1 The effects of caffeine on both levcromakalim-induced macroscopic and unitary currents in pig proximal urethra were investigated by the use of patch-clamp techniques (conventional whole-cell configuration and cell-attached configuration). The effects of caffeine were also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K+ channel (Kir6.2) subunits (i.e. Kir6.2ΔC36) which form ATP-sensitive K+ channels (K(ATP) channels). 2 In conventional whole-cell configuration, the levcromakalim (100 μM)-induced inward current (symmetrical 140 mM K+ conditions) was inhibited by caffeine (≥1 mM) at a holding potential of -50 mV. In contrast, ryanodine (10 μM) caused no significant inhibitory effect on the gradual decay of the levcromakalim-induced current at -50 mV. 3 The amplitude of the 30 μM levcromakalim-induced current was enhanced by 3-isobutyl-1-methylxanthine (IBMX, 100 μM). 4 In cell-attached configuration, the levcromakalim-induced K+ channel openings were inhibited by subsequent application of 10 mM caffeine, decreasing the channel open probability at -50 mV. 5 Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of Kir6.2 transcript in pig urethra. 6 Caffeine (≥3 mM) inhibited the channel activity of Kir6.2ΔC36 expressed in COS7 cells (3 mM caffeine, 65 ± 6%, n = 4; 10 mM caffeine, 29 ± 2%, n = 4). 7 These results suggest that caffeine can inhibit the activity of K(ATP) channels through a direct blocking effect on the pore-forming Kir subunit.
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