The effects of caffeine on ATP-sensitive K+ channels in smooth muscle cells from pig urethra

1 The effects of caffeine on both levcromakalim-induced macroscopic and uni tary 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 i nside-out patches of COS7 cells expressing carboxy terminus truncated inwar dly rectifying K+ channel (Kir6.2) subunits (i.e. Kir6.2 Delta C36) which f orm ATP-sensitive K+ channels (K-ATP channels). 2 In conventional whole-cell configuration, the levcromakalim (100 mu M)-in duced inward current (symmetrical 140 mM K+ conditions) was inhibited by ca ffeine (greater than or equal to 1 mM) at a holding potential of -50 mV. In contrast, ryanodine (10 mu M) caused no significant inhibitory effect on t he gradual decay of the levcromakalim-induced current at - 50 mV. 3 The amplitude of the 30 mu M levcromakalim-induced current was enhanced b y 3-isobutyl-1-methylxanthine (IBMX, 100 mu M). 4 In cell-attached configuration, the levcromakalim-induced K+ channel open ings 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 reveale d the presence of Kir6.2 transcript in pig urethra. 6 Caffeine (greater than or equal to 3 mM) inhibited the channel activity o f Kir6.2 Delta C36 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 KATP chan nels through a direct blocking effect on the pore-forming Kir subunit.