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Redox regulation of large conductance Ca(2+)-activated K+ channels in smooth muscle cells.

Wang ZW, Nara M, Wang YX, Kotlikoff MI - J. Gen. Physiol. (1997)

Bottom Line: The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes.Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP0 relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP0 following reduction did not result from recruitment of more functional channels but rather from changes of channel gating.We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.

ABSTRACT
The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, beta-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2'-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP0 relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP0 following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.

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The thiol alkylating agent NEM inhibits maxi-K channels and blocks the effect of oxidation. (A) Continuous trace showing the effects of N-ethylmaleimide (NEM (1 mM)) and subsequent  diamide (0.5 mM) on maxi-K activity in an inside-out patch (holding potential = 10 mV). After recording control (control 1) activity,  the patch was exposed to NEM for 5 min and then washed. Channel activity was recorded for about 3 min (control 2) before addition of diamide (0.5 mM). (B) Mean data from six similar experiments. NEM inhibited maxi-K channel activity significantly (indicated by *), and diamide had no additional effect.
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Figure 9: The thiol alkylating agent NEM inhibits maxi-K channels and blocks the effect of oxidation. (A) Continuous trace showing the effects of N-ethylmaleimide (NEM (1 mM)) and subsequent diamide (0.5 mM) on maxi-K activity in an inside-out patch (holding potential = 10 mV). After recording control (control 1) activity, the patch was exposed to NEM for 5 min and then washed. Channel activity was recorded for about 3 min (control 2) before addition of diamide (0.5 mM). (B) Mean data from six similar experiments. NEM inhibited maxi-K channel activity significantly (indicated by *), and diamide had no additional effect.

Mentions: We reasoned that the action of redox agents on maxi-K channel activity was due to the modification of the sulfhydryl group of cysteine residues, and the formation or breakdown of one or more disulfide bonds. To confirm that the modification of reactive thiols underlies the observed effect of redox agents, we examined the response of maxi-K channels to oxidizing agents in NEM-treated patches. NEM alkylates free sulfhydryl groups (Creighton, 1993). If free thiols are involved in the responses to oxidizing agents, covalent modification of the free thiols by NEM should prevent disulfide bond formation, and attendant alterations in channel activity, following exposure to oxidizing agents. As shown in Fig. 9 A, addition of NEM (1 mM) to the bath solution rapidly inhibited maxi-K channel opening in inside-out patches and prevented the subsequent inhibition of channel activity upon exposure to diamide (0.5 mM). The concentration of diamide applied was 100-fold greater than that required to significantly inhibit channel activity in nonalkylated patches (compare Fig. 4). The results of six similar experiments are summarized in Fig. 9 B.


Redox regulation of large conductance Ca(2+)-activated K+ channels in smooth muscle cells.

Wang ZW, Nara M, Wang YX, Kotlikoff MI - J. Gen. Physiol. (1997)

The thiol alkylating agent NEM inhibits maxi-K channels and blocks the effect of oxidation. (A) Continuous trace showing the effects of N-ethylmaleimide (NEM (1 mM)) and subsequent  diamide (0.5 mM) on maxi-K activity in an inside-out patch (holding potential = 10 mV). After recording control (control 1) activity,  the patch was exposed to NEM for 5 min and then washed. Channel activity was recorded for about 3 min (control 2) before addition of diamide (0.5 mM). (B) Mean data from six similar experiments. NEM inhibited maxi-K channel activity significantly (indicated by *), and diamide had no additional effect.
© Copyright Policy
Related In: Results  -  Collection

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Figure 9: The thiol alkylating agent NEM inhibits maxi-K channels and blocks the effect of oxidation. (A) Continuous trace showing the effects of N-ethylmaleimide (NEM (1 mM)) and subsequent diamide (0.5 mM) on maxi-K activity in an inside-out patch (holding potential = 10 mV). After recording control (control 1) activity, the patch was exposed to NEM for 5 min and then washed. Channel activity was recorded for about 3 min (control 2) before addition of diamide (0.5 mM). (B) Mean data from six similar experiments. NEM inhibited maxi-K channel activity significantly (indicated by *), and diamide had no additional effect.
Mentions: We reasoned that the action of redox agents on maxi-K channel activity was due to the modification of the sulfhydryl group of cysteine residues, and the formation or breakdown of one or more disulfide bonds. To confirm that the modification of reactive thiols underlies the observed effect of redox agents, we examined the response of maxi-K channels to oxidizing agents in NEM-treated patches. NEM alkylates free sulfhydryl groups (Creighton, 1993). If free thiols are involved in the responses to oxidizing agents, covalent modification of the free thiols by NEM should prevent disulfide bond formation, and attendant alterations in channel activity, following exposure to oxidizing agents. As shown in Fig. 9 A, addition of NEM (1 mM) to the bath solution rapidly inhibited maxi-K channel opening in inside-out patches and prevented the subsequent inhibition of channel activity upon exposure to diamide (0.5 mM). The concentration of diamide applied was 100-fold greater than that required to significantly inhibit channel activity in nonalkylated patches (compare Fig. 4). The results of six similar experiments are summarized in Fig. 9 B.

Bottom Line: The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes.Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP0 relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP0 following reduction did not result from recruitment of more functional channels but rather from changes of channel gating.We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.

ABSTRACT
The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, beta-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2'-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP0 relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP0 following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.

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