Inoue, Ryuji, and Yushi Ito. Intracellular ATP slows time-dependent decline of muscarinic cation current in guinea pig ileal smooth muscle. Am J Physiol Cell Physiol 279: C1307-C1318, 2000.-The effects of intracellular nucleotide triphosphates on time-dependent changes in muscarinic receptor cation currents (I cat ) were investigated using the whole cell patch-clamp technique in guinea pig ileal muscle. In the absence of nucleotide phosphates in the patch pipette, I cat evoked every 10 min

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... ed progressively. This decay was slowed dose dependently by inclusion of millimolar concentrations of ATP in the pipette. This required a comparable concentration of Mg 2ϩ , was mimicked by UTP and CTP, and was attenuated by simultaneous application of alkaline phosphatase or inhibitors of tyrosine kinase. In contrast, a sudden photolytic release of millimolar ATP (probably in the free form) caused a marked suppression of I cat . Submillimolar concentrations of GTP dose dependently increased the amplitude of I cat as long as ATP and Mg 2ϩ were in the pipette, but, in their absence, GTP was ineffective at preventing I cat decay. The decay of I cat was paralleled by altered voltage-dependent gating, i.e., a positive shift in the activation curve and reduction in the maximal conductance. It is thus likely that ATP exerts two reciprocal actions on I cat , through Mg 2ϩ -dependent and -independent mechanisms, and that the enhancing effect of GTP on I cat is essentially different from that of ATP. nucleotide phosphate; cation channels; muscarinic receptor; rundown ATP IS INVOLVED IN A VARIETY of fundamental cellular reactions associated with, e.g., synthesis of many bioactive molecules, performance of mechanical work, and active and passive transport of molecules and ions. Modification of ionic channel activity by ATP is a widespread mode of regulating cellular functions. For instance, phosphorylation of channel protein and associated regulatory subunits by protein kinases leads to altered kinetics of activation, inactivation, or desensitization in many different types of channel (24). In some, ATP is required through its hydrolysis, whereas in others it inhibits the channel activity through direct interaction with intracellular sites [ATP-sensitive K ϩ channels (31, 34); voltage-dependent Ca 2ϩ channels, (17, 28, 41); Ca 2ϩ -activated nonselective cation channels (33, 35); ion transporters and exchangers (10) ]. The muscarinic receptor-activated cation channel (or current; I cat ) has been found ubiquitously over the whole gastrointestinal tract and in chromaffin cells and some neuronal tissues (for review see Refs. 4 and 23). This channel is activated through a pertussis toxin-sensitive G protein (G i /G o or G o ) (14, 20, 37, 42) and undergoes effective regulation by the membrane potential (2, 12, 42), intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) (13, 29), and mechanical distortion (38). The functional significance of I cat regulation by intracellular ATP and other high-energy phosphates has recently been investigated in some detail. In the guinea pig gastric muscle, phosphorylation of the muscarinic receptor by protein kinase C, Ca 2ϩ /calmodulin, and myosin light chain kinase has been implicated in desensitization and maintenance of I cat activity (18, 19, 21) . In guinea pig ileal muscle, a strong enhancing effect of phosphocreatine and an inhibitory effect of ATP were observed on I cat , and the sites of these actions appeared to be downstream of the receptor (1). However, whether protein phosphorylation is involved in these effects remains equivocal, and, more importantly, the results of this study seem inconsistent in several critical points with those of previous studies including our own, particularly as to the actions of ATP, GTP, and Mg 2ϩ (15, 42; for details see DISCUSSION). We reasoned that this may be due to problems with the protocol for evaluating I cat activity and the efficacy of internal perfusion, which depends critically on the properties of intracellular perfusates as well as the accessibility between the patch pipette and the cell interior (see below). Failure to allow for the complex interactions of metals (e.g., Mg 2ϩ , Ca 2ϩ ) with their chelators [1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈtetraacetic acid (BAPTA), ATP, GTP] in different concentrations and combinations might have also affected the results, since divalent cations are potentially effective regulators of I cat activity. The goal of the present study was therefore twofold. First, we sought appropriate conditions for assessing the efficacy of intracellular perfusion on I cat . This was