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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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Structures of SNDTT and DTT compared with those of several QA and tertiary amine open-channel blockers. Open-channel block of squid axonal Kv channels by C9 has been described by Armstrong 1971 and Swenson 1981. Methadone is an opioid receptor agonist that has been shown to block IK in squid giant fiber lobe (GFL) neurons (Horrigan and Gilly 1996). The antiarrhythmic drug quinidine produces open-channel block in the cardiac Kv1.5 channel (Snyders et al. 1992). The antihistamine terfenadine (Yang et al. 1995), and the Ca2+ channel blocker verapamil (Rampe et al. 1993a) also produce open-channel block in Kv1.5. All tertiary amines are shown in protonated form, which predominates at neutral pH.
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Figure 1: Structures of SNDTT and DTT compared with those of several QA and tertiary amine open-channel blockers. Open-channel block of squid axonal Kv channels by C9 has been described by Armstrong 1971 and Swenson 1981. Methadone is an opioid receptor agonist that has been shown to block IK in squid giant fiber lobe (GFL) neurons (Horrigan and Gilly 1996). The antiarrhythmic drug quinidine produces open-channel block in the cardiac Kv1.5 channel (Snyders et al. 1992). The antihistamine terfenadine (Yang et al. 1995), and the Ca2+ channel blocker verapamil (Rampe et al. 1993a) also produce open-channel block in Kv1.5. All tertiary amines are shown in protonated form, which predominates at neutral pH.

Mentions: Inactivation of voltage-gated potassium (Kv) channels by large organic cations was first described by Armstrong 1969, Armstrong 1971, who observed that injection of long-chain TEA derivatives (see Fig. 1) into voltage-clamped squid giant axons causes an exponential decay of IK during a strong depolarization. This decay follows a normal activation time course, suggesting that block by these quaternary ammonium (QA) ions occurs only after the channel activation gates have opened (i.e., “open-channel block”). Also in agreement with a gated access mechanism, bound QA ions inhibit closing of channel activation gates after repolarization by what was later termed a “foot-in-the-door” effect (Yeh and Armstrong 1978). This effect can be overcome at very hyperpolarized potentials, where channels can apparently be forced to close with blocker still bound. Unbinding (or recovery) is very slow at these voltages, leading to the concept that QA ions can be trapped in an internal cavity located between the channel selectivity filter and activation gate. The interpretation of these results as summarized above has become fundamental to the field (for reviews see Armstrong 1972; Yellen 1998)


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

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

Structures of SNDTT and DTT compared with those of several QA and tertiary amine open-channel blockers. Open-channel block of squid axonal Kv channels by C9 has been described by Armstrong 1971 and Swenson 1981. Methadone is an opioid receptor agonist that has been shown to block IK in squid giant fiber lobe (GFL) neurons (Horrigan and Gilly 1996). The antiarrhythmic drug quinidine produces open-channel block in the cardiac Kv1.5 channel (Snyders et al. 1992). The antihistamine terfenadine (Yang et al. 1995), and the Ca2+ channel blocker verapamil (Rampe et al. 1993a) also produce open-channel block in Kv1.5. All tertiary amines are shown in protonated form, which predominates at neutral pH.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233744&req=5

Figure 1: Structures of SNDTT and DTT compared with those of several QA and tertiary amine open-channel blockers. Open-channel block of squid axonal Kv channels by C9 has been described by Armstrong 1971 and Swenson 1981. Methadone is an opioid receptor agonist that has been shown to block IK in squid giant fiber lobe (GFL) neurons (Horrigan and Gilly 1996). The antiarrhythmic drug quinidine produces open-channel block in the cardiac Kv1.5 channel (Snyders et al. 1992). The antihistamine terfenadine (Yang et al. 1995), and the Ca2+ channel blocker verapamil (Rampe et al. 1993a) also produce open-channel block in Kv1.5. All tertiary amines are shown in protonated form, which predominates at neutral pH.
Mentions: Inactivation of voltage-gated potassium (Kv) channels by large organic cations was first described by Armstrong 1969, Armstrong 1971, who observed that injection of long-chain TEA derivatives (see Fig. 1) into voltage-clamped squid giant axons causes an exponential decay of IK during a strong depolarization. This decay follows a normal activation time course, suggesting that block by these quaternary ammonium (QA) ions occurs only after the channel activation gates have opened (i.e., “open-channel block”). Also in agreement with a gated access mechanism, bound QA ions inhibit closing of channel activation gates after repolarization by what was later termed a “foot-in-the-door” effect (Yeh and Armstrong 1978). This effect can be overcome at very hyperpolarized potentials, where channels can apparently be forced to close with blocker still bound. Unbinding (or recovery) is very slow at these voltages, leading to the concept that QA ions can be trapped in an internal cavity located between the channel selectivity filter and activation gate. The interpretation of these results as summarized above has become fundamental to the field (for reviews see Armstrong 1972; Yellen 1998)

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