Although a critical factor causing lethal ischemic ventricular arrhythmias, net cellular K loss during myocardial ischemia and hypoxia is poorly understood. We investigated whether selective activation of ATP-sensitive K (K ATP ) channels causes net cellular K loss by examining the effects of the K ATP channel agonist cromakalim on unidirectional K efflux, total tissue K content, and action potential duration (APD) in isolated arterially perfused rabbit interventricular septa. Despite increasing unidirectional K efflux and shortening APD to a comparable degree as hypoxia, cromakalim failed to induce net tissue K loss, ruling out activation of K ATP channels as the primary cause of hypoxic K loss. Next, we evaluated a novel hypothesis about the mechanism of hypoxic K loss, namely that net K loss is a passive reflection of intracellular Na gain during hypoxia or ischemia. When the major pathways promoting Na influx were inhibited, net K loss during hypoxia was almost completely eliminated. These findings show that altered Na fluxes are the primary cause of net K loss during hypoxia, and presumably also in ischemia. Given its previously defined role during hypoxia and ischemia in promoting intracellular Ca overload and reperfusion injury, this newly defined role of intracellular Na accumulation as a primary cause of cellular K loss identifies it as a central pathogenetic factor in these settings. ( J. Clin. Invest. 1997. 100:1782-1788.)

Although previous work has implicated activation of ATP-sensitive K+ currents (IK,ATP) in action potential duration (APD) shortening and increased cellular K+ efflux during hypoxia, ischemia, and metabolic inhibition, no prior study has directly assessed the tissue levels of ATP at which IK,ATP activates in intact cardiac muscle. Accordingly, we correlated changes in tissue high-energy phosphate levels during substrate-free hypoxia with activation of IK,ATP in intact voltage-clamped rabbit papillary muscles. During 10 min of hypoxia, the outward K+ current measured in response to a voltage-clamp pulse step from -50 to 0 mV increased from 8.57 +/- 0.27 to 15.67 +/- 1.41 microA (P less than 0.05, n = 6), and APD decreased from 452 +/- 54 to 292 +/- 56 ms (P less than 0.05, n = 6). Glibenclamide (10 microM), a specific IK,ATP blocker, prevented both of these changes. In a parallel set of experiments, papillary muscles were freeze-clamped and assayed for tissue ATP. In these muscles, 10 min of hypoxia resulted in a comparable degree of APD shortening (441 +/- 24 to 297 +/- 18 ms, P less than 0.05, n = 12), and tissue ATP levels fell from 13.2 +/- 1.3 to 9.7 +/- 0.7 mumol/g dry wt (P less than 0.05, n = 12). These results directly demonstrate that IK,ATP is activated and causes APD shortening during hypoxia in intact cardiac muscle despite only a modest (approximately 25%) decline in tissue ATP content.

ATP-sensitive K + (KATP) channels are present in a wide variety of tissues. The sensitivity of these channels to closure by cytosolic ATP (ATPi) varies significantly among different tissues and even within the same tissue. The purpose of this study was to test the hypothesis that negative surface charges modulate the sensitivity of the KATe channels to ATPi by influencing surface potential in the vicinity of the ATP-binding site(s) of the channel. Unitary currents through KATe channels were measured in inside-out membrane patches excised from rabbit ventricular myocytes using the patch-clamp technique. Agents known to be effective at screening negative surface charges were applied to the cytosolic surface of the patches, and their effects on ATP sensitivity were examined. These agents included Mg 2+ (2-15 mM), Ba 2+ (2-10 mM), and the polycations protamine (0.01-10 ~,M), poly-L-lysine (500 p,M), and poly-L-arginine (0.5 I~M). The divalent cations and the various polycations all dramatically reduced the concentration of ATP i required to half-maximally suppress current through KATe channels (Kd), from ~ 100 IxM in the absence of these agents to 1.6-8 ~M in their presence. The effects were dose dependent. Protamine also reduced the sensitivity of KA~ channels to block by cytosolic ADP. The sensitivity of KATV channels to block by ATP was independent of membrane potential, suggesting that the ATP-binding site is not located within the transmembrane voltage field. The effects of the polycation poly-L-lysine on ATP sensitivity were also independent of membrane potential or the direction (inward or outward) of current through KATe channels. In addition to increasing ATP sensitivity, Mg 2+, Ba 2+, and the polycations all caused dose-dependent block of inward and outward currents through KAav channels over similar concentration ranges as their effects on ATP sensitivity. The block of inward current by polycations was not associated with reduction of single-channel conductance or evidence of fast open channel block. However, the polycations did cause a modest reduction in singlechannel conductance of outward current. These results are consistent with the presence of negative surface charges that reduce the local ATP concentration at the ATP-binding site(s) on the channel, relative to the bulk cytosolic ATP concentration. Screening these negative surface charges with divalent cations or polycations on May 12, 2018 jgp.rupress.org Downloaded from http://doi.org/10.1085/jgp.104.4.773 Published Online: 1 October, 1994 | Supp Info: 774THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 104 9 1994 decreases the local ATP gradient, resulting in a decrease in the apparent Kd for ATP. Applying surface potential theory to the Mg 2+ data, a rough calculation of the surface charge density necessary to account for these effects yielded a value of 1 negative charge per 199/~2, corresponding to surface potentials in the range of -50 to -70 inV. The putative negative surface charges are most likely located on the KATe channel protein it...

SUMMARY1. ATP-sensitive K+ (KATP) channels are believed to make an important contribution to the increased cellular K+ efflux and shortening of the action potential duration (APD) during metabolic inhibition, hypoxia, and ischaemia in the heart. The mechanisms by which the activity of the KA channel is regulated during conditions of metabolic impairment are not completely clear.

No abstract