Sarcolemmal KATP channels: what do we really know?

Focused issue on KATP channels

Sarcolemmal KATP channels: what do we really know?

Thomas P. Flagg, Ph.D., Colin G. Nichols, Ph.D. *

Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA

Received 8 July 2004; received in revised form 8 January 2005; accepted 12 January 2005

Available online 08 March 2005

Abstract

KATP channels are present at an extremely high density in the heart, and we know from in vitro studies that channel activation causesdramatic action potential shortening and contractile failure. But, if and when this happens in vivo is still a matter of debate. Twenty one yearsof intense study have led to a well-developed understanding of the molecular basis of KATP channel activity. Structure-function studies,together with cellular experiments probing regulatory molecules have told us much about the way the KATP channel can activate, and gene-targeting and proteomic tools have further elucidated determinants of in vivo function. However, the true physiological determinants ofsarcolemmal KATP activity remain elusive, we still await full illumination of the role of the channel in the intact heart.© 2005 Elsevier Ltd. All rights reserved.

Keywords: KATP channel; Action potential shortening; Contractile failure

1. Introduction: where are we coming from?

In 1983, Akinori Noma first described “specific K+ chan-nels which are depressed by intracellular ATP (ATPi) at lev-els greater than 1 mM” [1]. Because the KATP channel wasgated directly by intracellular ATP, it appeared a perfect can-didate for coupling the cell’s metabolic state with its electri-cal activity and function. Similar channels were subse-quently identified in numerous excitable tissues, includingthe pancreatic b-cell [2]. In this coming of age year, we canlook back to having learned a lot about the nucleotide depen-dence of the native channel and the molecular basis of KATP

activity. The genes encoding the KATP channel have beencloned. Structure-function studies have identified many of theamino acids that control nucleotide-dependent gating and sec-ond messenger modulation [3,4]. Residues that determinechannel rectification have been isolated [5,6], and detailedkinetic models of channel function have been constructed[7,8]. In spite of this, the answers to two fundamentalquestions—what molecular components comprise the sar-colemmal KATP channel in the heart, and what molecularevents trigger the opening of the sarcolemmal KATP channelin the heart—are still incomplete.

The defining property of the KATP channel is its regulationby intracellular nucleotides, and ATP-dependent inhibitionof native cardiac channels has been extensively studied inexcised membrane patches from a number of different ani-mal models [1,9–12]. On average, KATP channels are half-maximally inhibited by ~10–50 µM ATP. AMP−PNP andother nonhydrolyzable analogues of ATP [9,13,14] are alsoeffective, indicating that phosphorylation is not required forchannel inhibition. In the absence of magnesium, ADP alsoinhibits the channel with a K1/2 about 275 µM [9], most likelyby binding at the same inhibitory site as ATP. When magne-sium is present, however, ADP has the opposite effect, acti-vating channels that have rundown or have been inhibited byATP [9,15,16]. These realizations were made within 5 yearsof the discovery of the KATP channel. After another 16 years,what has our understanding graduated to?

2. What is the make-up of the sarcolemmal KATP

channel?

The cloning and expression of the molecular componentsof the b-cell KATP channel in 1995 introduced a structuralparadigm for all KATP [17]. Coexpression of an inward recti-fying K+ channel (Kir) with a sulfonylurea receptor (SUR), amember of the ATP-binding cassette family of proteins, reit-

* Corresponding author. Tel.: +1 314 362 6630; fax: +1 314 362 7463.E-mail address: [email protected] (C.G. Nichols).

Journal of Molecular and Cellular Cardiology 39 (2005) 61–70

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erates the defining properties of the KATP channel, includingsensitivity to both ATP and ADP, and to sulfonylureas, thefamily of drugs that specifically block KATP channels. Thechannels form as a complex of four Kir6 subunits each asso-ciated with one SUR subunit [18,19]. One early observationthat framed the process of defining the structural elements ofnucleotide regulation was the expression of Kir6.2-only chan-nels. While the full-length Kir6.2 is largely retained insidethe cell, deletion of the distal COOH-terminus (Kir6.2DC36)enabled the channel to reach the plasma membrane withoutSUR [20]. These channels were still inhibited by ATP, imply-ing that the determinants of ATP-inhibition were containedon the Kir subunit of the channel [20]. Subsequently, a num-ber of residues on the Kir6.2 subunit that regulated channelinhibition by ATP were identified [20–23]. In parallel, muta-tions were identified within the nucleotide binding foldsequences of SUR1 that cause persistent hyperinsulinemichypoglycemia of infancy. When these mutations are intro-duced in heterologously expressed Kir6.2/SUR1 channels,there is no effect on the ATP sensitivity of the channel, butthe ability of MgADP to stimulate the channel is inhibited,indicating the importance of the SUR subunit in the MgADP-dependent channel activation [24,25]. The structural ele-ments in the SUR molecule that control channel function arenot as well explored or understood as they are for the pore-forming subunit, however, the essential role of the nucleotidebinding folds (NBF1 and NBF2) in KCO- and MgADP-dependent channel activation [26–30] is clear. Together, thesekinds of molecular and genetic approaches have defined manyof the residues that are involved nucleotide regulation of KATP.

While this general Kir/SUR structural paradigm seems toapply for all types of KATP channels [31], studies of nativecardiac and pancreatic KATP channels consistently reveal dif-ferences in cellular regulation and function. For example,b-cell KATP channels respond to changes in [glucose], whilecardiac channels only appear to activate during more severemetabolic stresses. This difference may reflect the tissue-specific channel structure. The cloning of additional Kir6.1[32] and SUR2 [17,33] genes along with splice variants[33–35] gives rise to multiple possible subunit combinations.Clearly, the subunit composition has important conse-quences for the pharmacological properties of the differentchannels [36,37]. In addition, the distinct SUR subunits deter-mine, in part, the channel kinetics [38], which can have pro-found effects on channel regulation by nucleotides [39]. Twosubunits, Kir6.2 and SUR2A, have been shown to function-ally [40] and physically [41] interact, with a pharmacologi-cal profile that most closely fits the cardiac sarcolemmal KATP

[40,42]. This has led to the widely accepted notion that thesarcolemmal KATP channel is a heteromultimer of Kir6.2 andSUR2A, however, in recent years this conclusion has beenchallenged (Fig. 1A).

The challenges arise from the fact that at least two Kirsubunits (Kir6.1 and Kir6.2) and two SUR subunits(SUR1 and SUR2A) are expressed in the heart [17,32,35,42–45]. Dominant negative coexpression strategies to probe the

nature of Kir channel assembly have demonstrated thatKir6.1 and Kir6.2 can assemble into functional channel com-plexes in some studies [46] but not others [47]. TandemKir6.1–Kir6.2 channel constructs are also functional, whetherexpressed with SUR2A [48] or SUR2B [46]. Moreover,Kir6.1 and Kir6.2 have been shown to physically associate[46,49]. But does this mean that co-assembly of Kir6.1 andKir6.2 is relevant in the myocardium? Using similardominant-negative overexpression strategies in native cellsthere are again mixed results. In some studies [45], dominantnegative Kir6.1 suppresses cardiac KATP, while in others [47]it does not. Studies of mice lacking Kir6.2 and Kir6.1 indi-cate that the former is essential (and therefore likely to be theprincipal component of the sarcolemmal KATP) and that the

Fig. 1. The complex sarcolemmal KATP channel. (A) Complexity of KATP

channel structure. The KATP channel is an octameric structure of Kir6 andSUR subunits. While Kir6.2 and SUR2A subunits are clearly required togenerate sarcolemmal KATP channels, there are nagging indications thatKir6.1 and SUR1 subunits are also present in the sarcolemma. It has longbeen argued that the channels interact with the cytoskeleton, and biochemi-cal and pharmacological assays indicate that metabolic enzymes, includingAK, CK and LDH are also present in functional KATP complexes. (B) Com-plexity of KATP channel regulation. Carbohydrate metabolism leads to gene-ration of ATP from glucose, the signature inhibitory molecule, which acts atthe Kir6 subunit. ATP is hydrolyzed at the SUR nucleotide-binding folds,and MgADP activates channel activity by interaction with these sites. Inresponse to receptor activation, protein phosphorylation (P) may have bothstimulatory and inhibitory effects at Kir6 and SUR. Regulation by lipids iscomplex and powerful. PIP2 and acyl CoAs have powerful stimulatory effects,antagonistic to ATP inhibition. Secondary complexity arises from the inter-play of these three major metabolic effectors. [ATP] as a substrate will affectboth PIP2 levels and degree of phosphorylation; acyl-CoA may indirectlyaffect PKC activity. (Adapted with permission from Ref. [4]).

62 T.P. Flagg, C.G. Nichols / Journal of Molecular and Cellular Cardiology 39 (2005) 61–70

latter forms vascular channels [44,50]. In addition, the iden-tity of the SUR subunit of the sarcolemmal channel has beenquestioned. Again, sarcolemmal channels are significantlysuppressed in myocytes from mice with a disruption of theSUR2 gene [51], but the picture is again muddied by a recentreport that some KATP channels are still present [52]. Anti-sense oligonucleotides specific for either SUR1 or SUR2A/Bsuppress KATP current in neonatal rat ventricular myocytes,suggesting that SUR1 can participate in forming the channel,either alone or in conjunction with SUR2A [53]. However,others have presented evidence that SUR1 and SUR2A donot coassemble when they are expressed in the same cell line[54], and the lack of any reports of altered cardiac KATP inSUR1–/– animals [55,56] might indicate no functional rel-evance. The future reconciliation of these studies and com-plete determination of the molecular structure of the sarcolem-mal channels will be necessary to fully understand theirregulation and role in cardiac physiology.

In order to probe the significance of channel compositionin KATP function in cardiac myocytes, we recently generatedtransgenic mouse lines that overexpress either an ATP-insensitive Kir6.2 [57] (see below) or SUR1 [58] under aMHC[59] control. Interestingly, overexpression of either theKir6.2 or SUR1 subunits in the myocardium has little effecton normal heart function. However, the transgenes areexpressed in a sarcomeric pattern, and do contribute to sur-face channel composition (Fig. 2A, B). Surprizingly, trans-genic expression of either subunit significantly suppresses sar-colemmal KATP channel density (Fig. 2B) [57,58]. While thissuppression may reflect an ability to dynamically regulate totalKATP conductance, the most reasonable explanation seems tobe that overexpressed SUR1 or Kir6.2 subunits interact withendogenous subunits, thereby disrupting the stoichiometry ofthe channel [60] and presumably affecting assembly and traf-ficking [61].

3. What are the regulators of cardiac KATP activity?

While the assembly of Kir and SUR subunits are sufficientto reiterate hallmark nucleotide-sensitivities of KATP chan-nels, there may be any number of other accessory subunitsthat fine tune channel function in native tissues (Fig. 1A), ashas been shown for other cardiac potassium channels (e.g.KCNQ1/KCNE1 [62]). Recent studies have suggested a moreelaborate b-cell KATP channel complex [63,64], and whilethis possibility remains relatively unexplored for cardiac KATP,there is mounting evidence that other metabolic enzymes,including adenylate kinase (AK) [65], creatine kinase (CK)[66] and lactate dehydrogenase (LDH) [67], form part of, andregulate, the KATP channel complex. Localization of the chan-nel in a multi-protein complex including both phosphotrans-fer and glycolytic enzymes might explain early studies of thenative channel, where glycolytic substrates applied to excisedpatches can inhibit channel activity [68]. Indeed, there arenow studies demonstrating the importance of the phospho-

transfer network in regulating channel activity [65,66,69]. Byamplifying small changes in cytoplasmic ATP concentration,AK and CK can play an integral role in regulating the nucle-otide concentration in the localized space surrounding thechannel, and fine-tuning the response of KATP to cellularevents.

A more complete understanding of the functional ele-ments within the known channel structure is still required.The disparate effects of ATP-insensitive Kir6.2 transgenes incardiac myocytes and pancreatic b-cells suggest that differ-

Fig. 2. The surprising sarcolemmal KATP channel. (A) Confocal fluores-cence images of an isolated ventricular Kir6.2[DN,K185Q]-GFP line 4 TGmyocyte, loaded with MitoTracker RedTM. GFP fluorescence (left) locali-zes in a sarcomeric pattern and in discrete ~5 mm ‘hot spots’. Mitochondria,labeled with MitoTracker RedTM (center) are localized in interfibrillar chains,and there is no obvious overlap of mitochondrial and Kir6.2[DN,K185Q]-GFP fluorescence (right). (B) KATP current recorded from inside-out mem-brane patches from control (left) and line 4 Kir6.2[DN,K185Q]-GFP trans-genic (right) myocytes. In each case the patch was isolated at the verticalarrow. [ATP] was applied as indicated. Transgenic channels are ~40-foldless sensitive to inhibitory ATP than control channels, yet remain predomi-nantly closed in the intact cell. (C) Typical transmembrane action potentialsfrom wild type and transgenic hearts, show that there is no action potentialshortening in the transgenic heart. (D) Instead, a small basal activation ofKATP current may be compensated by a chronic stimulation of L-type Ca2+

current, in transgenic myocytes (Adapted with permission from Refs.[57,120]).

63T.P. Flagg, C.G. Nichols / Journal of Molecular and Cellular Cardiology 39 (2005) 61–70

ences in subunit composition of KATP channels in the twotissues may be an important determinant of tissue-specificfunction, i.e. of when the channels are turned on and off. It isnow recognized that the nucleotide binding folds of SUR1 areenzymatically active and that ATP hydrolysis is crucial forchannel regulation by nucleotides and pharmacological agents[70,71]. A network of phosphotransfer enzymes combinedwith ATP hydrolysis by the channel itself provides a molecu-lar framework by which nucleotide concentration can be regu-lated in the subsarcolemmal space to tightly control the chan-nel. This may provide the answer to the long-standing questionof how small changes in cytoplasmic [ATP] can be sensed bychannels that are largely closed by millimolar concentrationsin excised patch clamp experiments.

While sensitivity to intracellular nucleotides is the defin-ing characteristic of the KATP channel, it is now apparent thatATP inhibition is a dynamic property that is modulated bothby post-translational modification and by interactions withnon-nucleotide factors (Fig. 1B). Phosphorylation of recom-binant KATP channels by both protein kinase A [72,73] andprotein kinase C [74] have been shown to modulate the nucle-otide sensitivity of the channel. The application of the phos-pholipid PIP2 to the cytoplasmic side of inside-out patchescauses an increase in channel open probability and a decreasein sensitivity toATP [75–77]. Fatty acyl CoA esters have beenshown to have a similar effect on recombinant [78] and nativeKATP [79], and minute-to-minute modulation of lipid contentin the cells may regulate the channel in vivo. One recent studydemonstrated a rapid decrease in recombinant KATP currentas a result of PIP2 hydrolysis following activation of M1 mus-carinic receptors, as well as inhibition of recovery by wort-mannin, a phosphoinositol-3-kinase inhibitor [80]. Takentogether (Fig. 1B), these findings demonstrate the complexdeterminants of channel activity over and above the level ofintracellular nucleotides alone. Interestingly, in a study ofATPinhibition of native KATP channels, Findlay showed K1/2 val-ues ranged from 9 to 580 µM in 102 individual excised patches[11]. The basis of this patch-to-patch variability and its sig-nificance is not clear, but reflects the complex regulation ofKATP activity. Similarly, in experiments where KATP was acti-vated in isolated myocytes by anoxia, the latency to channelactivation was variable and the duration of channel activitywas short-lived [81]. Similarly, in intact cells, activation ofrecombinant channels by metabolic inhibition is short-lived[82]. Following an initial activation as a result of falling ATPlevels, channel activity declines, in parallel with a fall in thelevels of PIP and PIP2, suggesting that the levels of these twophospholipids act in concert with the intracellular nucle-otides to control channel function [82]. Finally, in transgenicmice that express an ATP-insensitive Kir6.2 subunit(Kir6.2[DN30,K185Q]) in the heart, sarcolemmal KATP chan-nels are extremely insensitive to ATP-dependent inhibition(K1/2 = 1.4 mM c.f. 25 µM in WT), but in the on-cell configu-ration channels remain largely closed [57]. One likely expla-nation for this surprising finding is that non-nucleotide fac-tors act to maintain sarcolemmal channels in a closedconformation.

An understanding of the interplay between metabolic andother signaling intermediates in determining channel func-tion is increasingly important as KATP channels, and theirregulation, are clearly distinct and optimized for the cell inwhich they are expressed. In the b-cell, KATP channel activ-ity varies with changes in blood [glucose], matching insulinsecretion with glucose intake to maintain glucose homeosta-sis. By contrast, the sarcolemmal KATP channel is not acti-vated by changes in blood glucose, and requires a differenttype of stimulus. It has been known for at least 50 years thatischemia and metabolic inhibition lead to the activation of aK+ conductance [83]. While we still do not have a clear ideaabout the precise cellular components that trigger KATP open-ing in vivo, the application of pharmacological inhibitors (e.g.glibenclamide, tolbutamide, 5-hydroxydecanoic acid) or acti-vators (e.g. pinacidil, diazoxide, cromakalim) at differenttimes during ischemia, hypoxia or metabolic inhibition hasbeen used to dissect the role of cardiac KATP. More recently,the advent of functional genomics and transgenic technologyhas led to another means of assessing the role of sarcolem-mal KATP in cardiac function. While the consensus appearsto be that KATP channels are important in protecting the myo-cardium during and following an ischemic insult, the detailsare still murky.

4. Pathophysiological activation of sarcolemmalKATP—what is the role in ischemic protectionand preconditioning?

Under normal conditions, sarcolemmal KATP channels arepredominantly closed, thus channel blockers have little, if any,effect on the resting membrane potential and the cardiac actionpotential [84]. During a metabolic insult, however, it has beendemonstrated in a number of different experimental prepara-tions, from single myocytes to whole heart, that KATP chan-nels are activated and protect the cell or tissue from damage.When KATP channels open, they shorten the cardiac actionpotential and cause contractile failure, and each of theseeffects is attenuated by the application of inhibitors and mim-icked by channel openers [85]. By reducing the action poten-tial duration (APD), calcium entry is reduced, and by reduc-ing calcium entry, myocyte contraction fails [86], thuspreserving the energy stores that would otherwise be used upin the contracting cell. In support of this model, it has beendemonstrated that pinacidil enhances the preservation of ATP[87]. More recently, convincing evidence for the role of sar-colemmal KATP in cardioprotection has been obtained inKir6.2–/– mice. In these mice, completely lacking the sar-colemmal KATP, metabolically compromised ventricular myo-cytes exhibit no increase in outward current [88]. In additionthere is no APD shortening, there is a longer delay untilischemic hearts stop contracting and there is reduced func-tional recovery following reperfusion [88]. Thus, it remains atruism that activation of KATP channels during an ischemicchallenge is a relevant endogenous defense mechanism thatprotects the myocardium and enhances recovery.


The cardioprotective functions of the KATP channel havebeen most characterized in studies of the phenomenon of pre-conditioning. It was first recognized in the late 1980s thatbrief ischemic periods could improve the recovery of contrac-tile function and reduce infarct size resulting from a subse-quent prolonged metabolic insult [89]. This “precondition-ing” of the heart can be mimicked by a number of humoralfactors, including adenosine and acetylcholine. The precisemolecular mechanisms underlying preconditioning are stillbeing sorted out, but it appears that in each case the commonfunctional endpoint is a KATP channel [90]. In support of this,glibenclamide can inhibit ischemic as well as adenosine- andacetylcholine-induced preconditioning, and the cardioprotec-tive effects of preconditioning can be reproduced by pretreat-ment with a number of KATP channel openers [91]. A numberof observations implicate the proposed mitochondrial KATP,rather than the sarcolemmal KATP. First, neither the cardio-protective effects of cromakalim, nor of pinacidil [92], cor-relate with action potential shortening, as might be predictedif only the sarcolemmal KATP is activated during precondi-tioning. Second, the sarcolemmal KATP channel has gener-ally been considered insensitive to diazoxide, so the observa-tion that diazoxide mimics ischemic preconditioning [93]suggests a non-sarcolemmal (i.e. mitochondrial) channelinvolvement. Finally, 5-hydroxydecanoic acid (5-HD), whichappears to inhibit only the mitochondrial KATP channel, effec-tively abolishes ischemic preconditioning [94], and a similardifferential effect has been reported for MCC-134 [95].

Each of these pharmacological studies has to be inter-preted carefully, however, since it is clear that, like ATP-dependent regulation, the pharmacology of KATP channels isnot static. For example, a newer study disproves the criticalassumption that the sarcolemmal channelit is insensitive todiazoxide [96]. It has also been reported that sulfonylureaefficacy changes when cardiac KATP channels are activatedby metabolic inhibition [97], which may reflect the rise ofADP obviating the sulfonylurea sensitivity [98–100]. Otherrecent studies suggest that the channel pharmacology, likethe ATP sensitivity, is modulated by the level of phospholip-ids in the membrane [101]. Further complications arise fromthe fact that KATP is a prominent feature of the vasculature ofthe heart [102], making it difficult to separate drug modula-tion of the myocardium and vascular smooth muscle. Func-tional genomic approaches have made it possible to circum-vent some of the limitations of pharmacology to examine genefunction directly. The molecular structure of the mitochon-drial KATP channel is at present unknown, but an intriguingnew report implicates a multi-protein complex consisting ofsuccinate dehydrogenase, mitochondrial ATP-binding cas-sette protein 1 (mABC1), phosphate carrier, adenine nucle-otide translocator, and ATP synthase in reconstitution of afunctional mitochondrial KATP [103].

Kir6.2 does not appear to be a component of the mitochon-drial channel [104]. Therefore, by genetically controlling onlythe sarcolemmal KATP channel, it is now possible to moredirectly assess its role in preconditioning. Interestingly, stud-

ies indicate that ischemic preconditioning is abolished in theKir6.2 knockout mouse [88], as is diazoxide induced precon-ditioning [105], suggesting an important, though not neces-sarily direct, role of the sarcolemmal channel in mediatingcardioprotection. In addition, in our own mice expressingATP-insensitive sarcolemmal channels, preconditioning isabolished [106]. Though the precise mechanisms still remainto be worked out, it is increasingly clear that the sarcolemmalKATP channel is at least an essential modulator of precondi-tioning.

5. Pathophysiological activation of sarcolemmalKATP—is there a role in cardiac rhythm?

While many studies demonstrate that KATP channels canprotect cardiac function following global ischemia or com-plete metabolic inhibition, none have really pinpointed theminimal metabolic events that cause the channel to open. Arethe protective effects of KATP only relevant during an extrememetabolic event, like global ischemia? Or do KATP channelsplay a role during periods of increased workload, like normalexercise? One study suggests the latter. In addition to the lackof protection during ischemia–reperfusion, KATP-deficientKir6.2–/– mice exhibit reduced tolerance for vigorous exer-cise. Exercise or stress (mimicked by high-dose isoproter-enol) may be sufficient to activate KATP channels and causeAPD shortening [107]. In addition, there is a disruption ofcalcium cycling dynamics with deleterious effects on the ani-mal [107]. While further study is warranted, it is intriguing tospeculate that KATP channels may play a more widespreadrole in cardiac physiology than generally appreciated, andare not limited to roles in pathological ischemic states.

Cardiac arrhythmias frequently accompany myocardialischemia and reperfusion. In principle, the activation of KATP

could be either antiarrhythmic or proarrhythmic. On one hand,increased potassium conductance during a metabolic insultshould stabilize the resting membrane potential, reducinginjury current between cells and reducing ectopic pacemakeractivity. On the other hand, because KATP channel activationaccelerates the repolarization of the action potential, shorten-ing the QT-interval and reducing the refractory period of thecell, KATP channel activation may predispose the heart tore-entrant arrhythmias. Pharmacological interventions haveproven inconclusive in elucidating the role of KATP in arrhyth-mogenesis. Glibenclamide-treatment during ischemia–reperfusion appears to decrease the incidence of sustainedtachycardia and ventricular fibrillation [108,109], but thereare contradictory reports of increased occurrence of tachy-cardia [110]. Similarly, in some studies, KATP channel open-ers have been shown to promote tachycardia and fibrillation[109–111], while in others, channel openers reduce the fre-quency of arrhythmias [112]. It should also be pointed outthat this latter study [112] showed contrasting effects of cro-makalim in anesthetized dogs (where it inhibited fibrillation)and in isolated rat hearts (where it induced fibrillation), illus-


trating the significant differences that can arise as a functionof the experimental model used to assess potential arrhyth-mic effects. Abnormalities in the repolarization phase of theelectrocardiogram (ECG) are also a prominent feature of myo-cardial ischemia. Both the peaked T wave [113] and ST-elevation [114] that are characteristic of ischemia are blockedby glibenclamide, consistent with activation of sarcolemmalKATP underlying the changes in the ECG. The mouse ECG issignificantly different from that of larger animals owing tothe difference in the shape of the ventricular action potential;however, equivalent ‘ST-segment’ elevation during ischemiais absent in Kir6.2–/– mice, demonstrating that the sarcolem-mal KATP channel activation can cause this electrocardio-graphic abnormality [115]. While a definitive mechanistic linkbetween ST-segment elevation and ventricular tachycardia/fibrillation during ischemia is not established in humans orother large aninmals, many results are consistent with such alink. For instance pinacidil has been shown to cause a hetero-geneous loss of the action potential plateau leading to a trans-mural voltage gradient during repolarization and ST segmentelevation [116], sufficient to trigger extrasystolic beats lead-ing to ventricular fibrillation by a phase 2 reentry mecha-nism. As an aside, the ECG abnormalities and ventricularfibrillation induced by pinacidil were blocked with4-aminopyridine, a specific Ito antagonist [116], highlightingan important point—because the ECG results from the inte-gration of multiple conductances, the blockade of one cur-rent by a drug may alleviate a problem without pinpointingthe specific cause.

6. Disease mutations of KATP and cardiac remodeling

Transgenic tools provide an additional approach to com-bine traditional structure-function studies with in vivo stud-ies. We have generated transgenic animals expressing mutatedKir6.2[DN30,K185Q] subunits, which generate KATP chan-nels with reduced ATP sensitivity. The KATP channels in thesemice are very insensitive to inhibition byATP (K1/2 = 1.4 mM)[57], and it is predicted that at least some channels will beactive under normal circumstances. When similar ATP-insensitive Kir6.2 transgenes are introduced in the pancreaticb-cell, mice develop lethal neonatal diabetes, consistent withan essential role of KATP channels in regulating insulin secre-tion. Since computer modeling [10,117] and experimentalstudies [118–120] suggest that the activation of just 1% ofthe total KATP conductance in the heart will suffice to reducethe APD by 50%, it was predicted that transgenic expressionof these ATP-insensitive subunits would suppress excitabil-ity and contractility. In conventional whole cell recordingsthere is a very weak basal KATP channel activity in transgenicmyocytes, but there is no action potential shortening in eitherthe intact heart (Fig. 2C), or in isolated myocytes. Quite unex-pectedly, there is a significant elevation of L-type Ca2+ cur-rent (Fig. 2D) that may contribute to maintenance of APDand contractility [121]. The underlying mechanism of this

compensation is unclear but likely involves a “prestimula-tion” of the Ca2+ channel, not an upregulation of channelexpression. Thus, the transgenic phenotype is opposite whatis naively predicted for expression of a gain-of-function KATP

channel in the heart, and suggests that KATP activation mightnot only control cell excitability, but may also indirectly regu-late other ion channels. Whether the remodeling of E-C cou-pling is a specific adaptive mechanism to KATP activation, oris a common response when any “gain-of-function” K+ chan-nel is expressed in the myocardium, remains to be seen.

The identification of disease-causing mutations providesa powerful method to assess the function of a gene and itsphysiological role. Inactivating mutations in either Kir6.2 orSUR1 in humans cause persistent hypoglycemic hyperin-sulinemia of infancy (PHHI), a disease characterized byunregulated insulin secretion [122]. Activating mutationswould be expected to have the opposite effect, causinghypoexctability and a diabetic phenotype. This is strikinglyevident in mice, where an overactive Kir6.2 transgene causeslethal neonatal diabetes [123]. As predicted, there are now anumber of convincing human studies linking mutations andpolymorphisms in the Kir6.2 gene with the incidence of dia-betes [124] and very recently it has been demonstrated thatgain-of-function mutants also cause human neonatal diabe-tes [125]. It remains to be determined whether there is anycardiac manifestation of this disease but conceivably therecould be a short-QT syndromic effect. Until recently, no KATP

mutations have been linked with alterations in cardiac func-tion or disease [126], although mutations within theSUR2 gene locus have been reported to underlie humandilated cardiomyopathy [127]. The two identified mutations(Fs1524 and A1513T) both alter the catalytic activity of thesecond nucleotide binding domain. Both mutant channels areless sensitive to inhibition by ATP and the mutations affectthe ability of ADP to activate channels that are closed by ATP.It remains unclear how the resultant alteration of KATP chan-nel function causes the cardiac dysfunction, but the contin-ued identification of disease-causing mutations and charac-terization of known mutations provides a new path to explorethe function of the KATP channel in the heart.

7. Conclusions

Despite 21 years of intense study, and a detailed under-standing of the molecular basis of KATP channel activity thathas resulted, it has to be admitted that we remain largely inthe dark regarding the true physiological determinants, andrelevance of sarcolemmal KATP activity. KATP channels arepresent at an extremely high density in the heart, and we knowfrom in vitro studies that channel activation causes dramaticaction potential shortening and contractile failure. But, if andwhen this happens in vivo is still a matter of debate. Tradi-tional structure-function studies, together with cellular experi-ments probing regulatory molecules have told us much aboutthe way the KATP channel can activate in the heart. Gene-


targeting, genomic and proteomic tools will further elucidatedeterminants of in vivo function, yet it remains an unfulfilledpromise that such approaches will fully illuminate the role ofthe channel in the intact heart.

Acknowledgements

Over the years of studying the KATP channel in the heart,we are grateful to our colleagues in the field whose findingshave stimulated and directed our own work, as well as pro-viding the materials to take our studies to higher levels. Wehave tried above to liberally acknowledge major break-throughs, but apologize in advance for not having beenexhaustive in citing references. We would very much like toacknowledge our own immediate collaborators, too numer-ous to mention, who have joined us in our own experimentalstudies. We have been continually supported in these studiesby the National Institutes of Health (grants HL45742 andHL51909 to CGN), and we are in turn very grateful for thissupport.

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Sarcolemmal KATP channels: what do we really know?