - PubMed

Affiliation: Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, CA 93950, USA.

ABSTRACT
Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

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SNDTT block does not require channel cysteines, and is not simply an acceleration of endogenous C-type inactivation. (A) SNDTT blocks a Shaker mutant lacking all cysteine residues. Cys-less ShBΔ, in which all six wild-type cysteines have been substituted by serine, valine, or alanine (see materials and methods), was transiently expressed in HEK 293 cells. IK at +40 mV is shown before the application of SNDTT (unmarked), in the presence of 2 mM SNDTT (arrow), and after washout (dotted). (B) SNDTT block is distinct from inactivation. This experiment used ShBΔ with no additional mutations (see materials and methods) expressed in the Sf9 insect cell line. (panel i) Block versus C-type inactivation. Whole-cell IK during a 10-s depolarization to +40 mV is illustrated before (dotted) and after (solid) the addition of 1 mM SNDTT. Peak IK in the presence of SNDTT is indicated with an arrow. (panel ii) Recovery from block versus inactivation. Fraction recovered was calculated as in Fig. 3 from two-pulse recovery protocols initiated at the times indicated by the symbols in panel i. After a 10-s pulse in the absence of SNDTT (○), recovery at −80 mV is well described by a single exponential with τ = 1,055 ms (dotted line). After a 250-ms depolarization in the presence of 1 mM SNDTT (□), recovery is much faster (τ = 12 ms, solid line). After a 10-s depolarization in the presence of 1 mM SNDTT (▵), however, recovery is primarily slow. A double exponential fit (dashed line) reveals a major component (75% of amplitude) with τ = 1,175 ms, and a minor component (25%) with τ = 15 ms.
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Figure 10: SNDTT block does not require channel cysteines, and is not simply an acceleration of endogenous C-type inactivation. (A) SNDTT blocks a Shaker mutant lacking all cysteine residues. Cys-less ShBΔ, in which all six wild-type cysteines have been substituted by serine, valine, or alanine (see materials and methods), was transiently expressed in HEK 293 cells. IK at +40 mV is shown before the application of SNDTT (unmarked), in the presence of 2 mM SNDTT (arrow), and after washout (dotted). (B) SNDTT block is distinct from inactivation. This experiment used ShBΔ with no additional mutations (see materials and methods) expressed in the Sf9 insect cell line. (panel i) Block versus C-type inactivation. Whole-cell IK during a 10-s depolarization to +40 mV is illustrated before (dotted) and after (solid) the addition of 1 mM SNDTT. Peak IK in the presence of SNDTT is indicated with an arrow. (panel ii) Recovery from block versus inactivation. Fraction recovered was calculated as in Fig. 3 from two-pulse recovery protocols initiated at the times indicated by the symbols in panel i. After a 10-s pulse in the absence of SNDTT (○), recovery at −80 mV is well described by a single exponential with τ = 1,055 ms (dotted line). After a 250-ms depolarization in the presence of 1 mM SNDTT (□), recovery is much faster (τ = 12 ms, solid line). After a 10-s depolarization in the presence of 1 mM SNDTT (▵), however, recovery is primarily slow. A double exponential fit (dashed line) reveals a major component (75% of amplitude) with τ = 1,175 ms, and a minor component (25%) with τ = 15 ms.

Mentions: To avoid directional bias due to SNDTT breakdown, the sequence of voltage amplitudes for IK families (see Fig. 5) was randomized, as was the order of recovery intervals in two-pulse protocols (see Fig. 3 and Fig. 10).


Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Brock MW, Mathes C, Gilly WF - J. Gen. Physiol. (2001)

SNDTT block does not require channel cysteines, and is not simply an acceleration of endogenous C-type inactivation. (A) SNDTT blocks a Shaker mutant lacking all cysteine residues. Cys-less ShBΔ, in which all six wild-type cysteines have been substituted by serine, valine, or alanine (see materials and methods), was transiently expressed in HEK 293 cells. IK at +40 mV is shown before the application of SNDTT (unmarked), in the presence of 2 mM SNDTT (arrow), and after washout (dotted). (B) SNDTT block is distinct from inactivation. This experiment used ShBΔ with no additional mutations (see materials and methods) expressed in the Sf9 insect cell line. (panel i) Block versus C-type inactivation. Whole-cell IK during a 10-s depolarization to +40 mV is illustrated before (dotted) and after (solid) the addition of 1 mM SNDTT. Peak IK in the presence of SNDTT is indicated with an arrow. (panel ii) Recovery from block versus inactivation. Fraction recovered was calculated as in Fig. 3 from two-pulse recovery protocols initiated at the times indicated by the symbols in panel i. After a 10-s pulse in the absence of SNDTT (○), recovery at −80 mV is well described by a single exponential with τ = 1,055 ms (dotted line). After a 250-ms depolarization in the presence of 1 mM SNDTT (□), recovery is much faster (τ = 12 ms, solid line). After a 10-s depolarization in the presence of 1 mM SNDTT (▵), however, recovery is primarily slow. A double exponential fit (dashed line) reveals a major component (75% of amplitude) with τ = 1,175 ms, and a minor component (25%) with τ = 15 ms.
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Figure 10: SNDTT block does not require channel cysteines, and is not simply an acceleration of endogenous C-type inactivation. (A) SNDTT blocks a Shaker mutant lacking all cysteine residues. Cys-less ShBΔ, in which all six wild-type cysteines have been substituted by serine, valine, or alanine (see materials and methods), was transiently expressed in HEK 293 cells. IK at +40 mV is shown before the application of SNDTT (unmarked), in the presence of 2 mM SNDTT (arrow), and after washout (dotted). (B) SNDTT block is distinct from inactivation. This experiment used ShBΔ with no additional mutations (see materials and methods) expressed in the Sf9 insect cell line. (panel i) Block versus C-type inactivation. Whole-cell IK during a 10-s depolarization to +40 mV is illustrated before (dotted) and after (solid) the addition of 1 mM SNDTT. Peak IK in the presence of SNDTT is indicated with an arrow. (panel ii) Recovery from block versus inactivation. Fraction recovered was calculated as in Fig. 3 from two-pulse recovery protocols initiated at the times indicated by the symbols in panel i. After a 10-s pulse in the absence of SNDTT (○), recovery at −80 mV is well described by a single exponential with τ = 1,055 ms (dotted line). After a 250-ms depolarization in the presence of 1 mM SNDTT (□), recovery is much faster (τ = 12 ms, solid line). After a 10-s depolarization in the presence of 1 mM SNDTT (▵), however, recovery is primarily slow. A double exponential fit (dashed line) reveals a major component (75% of amplitude) with τ = 1,175 ms, and a minor component (25%) with τ = 15 ms.
Mentions: To avoid directional bias due to SNDTT breakdown, the sequence of voltage amplitudes for IK families (see Fig. 5) was randomized, as was the order of recovery intervals in two-pulse protocols (see Fig. 3 and Fig. 10).

Bottom Line: SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect.Finally, SNDTT is remarkably selective for Kv1 channels.When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

View Article: PubMed Central - PubMed

Affiliation: Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, CA 93950, USA.

ABSTRACT
Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

Show MeSH
Related in: MedlinePlus