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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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Voltage dependence of SNDTT block. (A) ShBΔ IK records are illustrated at the indicated voltages in the presence of the indicated concentrations of SNDTT. All records are from a single cell. The application of each concentration was preceded by the acquisition of control records in normal external solution, and control IK at a given voltage varied by <10% over the course of the experiment. Symbols associated with concentrations are used in C and D. (B) The IK family for 0.5 mM SNDTT was normalized to controls as described for Fig. 4 B. Single exponential fits are superimposed. (C) The ratio of blocked to open channels (B/O) at the end of a 250-ms depolarization was calculated at voltages between 0 and +80 mV using the equation, B/O = (Icontrol − ISNDTT)/ISNDTT, where Icontrol and ISNDTT represent IK in the absence and presence of SNDTT, respectively. (C) τblock was derived from least-squares fits of single exponentials to normalized IK traces at voltages between 0 and +80 mV, as shown in B. Symbols (corresponding to SNDTT concentrations in A) and associated error bars represent means ± SEM for six cells. Numbers in parentheses in C and D represent equivalent charge movement q for least-squares fits (solid lines) to the generic equation: A = A(0) eqVF/RT, where A represents y-axis amplitude, A(0) represents amplitude at 0 mV, F is Faraday's constant, R is the universal gas constant, and T is absolute temperature (293°K).
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Figure 5: Voltage dependence of SNDTT block. (A) ShBΔ IK records are illustrated at the indicated voltages in the presence of the indicated concentrations of SNDTT. All records are from a single cell. The application of each concentration was preceded by the acquisition of control records in normal external solution, and control IK at a given voltage varied by <10% over the course of the experiment. Symbols associated with concentrations are used in C and D. (B) The IK family for 0.5 mM SNDTT was normalized to controls as described for Fig. 4 B. Single exponential fits are superimposed. (C) The ratio of blocked to open channels (B/O) at the end of a 250-ms depolarization was calculated at voltages between 0 and +80 mV using the equation, B/O = (Icontrol − ISNDTT)/ISNDTT, where Icontrol and ISNDTT represent IK in the absence and presence of SNDTT, respectively. (C) τblock was derived from least-squares fits of single exponentials to normalized IK traces at voltages between 0 and +80 mV, as shown in B. Symbols (corresponding to SNDTT concentrations in A) and associated error bars represent means ± SEM for six cells. Numbers in parentheses in C and D represent equivalent charge movement q for least-squares fits (solid lines) to the generic equation: A = A(0) eqVF/RT, where A represents y-axis amplitude, A(0) represents amplitude at 0 mV, F is Faraday's constant, R is the universal gas constant, and T is absolute temperature (293°K).

Mentions: Possible contributions of series resistance to the observed voltage dependence of SNDTT binding were investigated in the following manner. First, the corrected voltage (VSNDTT) for each measurement in the presence of SNDTT was calculated as VSNDTT = Vcom − ISNDTTRS, where Vcom is the command voltage, ISNDTT is steady-state IK in SNDTT, and RS is the effective series resistance. Second, voltages (Vcontrol) for control IK (Icontrol) were corrected in the same manner, and a corrected Icontrol − Vcontrol relationship was plotted. This relationship was linear above 0 mV for all cells, and the proper Icontrol at VSNDTT was determined by extrapolation. Corrected values for the ratio of blocked to open channels (B/O) could then be calculated and plotted as in Fig. 5 C. This correction procedure did not detectably alter the apparent valence of approximately −0.3 e0.


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

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

Voltage dependence of SNDTT block. (A) ShBΔ IK records are illustrated at the indicated voltages in the presence of the indicated concentrations of SNDTT. All records are from a single cell. The application of each concentration was preceded by the acquisition of control records in normal external solution, and control IK at a given voltage varied by <10% over the course of the experiment. Symbols associated with concentrations are used in C and D. (B) The IK family for 0.5 mM SNDTT was normalized to controls as described for Fig. 4 B. Single exponential fits are superimposed. (C) The ratio of blocked to open channels (B/O) at the end of a 250-ms depolarization was calculated at voltages between 0 and +80 mV using the equation, B/O = (Icontrol − ISNDTT)/ISNDTT, where Icontrol and ISNDTT represent IK in the absence and presence of SNDTT, respectively. (C) τblock was derived from least-squares fits of single exponentials to normalized IK traces at voltages between 0 and +80 mV, as shown in B. Symbols (corresponding to SNDTT concentrations in A) and associated error bars represent means ± SEM for six cells. Numbers in parentheses in C and D represent equivalent charge movement q for least-squares fits (solid lines) to the generic equation: A = A(0) eqVF/RT, where A represents y-axis amplitude, A(0) represents amplitude at 0 mV, F is Faraday's constant, R is the universal gas constant, and T is absolute temperature (293°K).
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Figure 5: Voltage dependence of SNDTT block. (A) ShBΔ IK records are illustrated at the indicated voltages in the presence of the indicated concentrations of SNDTT. All records are from a single cell. The application of each concentration was preceded by the acquisition of control records in normal external solution, and control IK at a given voltage varied by <10% over the course of the experiment. Symbols associated with concentrations are used in C and D. (B) The IK family for 0.5 mM SNDTT was normalized to controls as described for Fig. 4 B. Single exponential fits are superimposed. (C) The ratio of blocked to open channels (B/O) at the end of a 250-ms depolarization was calculated at voltages between 0 and +80 mV using the equation, B/O = (Icontrol − ISNDTT)/ISNDTT, where Icontrol and ISNDTT represent IK in the absence and presence of SNDTT, respectively. (C) τblock was derived from least-squares fits of single exponentials to normalized IK traces at voltages between 0 and +80 mV, as shown in B. Symbols (corresponding to SNDTT concentrations in A) and associated error bars represent means ± SEM for six cells. Numbers in parentheses in C and D represent equivalent charge movement q for least-squares fits (solid lines) to the generic equation: A = A(0) eqVF/RT, where A represents y-axis amplitude, A(0) represents amplitude at 0 mV, F is Faraday's constant, R is the universal gas constant, and T is absolute temperature (293°K).
Mentions: Possible contributions of series resistance to the observed voltage dependence of SNDTT binding were investigated in the following manner. First, the corrected voltage (VSNDTT) for each measurement in the presence of SNDTT was calculated as VSNDTT = Vcom − ISNDTTRS, where Vcom is the command voltage, ISNDTT is steady-state IK in SNDTT, and RS is the effective series resistance. Second, voltages (Vcontrol) for control IK (Icontrol) were corrected in the same manner, and a corrected Icontrol − Vcontrol relationship was plotted. This relationship was linear above 0 mV for all cells, and the proper Icontrol at VSNDTT was determined by extrapolation. Corrected values for the ratio of blocked to open channels (B/O) could then be calculated and plotted as in Fig. 5 C. This correction procedure did not detectably alter the apparent valence of approximately −0.3 e0.

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