Surface charge and properties of cardiac ATP-sensitive K+ channels

Abstract

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 Mg2+ (2-15 mM), Ba2+ (2-10 mM), and the polycations protamine (0.01-10 µM), poly-L-lysine (500 µM), and poly-L-arginine (0.5 µM). The divalent cations and the various polycations all dramatically reduced the concentration of ATPi required to half-maximally suppress current through KATe channels (Kd), from ~ 100 µM in the absence of these agents to 1.6-8 µM 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, Mg2+, Ba2+, 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 decreases the local ATP gradient, resulting in a decrease in the apparent Kd for ATP. Applying surface potential theory to the Mg2+ data, a rough calculation of the surface charge density necessary to account for these effects yielded a value of 1 negative charge per 1992, 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 itself rather than in the lipid bilayer because the negatively charged amphiphile SDS, which inserts into the lipid bilayer and increases negative surface charge sensed by voltage-dependent ion channels, had no effect on the ATP sensitivity of KAVe channels when applied to the cytoplasmic surface. Our results suggest that variability in negative surface charge density near the ATP-binding site(s) of KATP channels may in part explain the large differences in the ATP sensitivity of KATP channels in various tissues. © 1994, Rockefeller University Press., All rights reserved.