Effects of Thiol-Modifying Agents on a K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

Effects of Thiol-Modifying Agents on a K(Ca2+) Channel of Intermediate Conductance in Bovine... Ca2+-activated K+ channels (K(Ca2+)) constitute key regulators of the endothelial cell electrophysiological response to InsP3-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca2+) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mm) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mm) reduces gradually the K(Ca2+) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mm) or thimerosal (1 to 5 mm) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mm) or reduced glutathione (GSH) (5 mm) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H2O2 at concentrations ranging from 500 μm to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4′-dithiodipyridine (4-PDS) (1 mm) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca2+) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mm) and thimerosal (5 mm) were unable to inhibit the K(Ca2+) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca2+) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca2+) channels showed significant rundown. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Effects of Thiol-Modifying Agents on a K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

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Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 1997 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002329900252
Publisher site
See Article on Publisher Site

Abstract

Ca2+-activated K+ channels (K(Ca2+)) constitute key regulators of the endothelial cell electrophysiological response to InsP3-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca2+) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mm) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mm) reduces gradually the K(Ca2+) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mm) or thimerosal (1 to 5 mm) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mm) or reduced glutathione (GSH) (5 mm) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H2O2 at concentrations ranging from 500 μm to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4′-dithiodipyridine (4-PDS) (1 mm) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca2+) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mm) and thimerosal (5 mm) were unable to inhibit the K(Ca2+) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca2+) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca2+) channels showed significant rundown.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Jul 15, 1997

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