The role of ryanodine receptors in the cyclic ADP ribose modulation of the M‐like current in rodent m1 muscarinic receptor‐transformed NG108‐15 cells

1 The role of cyclic ADP ribose and ryanodine receptors in the inhibition of the M‐like current ( I K(M,ng) ) by acetylcholine was investigated in m1 muscarinic receptor‐transformed mouse neuroblastoma‐rat glioma hybrid (NG108‐15) cells using patch‐clamp techniques and calcium microfluorimetry. 2 Acetylcholine (1–100 μM) decreased I K(M,ng) by up to 55 %. Application, via the patch pipette, of the cyclic ADP ribose antagonists 8‐amino‐cyclic ADP ribose (10–100 μM) and 8‐bromo‐cyclic ADP ribose (100–1000 μM) reduced this inhibition of I K(M,ng) in a concentration‐dependent manner. The half‐maximal inhibition concentrations for 8‐amino‐ cyclic ADP ribose and 8‐bromo‐cyclic ADP ribose were around 40 μM and 1 mM, respectively. 3 Neither of the cyclic ADP ribose antagonists altered the amplitude of I K(M,ng) per se , or the incidence of the concurrent Ca 2+ ‐activated K + current ( I IK(Ca) ) activation, also mediated by acetylcholine. 4 The ryanodine receptor modulators ryanodine (1–10 μM) and Ruthenium Red (10 μM) did not alter I K(M,ng) amplitude or I K(M,ng) inhibition mediated by acetylcholine. There was a statistically significant increase in the proportion of cells showing outward currents in the presence of Ruthenium Red. 5 Intracellular calcium levels measured with fura‐2 microfluorimetry were increased with low concentrations of ryanodine (1 μM), more consistently with caffeine (10 mM), and in almost every case with both bradykinin (300 nM) and acetylcholine (100 μM). Caffeine‐, but not bradykinin‐evoked responses were abolished by preincubation with ryanodine (10 μM). 6 The fast ‘rundown rate’ of the M‐current recorded in rat superior cervical ganglion cells under whole‐cell conditions precluded an investigation of the effects of intracellular dialysis of cyclic ADP ribose. However, when cyclic ADP ribose (5 μM) was applied directly to the cytoplasmic face of inside‐out membrane patches excised from rat superior cervical ganglion cells containing M‐channels, it had no effect on the main parameters of single channel activity (conductance, mean open time or frequency of opening). 7 These results indicate that cyclic ADP ribose acts on a specific intracellular site to mediate I K(M,ng) inhibition. However, unlike previously established effects of cyclic ADP ribose, the ryanodine receptor is not required, suggesting that another molecular target may be involved. Studies at the single channel level indicate that cyclic ADP ribose may not act directly on the M‐channels in inside‐out patches.