Different glibenclamide-sensitivity of ATP-sensitive K + currents using different patch-clamp recording methods

Abstract

Electorophysiological and pharmacological properties of the levcromakalim-induced inward ATP-sensitive K + currents (K ATP currents) in pig proximal urethra were investigated by use of two different whole-cell patch-clamp techniques, namely conventional whole-cell and nystatin-perforated patch recordings. In conventional whole-cell configuration, the levcromakalim (100 μM)-induced K ATP current decayed by about 30% in 8 min at a holding potential of − 50 mV. In contrast, with the nystatin-perforated patch, 96% of the levcromakalim-induced K ATP current still remained even after 8 min application of levcromakalim. The peak amplitude of the levcromakalim-induced inward K ATP currents in nystatin-perforated patch was approximately half of those observed in conventional whole-cell configuration. When cytosolic extract of pig urethra was included in the pipette solution, approximately 90% of the levcromakalim (100 μM)-induced K ATP current remained at 8 min, even after the establishment of conventional whole-cell configuration. In conventional whole-cell configuration, glibenclamide suppressed the levcromakalim-induced K ATP currents in a concentration-dependent manner ( K i = 175 nM). Inclusion of 1 mM uridine 5′-diphosphate (UDP) in the pipette solution shifted the glibenclamide-sensitivity ( K i = 640 nM) to the right in comparison with that in the absence of UDP (i.e., control). In contrast, using nystatin-perforated patch, glibenclamide inhibited the levcromakalim-induced K ATP currents with two affinity sites (high-affinity site, K i1 = 10 nM; low-affinity site, K i2 = 9 μM). The concentration response curves regarding the inhibitory effects of K ATP channel pore blockers (Ba 2+ and flecainide) on the levcromakalim-induced K ATP currents in conventional whole-cell recording nearly overlapped with those in nystatin-perforated patch recording. These results indicate that the glibenclamide-sensitivity of pig urethral K ATP channels in nystatin-perforated patch recording was significantly different from that in a conventional whole-cell configuration, and that the glibenclamide-sensitivity may be modified by some cytosolic factor(s).