A B S T R A C T Inactivation of a dihydropyridine-sensitive calcium current was studied in a cell line (A7rS) derived from smooth muscle of the rat thoracic aorta. Inactivation is faster with extracellular Ca ~÷ than with Ba ~÷. In Ba 2÷, inactivation increases monotonically with depolarization. In Ca 2÷, inactivation is related to the amount of inward current, so that little inactivation is seen in Ca 2÷ for brief depolarizations approaching the reversal potential. Longer depolarizations in Ca 2÷ reveal two components of inactivation, the slower component behaving like that observed in Ba ~÷. Furthermore, lowering extracellular Ca ~÷ slows inactivation. These results are consistent with the coexistence of two inactivation processes, a slow voltage-dependent inactivation, and a more rapid current-dependent inactivation which is observable only with Ca 2÷. Ca~÷-dependent inactivation is decreased but not eliminated when intraceUular Ca 2÷ is buffered by 10 mM BAPTA, suggesting that Ca 2÷ acts at a site on or near the channel. We also studied recovery from inactivation after either a short pulse (able to produce significant inactivation only in Ca 2÷) or a long pulse (giving similar inactivation with either cation). Surprisingly, recovery from Ca~÷-dependent inactivation was voltage dependent. This suggests that the pathways for recovery from inactivation are similar regardless of how inactivation is generated. We propose a model where Ca ~÷-and voltage-dependent inactivation occur independently.

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Extracellular ATP receptors in rat ventricular myocyles wcrc investigated through intact cell photolabclling followed by protein isolation. S-Azido-ATP @AZ-ATP) was used for labclliny under specific conditions determined by parallel functional sludics. In those studies ATP-induced cytosolic Ca?' iransients were irreversibly and specifically inhibited by UV-photolyzed SAz-ATP, but not by 2.azido-ATP (2Az-ATP), even in the presence of high concentrations or phosphonucleotidcs not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of4548 kDa on a SDS gel. Labclling under the above conditions in the presence of ATDyS or I-mcthylthio-ATP (2.meSATP), which arc distinct for tw3 functionally different cardiac ATP receptors. shows two different proteins within the same band consistent with the possible labelling of these two receptors.ATP receptor; Photolabelling; Myocyte; S-Azido-ATP I e INTRODUCTIONThe exposure of many cell types to micromolar concentrations of extracellular ATP results in signals modulating several intracellular functions. Following the original finding that extracellular ATP serves as a neurotransmitter [1,2], the spectrum of activities elicited by ATP has significantly expanded and now includes regulation of most parenchymal and epithelial cells [3]. This resulted in the classification of PI-purinergic receptors which are ATP specific as compared to adenosine-specific P,-purinergic receptors (reviewed in [4]).Recently, several P2 receptor subtypes in various cells and within the same cell type have been classified on the basis of functional responses and agonist specificity. Accordingly, evidence has been provided for receptors linked to G-proteins, activating channels or forming pores (For review see . Heretofore, no receptor has been isolated and no information exists on even partial primary structure, a condition which confounds classilication of ATP receptors and limits our knowledge of ATP interaction with the receptor(s) and the resulting cellular signals.Previous studies from our laboratory [7,8] and others [9] have shown that extracellular ATP regulates transmembrane potential and cytosolic Ca" in ventricular myocytes. At least two types of ATP receptors described in these cells have been shown to be functionally distinct from ATP receptors in other cells. The first type has stringent requirements for MgATP, and responds to 2-meSATP and ATP, but not to ATPyS, UTP, GTP, ADP or adenosine. The major effect of Mg-ATP binding to this receptor is the activation of an inward nonselective cation depolarizing current. This event, which is not G-protein linked, is followed by the activation of voltage-sensitive Ca?' channels, increase in Ca" inflwr and Ca'+-induced Ca?' release from the sarcoplasmic reticulum [i']. The second type is activated by ATP or ATPy.5, but not 2-meSATP, probably requires G-proteins, and initiates uptake of both gi and Na' from the extracellular spaces [IO].

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