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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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DTDP inhibits maxi-K channel activity. (A) Addition  of the oxidizing agent 2,2′-dithiodipyridine (DTDP) also inhibited  channel activity. Continuous current trace of a representative experiment showing the effect of 50 μM DTDP on maxi-K channel  activity (holding potential = 10 mV). Large deflections were produced by a temporary increase of the gain of the patch-clamp amplifier to mark the drug addition event. (B) Average results from 7  patches exposed to 50 μM DTDP. * indicates statistical significance.
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Figure 5: DTDP inhibits maxi-K channel activity. (A) Addition of the oxidizing agent 2,2′-dithiodipyridine (DTDP) also inhibited channel activity. Continuous current trace of a representative experiment showing the effect of 50 μM DTDP on maxi-K channel activity (holding potential = 10 mV). Large deflections were produced by a temporary increase of the gain of the patch-clamp amplifier to mark the drug addition event. (B) Average results from 7 patches exposed to 50 μM DTDP. * indicates statistical significance.

Mentions: Conversely, application of sulfhydryl oxidizing agents to the patch membrane cytosolic surface reduced channel open-state probability. As shown in Fig. 4, concentrations as low as 5 μM diamide significantly inhibited channel activity, and nPo was only 10% of the control level at 5 mM of diamide (0.391 ± 0.085 to 0.040 ± 0.011, n = 7). Channel activity was similarly inhibited by DTDP; at a concentration of 50 μM, DTDP significantly reduced nPo from 0.383 ± 0.062 to 0.171 ± 0.033 (n = 7) (Fig. 5). To eliminate possible solvent effects, the same concentration (0.01%) of ethanol was included in the bath solution during the control period, and DTDP was applied by perfusing at least 10 times the bath volume.


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)

DTDP inhibits maxi-K channel activity. (A) Addition  of the oxidizing agent 2,2′-dithiodipyridine (DTDP) also inhibited  channel activity. Continuous current trace of a representative experiment showing the effect of 50 μM DTDP on maxi-K channel  activity (holding potential = 10 mV). Large deflections were produced by a temporary increase of the gain of the patch-clamp amplifier to mark the drug addition event. (B) Average results from 7  patches exposed to 50 μM DTDP. * indicates statistical significance.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229357&req=5

Figure 5: DTDP inhibits maxi-K channel activity. (A) Addition of the oxidizing agent 2,2′-dithiodipyridine (DTDP) also inhibited channel activity. Continuous current trace of a representative experiment showing the effect of 50 μM DTDP on maxi-K channel activity (holding potential = 10 mV). Large deflections were produced by a temporary increase of the gain of the patch-clamp amplifier to mark the drug addition event. (B) Average results from 7 patches exposed to 50 μM DTDP. * indicates statistical significance.
Mentions: Conversely, application of sulfhydryl oxidizing agents to the patch membrane cytosolic surface reduced channel open-state probability. As shown in Fig. 4, concentrations as low as 5 μM diamide significantly inhibited channel activity, and nPo was only 10% of the control level at 5 mM of diamide (0.391 ± 0.085 to 0.040 ± 0.011, n = 7). Channel activity was similarly inhibited by DTDP; at a concentration of 50 μM, DTDP significantly reduced nPo from 0.383 ± 0.062 to 0.171 ± 0.033 (n = 7) (Fig. 5). To eliminate possible solvent effects, the same concentration (0.01%) of ethanol was included in the bath solution during the control period, and DTDP was applied by perfusing at least 10 times the bath volume.

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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