Time-dependent fading of the activation of KATP channels, induced by aprikalim and nucleotides, in excised membrane patches from cardiac myocytes.

1. The effects of the potassium channel opener (KCO) aprikalim (RP 52891) on the nucleotide-induced modulation of ATP-sensitive K+ (KATP) channels in freshly dissociated ventricular myocytes of guinea-pig heart, were studied by use of the inside-out patch-clamp technique. The internal surface of the excised membrane patch was initially bathed with a standard solution (Mg(2+)-free with EDTA), then sequentially superfused with solutions containing nucleoside diphosphates (NDPs: 200 microM ADP and 50 microM GDP) and NDPs plus 1 mM MgCl2 (with EGTA; referred to as Mg-NDP solution). 2. The normalized concentration-response (channel closing) relationship to ATP was shifted to the right when the standard solution was replaced by the Mg-NDP solution. Hence, the internal concentration of ATP ([ATP]i) inhibiting the channel activity by half (Ki) increased from 56 microM to 180 microM, with an apparently constant slope factor (s = 2.37). NDPs in the absence of Mg2+ did not decrease the sensitivity of the channels to ATP. 3. In standard solution, aprikalim (100 microM) activated KATP channels in the presence of a maximally inhibitory [ATP]i (500 microM). This effect was strongly enhanced when aprikalim was applied to patches exposed to Mg-NDP solution, as demonstrated by the 9 fold increase in Ki for [ATP]i (from 180 microM to 1.5 mM and s = 2.37). 4. The ability of aprikalim to overcome the channel closing effects of ATP in Mg-NDP solution waned rapidly. Similarly, the NDP-induced activation of ATP-blocked channels was also time-dependent. Both activation processes disappeared before the channel run-down phenomenon appeared in ATP-free conditions. 5. In conclusion, aprikalim is much more potent in opening KATP channels in membrane patches bathed in Mg-NDP solution than in standard solution. However, under the former experimental conditions, the effect of aprikalim waned rapidly. It is proposed that the waning phenomenon results from changes in the intrinsic enzymatic activity of the KATP channel protein (possibly linked to the experimental conditions) which lead to the channel closure.