EFFECTS OF LEVCROMAKALIM AND NUCLEOSIDE DIPHOSPHATES ON GLIBENCLAMIDE-SENSITIVE K+ CHANNELS IN PIG URETHRAL MYOCYTES

1 Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were in vestigated by use of patch clamp techniques (conventional whole-cell c onfiguration, nystatin perforated patch, cell-attached configuration a nd inside-out patches). 2 Levcromakalim produced a concentration-depen dent outward current at a holding potential of -50 mV. The peak curren t amplitude showed little variation when measured by either convention al whole-cell or nystatin perforated patch configurations. 3 In conven tional whole-cell configuration, the levcromakalim (100 mu M)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-indu ced outward current still remained after 18 min. 4 The peak amplitude of the levcromakalim (100 mu M)-induced outward membrane current recor ded by the conventional whole-cell configuration was greatly reduced b y inclusion of 5 mM EDTA in the pipette. The much smaller but signific ant outward membrane current remaining was abolished by glibenclamide. 5 In conventional whole-cell recordings, inclusion of an NDP in the p ipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamid e. Addition of 100 mu M levcromakalim after the NDP-induced current ha d peaked activated a further outward current which was larger than tha t recorded in the absence of NDPs. Approximately 50% of this current s till remained at 18 min, even when conventional whole-cell configurati on was used. 6 In the cell-attached mode in symmetrical 140 mM K+ cond itions, glibenclamide inhibited the 100 mu M levcromakalim-activated 4 3 pS K+ channel in a concentration-dependent manner. showing an inhibi tory dissociation constant (K-i) of approximately 520 nM. 7 In inside- out patches in which the glibenclamide-sensitive K+ channel had run do wn after exposure levcromakalim, both uridine 5'-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application o f Mg2+ to the UDP-reactivated K+ channels enhanced the channel activit y reversibly. 8 In inside-out patches UDP was capable of activating th e 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 f urther reversible activation of channel opening. 9 In the presence of levcromakalim, application of adenosine 5'-triphosphate (ATP) to the i nner surface of the membrane patch inhibited UDP-reactivated channel o pening in a concentration-dependent manner. 10 Addition of an untreate d cytosolic extract of pig proximal urethra reactivated the glibenclam ide-sensitive K+ channel in the presence of 100 mu M levcromakalim in inside-out patches. 11 These results demonstrate the presence in the p ig proximal urethra of a glibenclamide-sensitive K+ channel that is bl ocked by intracellular ATP and can be activated by levcromakalim. Intr acellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channe l activity.