News Physiol Sci 13: 131-137, 1998;
1548-9213/98 $5.00
News in Physiological Sciences, Vol. 13, No. 3, 131-137,
June 1998
© 1998 Int. Union Physiol. Sci./Am. Physiol. Soc.
Functional Modulation of Cardiac ATP-Sensitive K+ Channels
Masayasu Hiraoka and
Tetsushi Furukawa
M. Hiraoka is in the Dept. of Cardiovascular Diseases and T. Furukawa is in the Dept. of Autonomic Physiology of the Medicalcal Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113 Japan.
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Abstract
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ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP, but MgATP is necessary to maintain the channel activity. Numerous cofactors modulate channel function. K+ channel openers activate and sulfonylureas inhibit KATP channels. The structure of cardiac KATP channel is a complex of mainly KIR6.2 and SUR2a. Activation of cardiac KATP channels contributes to action potential shortening during ischemia and plays a role in cardioprotection.
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Introduction
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The ATP-sensitive K+ (KATP) channels were discovered by Noma (11) in cardiac ventricular myocytes. Since then, these channels have been identified in various tissues, such as pancreatic ß-cells, skeletal muscle, vascular and nonvascular smooth muscle, and neuronal cells (1, 9), where they contribute to diverse cellular responses by linking the bioenergetic states of the cell to the membrane potential. The physiological function of the KATP channel is well recognized in pancreatic ß-cells. Inactivation of this channel in response to changes in external glucose levels that alter the intracellular ATP-to-ADP ratio leads to cell depolarization, opening of Ca2+ channels, action potential generation, Ca2+ entry, and insulin secretion (12). In cardiac cells, the physiological function of KATP channels is not known because these channels are believed to be closed in the normal, functioning heart; however, they may play significant roles in pathological conditions, especially in myocardial ischemia, where cellular ATP levels fall substantially. The opening of KATP channels in early ischemia causes shortening of the action potential duration and, to some extent, contributes to cellular K+ loss. This could promote the development of serious arrhythmias, on the one hand, but, on the other hand, might be beneficial in abbreviating excess Ca2+ entry, thus providing protection from cell injury (2, 9, 15). It has been shown that KATP channel openings may play a pivotal role in ischemic preconditioning, in which brief ischemia followed by reperfusion protects the myocardium against the damage produced by a second prolonged ischemic insult. The protective action of the KATP channel openings is not limited to the heart but is also observed in the brain (9). Therefore, modulations of the KATP channel functions have important implications in the physiology and pathophysiology of the heart as well as other organs.
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Biophysical properties of cardiac KATP channels
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KATP channels are distributed in almost all types of cardiac cells in different regions of the heart. The KATP channel is a highly K+-selective channel with a Na+-to K+ permeability ratio of ~0.01. The single-channel conductance at voltages below the K+ equilibrium potential is 70–90 pS under symmetrical 150 mM K+ conditions, which is the second largest conductance in various cardiac K+ channels. At positive voltages above +40 mV, the outward current is smaller than the inward current and exhibits a weak inward rectification. The inward rectification of the KATP channel is mainly attributed to a voltage-dependent block by intracellular cations such as Mg2+ and Na+. Channel openings are characterized by clusters of bursts separated by long closed periods. The channels are not activated in a voltage-dependent manner, but voltage may modulate the channel activity. Most of these biophysical properties are shared by KATP channels in cardiac cells, pancreatic ß-cells, and skeletal muscles, but some diversity of their properties is reported in various vascular smooth muscle cells (1, 10, 11).
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Functional modulations of cardiac KATP channels by various intra- and extracellular factors
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KATP channel activity is inhibited when the intracellular ATP concentration ([ATP]i), but not extracellular ATP concetration, is increased (Fig. 1
). This ATP-induced inhibition is achieved both in freeform as well as in a Mg2+-bound form of ATP; nonhydrolyzable ATP analogs can also inhibit the channels. Therefore, the phosphorylation or hydrolysis of ATP is not required for channel inhibition; inhibition is caused by a ligand action of ATP. The ability to inhibit these channels is shared by other adenine nucleotides such as ADP, AMP, CTP, GTP, UTP, and ITP, but ATP is the most effective inhibitor among them. The effect of increased [ATP]i on channel activity is to increase interburst intervals and to decrease burst durations without affecting the fast flickering kinetics in the bursts and the single-channel conductance. The exact number of ATP molecules required for this inhibition is not known. The relationship between [ATP]i and channel activity, however, can be fitted to a Hill equation with an apparent Hill coefficient of ~2 (range between 1 and 6), suggesting that there might be two or more binding sites on the channel protein (1, 10, 11, 13).
Cytoplasmic domains of the KATP channel protein have been assumed to be responsible for determining ATP sensitivity. The sensitivity of channel closure to [ATP]i is somewhat less in cardiac and skeletal KATP channels than in the pancreatic ß-cell channels. Cardiac KATP channels appear particularly responsive to changes in [ATP]i produced by glycolysis. Moreover, the ATP sensitivity of the channel may be different in epicardial and endocardial cells in the ventricular wall. In hypertrophied hearts, ATP sensitivity appears to be decreased, suggesting that functional remodeling of the channels may take place under pathological conditions.
In inside-out, cell-free conditions, a half-maximal inhibition of the channel activity is achieved at [ATP]i of <100 µM (usually 20–30 µM), and the channel is completely inhibited above millimolar levels of [ATP]i. This raises a serious question as to whether KATP channels can be opened in cardiac cells subjected to early myocardial ischemia, in which [ATP]i is still maintained at nearly physiological levels of ~5 mM. Several possible explanations have been proposed to account for the openings of KATP channels in living cells despite the low channel activity in the presence of [ATP]i observed in cell-free patches and the relatively high cellular ATP content (see Refs. 10 and 13). First, there are various intra- and extracellular cofactors that modulate ATP-induced openings or closings of the channels in the living cells, which are lost during cell excision. These cofactors include MgADP, intracellular H+, G proteins, extracellular adenosine, and so forth. Second, [ATP]i may not be equally distributed throughout the cell, but, instead, its distribution may be nonuniform; [ATP]i may be low near the surface membrane close to the channels but high in the core of the cell. Third, there is a wide variability of the 50% inhibitory concentration values for the [ATP]i-induced inhibition observed in different patches by different investigators, suggesting heterogeneity in the sensitivities among KATP channels. Fourth, because of the high density of the KATP channel distribution (~3,000 channels/ventricular cell) and the large conductance of the channels, activation of only 0.5% of KATP channels may be sufficient (spare channel hypothesis). Thus sufficient KATP channels may be active in living cells even in the absence of markedly reduced cellular ATP content.
Although KATP channels are inhibited by increased [ATP]i, under ATP-free conditions, their activity gradually decreases with time, a process known as "rundown" (1, 10). The rundown of KATP channels is prevented or slowed by treatment of the cytoplasmic side of patch membranes with proteolytic enzymes such as trypsin in guinea pig and rabbit, but not in rat, cardiac preparations. It is proposed that trypsin removes an inactivation particle or modifies some regulatory unit on the channel protein or an associated structure. However, the molecular identification of this particle or regulatory unit on the channel proteins is not known. Application of divalent cations such as Ca2+ or Mg2+ at relatively high concentrations to the internal face of patch membranes can also induce channel rundown. The sensitivity of the KATP channel rundown to Ca2+ suggests that its action is mediated through activation of Ca2+-dependent phosphatase, but the findings that application of neither phosphatase inhibitors nor protease inhibitors can inhibit Ca2+-induced channel inactivation, do not support this notion.
The spontaneous or Ca2+-induced rundown of KATP channel activity is restored by a short exposure to MgATP (1, 10, 13). Therefore, although ATP is a strong inhibitor of channel activity, it is also required for maintenance of channel activity. In contrast to channel inhibition, the process of channel reactivation can only be achieved by MgATP but not by the free form of ATP, and nonhydrolyzable ATP analogs seem to be ineffective. These findings suggest that phosphorylation may be involved in maintaining KATP channels in an operative state.
However, experiments using various protein kinase inhibitors do not support the notion that dephosphorylation and phosphorylation are involved in rundown and subsequent reactivation by MgATP. The results further indicate that ATP hydrolysis is involved in the reactivation process. Furthermore, the assembly and disassembly of actin cytoskeletal network, which also utilize ATP hydrolysis energy, regulate the processes of rundown and reactivation of cardiac KATP channels (5). More recently, a different view has been presented that states that phosphatidylinositol 4,5-bisphosphate, which inhibits various actin binding proteins promoting polymerization of long F-actin, can itself induce the channel reactivation (6). Functional significance of this rundown and reactivation is not known, and further study employing the molecular probe for KATP channels is necessary to clarify this important issue.
In addition to [ATP]i, KATP channel activity is modulated by various other factors, whose mechanisms are not completely understood. Among various cofactors that modulate KATP channel activity, ADP may be the most physiologically important. Although in the absence of Mg2+ ADP is a weak inhibitor of the channels compared with ATP, ADP stimulates the channel activity in the presence of Mg2+ at concentrations <250 µM. In the pancreatic ß-cell, the ATP-to-ADP ratio, rather than the ADP concentration itself, is an important determinant of the channel function, and it may also be applicable to the cardiac KATP channel. This stimulating action of ADP is achieved by antagonizing [ATP]i-induced inhibition, thus shifting the dose-response curve of ATP inhibition to the right (Fig. 1
). The exact mechanism of this ADP antagonism to ATP inhibition is not known. One proposed explanation is that there are two ATP binding inhibitory sites, one with a strong inhibitory capacity and the other with a weak inhibitory capacity but with a strong binding affinity to MgADP over ATP. Therefore, the binding of MgADP to the second binding site somewhat relieves the ATP-induced inhibition (10). However, other nucleoside diphosphates such as UDP can also produce a stimulatory action of varying degrees, apparently independent from the [ATP]i-induced inhibitory site (13). Another characteristic action of ADP is the restoration of channel activity after rundown. This restorative action of the channel activity also requires the presence of Mg2+; this action is shared by different nucleoside diphosphates, with UDP being the most effective among the various diphosphates. Restoration of the channel activity is specific for the action of nucleoside diphosphates among other adenine nucleosides, and the action is seen in the absence of ATP, which is in contrast to the MgATP-induced reactivation observed after the washout of ATP.
A decrease in intracellular pH from a normal range of ~7.2–7.4 to levels between 6.0 and 6.5 stimulates the channel activity (10, 13). The exact mechanism of this stimulation is not known but can be achieved possibly through antagonizing [ATP]i-induced inhibition. Further decreases in pH to <6.0 inhibit channel activity by decreasing the conductance, with frequent induction of subconductance states. An increase in ADP concentration and a pH drop to ~6.5 may develop rapidly during myocardial ischemia, and these two factors may contribute significantly to the early openings of KATP channels.
Neurotransmitters such as adenosine, acetylcholine, and epinephrine stimulate the KATP channel openings in cardiac cells (13). In isolated membrane patches, however, none of these agents is effective in stimulating the channels unless [ATP]i has been considerably depleted. Adenosine and acetylcholine activate KATP channels through a direct membrane-delimited pathway via activation of G proteins. This activating action is achieved through an antagonism against ATP-induced inhibition. ß-Adrenoreceptor stimulation enhances KATP channel activity by depleting subsarcolemmal ATP concentration but not by a direct action on the channels.
The K+ channel openers (KCOs) are compounds of diverse chemical structure that open KATP channels in cardiac and vascular smooth muscle as well as other tissues (2). A common feature of KCO action is its dependence on [ATP]i. At the single-channel level, KCO drugs increase the channel open probability without changing the single-channel conductance. The drugs do not influence rapid flickering kinetics in the bursts but affect the slow gatings by prolonging the burst durations and shortening the interburst intervals; the action seems opposite to that of increased [ATP]i (2, 3, 13). Furthermore, the activated current is inhibited either by increased [ATP]i or the application of glibenclamide, a blocker of KATP channels (see below). An apparent opposing action by KCOs against [ATP]i-induced inhibition suggests a competition at the ATP-binding inhibitory site of the channel proteins, but some of the drugs do not competitively antagonize with [ATP]i (3). Among the different KCOs, there is a specific group of agents that confine their actions to counteracting the effect of [ATP]i; nicorandil, a pyridine derivative, and some other agents require the presence of nucleoside diphosphates (NDPs) to activate KATP channels (see Ref. 13). Therefore, different KCO drugs have diverse activating profiles to KATP channels. In addition to these different activating profiles among various KCOs, the KATP channels in vascular smooth musle cells are generally more sensitive to the drugs than the cardiac KATP channels, whereas the latter generally have a higher sensitivity to KCOs than the counterpart channels in pancreatic ß-cells.
Cardiac KATP channels are blocked by glibenclamide and other antidiabetic sulfonylureas that are used to treat patients with non-insulin-dependent diabetes mellitus (1, 4, 10). The affinity of this class of drugs for cardiac KATP channels is lower than the affinity found in pancreatic ß-cells. The absorption of a nonionized form of drug molecules into the membrane lipid bilayer is the presumed access to the receptor sites (4). Whereas sulfonylureas usually show very efficient blocking action against the channel openings by KCOs, their efficacy may not be consistently seen on the KATP channels during metabolic inhibition and their effect is lost under severe metabolic stress. The mechanism of this loss of sensitivity is not known. Different groups have noted that the efficacy of glibenclamide in experiments with cell-free excised patches is considerably less than its effect on whole cell current recordings. Overall these results suggest that sulfonylureas produce not only a direct influence on the channel gating but also increased affinity of the channel for intracellular ATP, possibly through allosteric interaction (4). Figure 2 presents a schematic illustration of modulation of cardiac KATP channels by various factors.
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FIGURE 2. A schematic illustration of variable modulations of cardiac KATP channels by different intra- and extracellular factors.
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Molecular structures and functional expression of KATP channels
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The cloning of KATP channels from pancreatic islet and glucose-responsive insulin-secreting cell lines revealed a member of an inwardly rectifying K+ channel family, called BIR or KIR6.2. Coexpression of KIR6.2 with the sulfonylurea receptor cloned from pancreas (SUR1) reconstituted an inwardly rectifying K+ current that was inhibited by [ATP]i and sulfonylureas (7). Neither KIR6.2 nor SUR1 alone could express the functional K+ channels with ATP sensitivity. The results indicate that the pancreatic ß-cell KATP channels are a complex composed of at least two subunits: KIR6.2 and SUR, a member of the ATP-binding cassette superfamily. An isoform of SUR1, SUR2a, which was predominantly expressed in heart and skeletal muscle, was cloned in an additional study. Coexpression of KIR6.2 and SUR2a in COS-1 cells reconstitutes the properties of K+ channel current less sensitive to [ATP]i and glibenclamide which resembles KATP channels in native cardiac and skeletal musle cells. It is activated by the cardiac KCOs, cromakalim and pinacidil (8). Therefore, SUR seems to act as a regulator of KATP channel activity, conferring sensitivity to sulfonylureas and KCOs and also NDP-induced stimulation, whereas KIR6.2 forms the pore region to determine conductance property. More recently, KIR6.2 has been shown to have an ATP inhibition site (14) (Fig. 3).
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FIGURE 3. Proposed functional sites of the KATP channel structure. KATP channels express their functions with a complex of a member of the inwardly rectifying K+ channel family, KIR6.2, and the sulfonylurea receptor, SUR, with different subclones depending on cell types. Functional unit is distinctly divided into 2 sites, with the KIR6.2 portion determining conductance property (A) and ATP-sensitivity (B) and the SUR site determining pharmacological responses to K+ channel openers (KCOs) and glibenclamide (C) and stimulation by nucleoside diphosphates (NDPs) (D).
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Functional roles of cardiac KATP channel openings
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The opening of KATP channels during ischemia shortens the action potential duration markedly and plays some role in cellular K+ loss, both of which contribute to the development of arrhythmias (15). The shortening of the action potential, on the other hand, would prevent Ca2+ entry during the plateau phase, resulting in conservation of ATP by decreased twitch contraction as well as prevention of excess Ca2+ accumulation that could lead to cell death. Cardioprotective effects of the KATP channel opening are protection against cellular damage/injury, the limiting of infarction size, and the improved recovery of cardiac function during reperfusion. Furthermore, the openings of cardiac KATP channels mimic ischemic preconditioning (2). Adenosine, which is released from ischemic myocardium, could also contribute to cardioprotection, possibly through the activation of cardiac KATP channels, since the protective effect of preconditioning by adenosine can be mimicked by adenosine A1 receptor agonists and abolished by glibenclamide. There are still unanswered questions as to these cardioprotective actions in different experimental settings, and the exact molecular mechanism of action of the KATP openings needs to be further clarified. It is interesting to note that neuronal cell death associated with brain ischemia is prevented by pretreatment with the KCOs, which may activate neuronal KATP channels (9). Further clarification of the KATP channel modulation and its functional role will promote our understanding of the physiology and pathophysiology of the heart and other organs.
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Acknowledgments
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A portion of this work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to M. Hiraoka and T. Furukawa.
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Top
Introduction
Biophysical properties of...
, Data obtained by use of ATP alone; 
, data obtained in the presence of 0.1 mM MgADP. Addition of ADP shifted curve to the right, indicating stimulating effects of ADP. [Reproduced from M Hiraoka et al. in Pathophysiology of Heart Failure, edited by N.S. Dhalla et al. Norwell, MA: Kluwer Academic, 1996, p. 427–437. By permission of the publisher.]