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 microM)-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 microM)-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 (Ki=175 nM). Inclusion of 1 mM uridine 5'-diphosphate (UDP) in the pipette solution shifted the glibenclamide-sensitivity (Ki=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, Ki1=10 nM; low-affinity site, Ki2=9 microM). 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).
Teramoto
Noriyoshi
N
Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi Ward, Fukuoka, 812-8582, Japan. noritera@med.kyushu-u.ac.jp
Tomoda
Toshihisa
T
Yunoki
Takakazu
T
Ito
Yushi
Y
D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D000465:Algorithms; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002457:Cell Extracts / Q000494:pharmacology; D019806:Cromakalim / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005424:Flecainide / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology*; D054086:KATP Channels; D008564:Membrane Potentials / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D024661:Potassium Channels, Inwardly Rectifying / Q000502:physiology*; D013552:Swine; D013997:Time Factors; D014521:Urethra / Q000166:cytology
D018528:ATP-Binding Cassette Transporters; D002457:Cell Extracts; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; C092520:uK-ATP-1 potassium channel; D019806:Cromakalim; D001464:Barium; D005424:Flecainide; D005905:Glyburide