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

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 mu M)-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 mu M)-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 mu M). The concentration response curves regarding the inhibitory effects of K-ATP channel pore blockers (Ba2+ 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). (c) 2005 Elsevier B.V. All rights reserved.