Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K⁺ channels in pig urethral myocytes

Abstract

1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches).

2. Levcromakalim produced a concentration-dependent outward current at a holding potential of −50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations.

3. In conventional whole-cell configuration, the levcromakalim (100 μM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min.

4. The peak amplitude of the levcromakalim (100 μM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide.

5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 μM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used.

6. In the cell-attached mode in symmetrical 140 mM K⁺ conditions, glibenclamide inhibited the 100 μM levcromakalim-activated 43 pS K⁺ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (K_i) of approximately 520 nM.

7. In inside-out patches in which the glibenclamide-sensitive K⁺ channel had run down after exposure to levcromakalim, both uridine 5′-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg²⁺ to the UDP-reactivated K⁺ channels enhanced the channel activity reversibly.

8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K⁺ channel without levcromakalim, providing that there was free Mg²⁺ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg²⁺). Additional application of levcromakalim caused a further reversible activation of channel opening.

9. In the presence of levcromakalim, application of adenosine 5′-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner.

10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K⁺ channel in the presence of 100 μM levcromakalim in inside-out patches.

11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K⁺ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg²⁺ may play an important role in regulating the channel activity.

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