Large-scale analysis of ion channel gene expression in the mouse heart during perinatal development.

The immature and mature heart differ from each other in terms of excitability, action potential properties, contractility, and relaxation. This includes upregulation of repolarizing K(+) currents, an enhanced inward rectifier K(+) (Kir) current, and changes in Ca(2+), Na(+), and Cl(-) currents. At the molecular level, the developmental regulation of ion channels is scantily described. Using a large-scale real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, we performed a comprehensive analysis of ion channel transcript expression during perinatal development in the embryonic (embryonic day 17.5), neonatal (postnatal days 1-2), and adult Swiss-Webster mouse hearts. These data are compared with publicly available microarray data sets (Cardiogenomics project). Developmental mRNA expression for several transcripts was consistent with the published literature. For example, transcripts such as Kir2.1, Kir3.1, Nav1.5, Cav1.2, etc. were upregulated after birth, whereas others [e.g., Ca(2+)-activated K(+) (KCa)2.3 and minK] were downregulated. Cl(-) channel transcripts were expressed at higher levels in immature heart, particularly those that are activated by intracellular Ca(2+). Defining alterations in the ion channel transcriptome during perinatal development will lead to a much improved understanding of the electrophysiological alterations occurring in the heart after birth. Our study may have important repercussions in understanding the mechanisms and consequences of electrophysiological alterations in infants and may pave the way for better understanding of clinically relevant events such as congenital abnormalities, cardiomyopathies, heart failure, arrhythmias, cardiac drug therapy, and the sudden infant death syndrome.

Cloning of components of a novel subthreshold-activating K(+) channel with a unique pattern of expression in the cerebral cortex.

Potassium channels that are open at very negative membrane potentials govern the subthreshold behavior of neurons. These channels contribute to the resting potential and help regulate the degree of excitability of a neuron by affecting the impact of synaptic inputs and the threshold for action potential generation. They can have large influences on cell behavior even when present at low concentrations because few conductances are active at these voltages. We report the identification of a new K(+) channel pore-forming subunit of the ether-à-go-go (Eag) family, named Eag2, that expresses voltage-gated K(+) channels that have significant activation at voltages around -100 mV. Eag2 expresses outward-rectifying, non-inactivating voltage-dependent K(+) currents resembling those of Eag1, including a strong dependence of activation kinetics on prepulse potential. However, Eag2 currents start activating at subthreshold potentials that are 40-50 mV more negative than those reported for Eag1. Because they activate at such negative voltages and do not inactivate, Eag2 channels will contribute sustained outward currents down to the most negative membrane potentials known in neurons. Although Eag2 mRNA levels in whole brain appear to be low, they are highly concentrated in a few neuronal populations, most prominently in layer IV of the cerebral cortex. This highly restricted pattern of cortical expression is unlike that of any other potassium channel cloned to date and may indicate specific roles for this channel in cortical processing. Layer IV neurons are the main recipient of the thalamocortical input. Given their functional properties and specific distribution, Eag2 channels may play roles in the regulation of the behavioral state-dependent entry of sensory information to the cerebral cortex.

Regulation of ATP sensitive potassium channel of isolated guinea pig ventricular myocytes by sarcolemmal monocarboxylate transport.

OBJECTIVE: The aim was to describe the effects of extracellular application of monocarboxylates (pyruvate, lactate, or acetate) on current through KATP channels (iK,ATP) in isolated guinea pig ventricular myocytes. METHODS: The iK,ATP was elicited during whole cell voltage clamping by application of metabolic poisons, 2,4-dinitrophenol (150 microM) or glucose free cyanide (1 mM) and could be blocked by glibenclamide (3 microM). RESULTS: Extracellular application of monocarboxylates, pyruvate (0.1-10 mM), L-lactate (0.1-10 mM), and acetate (10 mM) led to a rapid inhibition of iK,ATP--an effect which was fully reversible upon washout. Substances without any effect on iK,ATP were (10 mM each) gluconate, citrate, glutamate, creatine, succinate, and glycine. The mechanism underlying the effects of monocarboxylates on iK,ATP was unlikely to be related to an increased ATP production, since D-lactate (10 mM) essentially had the same effect on iK,ATP as the L-isomer of lactate. Furthermore, with intracellular dialysis of alpha-cyano-4-hydroxycinnamate (0.1-0.5 mM), which inhibits pyruvate uptake into mitochondria, extracellular pyruvate exerted the same inhibitory effect on iK,ATP. High concentrations of extracellular alpha-cyano-4-hydroxycinnamate (4 mM), which blocks the sarcolemmal monocarboxylate carrier, prevented the effects on iK,ATP by pyruvate, L-lactate, D-lactate, and acetate. Furthermore, intracellular dialysis with D-lactate (10 mM) led to a more rapid onset of iK,ATP when activated by ATP free dialysis. Activity of isolated KATP channels, measured in isolated membrane patches in the inside out or outside out configuration, typically had a single channel conductance of around 80 pS and was blocked by glibenclamide (3-9 microM). No significant effect of pyruvate was observed in either patch configuration. CONCLUSIONS: In cardiac tissue there may be some modulatory role involving monocarboxylate transport on KATP channel activity, the nature of which is unclear at present but which may involve cytosolic pH changes. Physiological and pathophysiological implications of these findings are discussed.

Reperfusion damage: free radicals mediate delayed membrane changes rather than early ventricular arrhythmias.

STUDY OBJECTIVE - The aim of the study was to reassess the role of reactive oxygen species in causing reperfusion arrhythmias, which they might do either by directly generating free oxygen radicals or by using scavengers of free oxygen radicals. DESIGN - Ventricular arrhythmias were studied in isolated rat hearts (n = 8-15 per experiment) subjected to regional ischaemia and treated with various free radical scavengers and spin trap agents. Reoxygenation automaticity was similarly studied in isolated guinea pig papillary muscles (n = 6-13 per experiment). MEASUREMENTS and RESULTS - In isolated rat hearts early reperfusion ventricular arrhythmias were unaltered by superoxide dismutase (1 X 10(5) IU.litre-1), catalase (1 X 10(6) IU.litre-1), N-tert-butyl-alpha-phenylnitrone (30 mumols.litre-1), 5,5-dimethyl-1-pyrroline-N-oxide (1 mmol.litre-1), or the combination of superoxide dismutase 1 X 10(5) IU.litre-1, catalase 1 X 10(6) IU.litre-1, and mannitol 10 mol.litre-1, or by the generation of the free radical .OH (Fe:ADP plus dihydroxyfumerate). In the isolated reoxygenated guinea pig papillary muscle, the incidence of reoxygenation automaticity was significantly reduced by verapamil 5 mumols.litre-1 but not by the following free oxygen radical scavengers: reduced glutathione (0.5 mmol.litre-1), N-acetyl cysteine (1 mmol.litre-1), the combination of superoxide dismutase (3 X 10(4) IU.litre-1) and catalase (5 X 10(3) IU.litre-1), or by pretreatment with allopurinol (30 mg.kg-1). Generating systems of .O2- or .OH induced relatively slow electrophysiological changes, including a decreased action potential duration. Reperfusion ventricular fibrillation in the rat heart was increased by increasing the extracellular calcium concentration from 1.25 to 1.9 or 2.5 mmol.litre-1, or by prolongation of the ischaemic time. CONCLUSIONS - Because of (a) the lack of an arrhythmogenic effect of free radical generating systems or of scavengers of free radicals, (b) the calcium sensitivity of reperfusion arrhythmias, and (c) the relatively slow time course of electrophysiological changes induced by free radical generating systems, we propose that free radicals are unlikely to be the prime cause of early ventricular arrhythmias in the systems that we tested. The mechanism of such arrhythmias is more likely to be a calcium sensitive process. The relatively slow electrophysiological changes mediated by free radicals suggest that these agents can cause delayed membrane change.

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2.

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels.

Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K+ channel principal subunit.

We have identified and cloned a new member of the mammalian tandem pore domain K+ channel subunit family, TWIK-originated similarity sequence, from a human testis cDNA library. The 939 bp open reading frame encodes a 313 amino acid polypeptide with a calculated Mr of 33.7 kDa. Despite the same predicted topology, there is a relatively low sequence homology between TWIK-originated similarity sequence and other members of the mammalian tandem pore domain K+ channel subunit family group. TWIK-originated similarity sequence shares a low (< 30%) identity with the other mammalian tandem pore domain K+ channel subunit family group members and the highest identity (34%) with TWIK-1 at the amino acid level. Similar low levels of sequence homology exist between all members of the mammalian tandem pore domain K+ channel subunit family. Potential glycosylation and consensus PKC sites are present. Northern analysis revealed species and tissue-specific expression patterns. Expression of TWIK-originated similarity sequence is restricted to human pancreas, placenta and heart, while in the mouse, TWIK-originated similarity sequence is expressed in the liver. No functional currents were observed in Xenopus laevis oocytes or HEK293T cells, suggesting that TWIK-originated similarity sequence may be targeted to locations other than the plasma membrane or that TWIK-originated similarity sequence may represent a novel regulatory mammalian tandem pore domain K+ channel subunit family subunit.

The role of Kir2.1 in the genesis of native cardiac inward-rectifier K+ currents during pre- and postnatal development.

Our results demonstrate that (a) the Kir2.1 gene encodes a native K+ channel protein with a 21-pS conductance; (b) this channel has an important role in the genesis of adult ventricular 1K1; and (c) the contribution of Kir2.1 channel proteins to 1K1 changes during development. The lack of contribution of Kir2.1 to fetal 1K1 channels is interesting from the point of view of possible future generation of knockout mice lacking Kir2.1, since cardiac abnormalities would not be expected to result in fetal lethality. These observations provide further support for a generalized hypothesis that different genes may code for 1K1 channel proteins at various developmental stages. However, the effects of these AS-oligos must first be examined on native 1K1 channels in cardiac myocytes before definite conclusions can be reached.

Molecular diversity of K+ channels.

K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.

Anti-arrhythmic effects of levcromakalim in the ischaemic rat heart: a dual mechanism of action?

The action of pharmacological openers of K(ATP) channels depends on the availability and levels of various intracellular nucleotides. Since these are subject to change during myocardial ischaemia, K(ATP) channel openers may affect ischaemic and non-ischaemic tissue differentially. Using a recently developed dual coronary perfusion method, we investigated the effects on arrhythmias of the prototypical K(ATP) channel opener levcromakalim when applied selectively to ischaemic and/or non-ischaemic tissue. A novel perfusion cannula was used to independently perfuse the left and right coronary beds of hearts isolated from rats. Selective infusion of levcromakalim (3, 10 or 30 microM) into the left coronary bed in the absence of ischaemia did not induce ventricular arrhythmias. Regional zero-flow ischaemia was induced by cessation of flow to the left coronary bed and hearts received levcromakalim selectively into either the left, right, or both coronary beds. When applied selectively to the ischaemic left coronary bed, levcromakalim (3, 10 or 30 microM; n=10/group) delayed the onset of ventricular tachycardia in a dose-dependent manner (by 21*, 43* and 112%* at 3, 10 and 30 microM; *P<0.05 vs. control). When applied only to the non-ischaemic right coronary bed, levcromakalim reduced the incidence of ventricular tachycardia during later phases of ischaemia (from 100% in controls to 30%*). When present in both coronary beds, levcromakalim had a striking anti-arrhythmic effect--the overall incidence of ventricular tachycardia being reduced from 100% in controls to 20%*. We conclude that levcromakalim may have an anti-arrhythmic effect when applied either to ischaemic or non-ischaemic tissue but that the mechanisms may differ depending on the metabolic state of the heart.

The regulation of ion channels and transporters by glycolytically derived ATP.

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na(+)/K(+)-ATPase, the H(+)-ATPase, various types of Ca(2+)-ATPases, the Na(+)/H(+) exchanger, the ATP-sensitive K(+) channel, cation channels, Na(+) channels, Ca(2+) channels and other channels involved in intracellular Ca(2+) homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches.

ATP-sensitive potassium channels and myocardial ischemia: why do they open?

There is evidence that the "ATP-sensitive" potassium channel opens, at least during the early stages of myocardial ischemia, despite relatively high ATP levels. Thus, channel opening may partially contribute to potassium efflux and accumulation of extracellular potassium, but probably much more profoundly to electrical abnormalities associated with ischemia, including the development of lethal arrhythmias. Several factors are discussed that may promote a significant open-channel probability of the channel, in spite of relatively high levels of ATP. It is argued that, even with a very low open probability, the magnitude of total membrane current carried by these channels may be substantial (comparable to other potassium currents) because of the high density and conductance of the ATP-sensitive potassium channel. Finally, it is shown how the ATP-sensitive potassium channel may play a role in various tissue types, ranging from the physiological to the pathophysiological. This potassium channel is therefore increasingly targeted for drug development and research.

Channel-mediated calcium current in the heart.

Calcium ions play an important role in the regulation of heart functions. Calcium ions may enter or leave the myocardial cell through various mechanisms, including several exchange mechanisms and pumps. This review concentrates on the influx of calcium ions through channels in the sarcolemma, resulting in an electric current flow. The calcium current plays an important role in the maintenance of the action potential duration, in the generation of pacemaker activity, and in the initiation of contraction. The calcium current displays both activation and a subsequent inactivation when the membrane potential is changed in a stepwise fashion. Previously, the activation was thought to occur rather slowly, hence the name "slow inward current." Recent evidence suggests that the calcium current occurs much faster and that two types of calcium currents might exist, differing in their selectivity to other ions and in their sensitivity to membrane potential and to drugs. The calcium current is modulated by several factors. Beta-adrenergic stimulation increases the calcium current by increasing the opening probability of the calcium channel. The effects of acetylcholine are less well described. There also exists a class of drugs, called calcium channel blockers (or calcium antagonists) that decrease the flow of calcium ions through calcium channels. It is not quite clear how the calcium current is changed during myocardial ischemia. Factors that may reduce the calcium current during ischemia are the increased extracellular potassium concentration, metabolic inhibition and a decreased ATP level, and acidosis. Raised levels of intracellular cAMP, however, should lead to an increased calcium current.

Role of calcium ions in reperfusion arrhythmias: relevance to pharmacologic intervention.

Calcium ions may play a role in reperfusion arrhythmias, as suggested by 1) evidence favoring excess internal recycling of calcium during the reperfusion period; 2) electrophysiologic studies in Purkinje fibers and guinea pig papillary muscle in which calcium-dependent delayed after-depolarizations (DADs) have been found; 3) identification of the transient inward current as the basic mechanism underlying DADs; 4) the influence of cyclic adenosine monophosphate (cAMP) in the ischemic period on reperfusion electrophysiologic abnormalities; and 5) calcium oscillations in reoxygenated myocytes. More direct evidence for the role of calcium lies in the concordance between the factors influencing DADs and those associated with reperfusion arrhythmias, as well as the role of an elevated extracellular Ca2+ in causing reperfusion ventricular fibrillation. However, a role for Ca2+ does not necessarily imply that calcium antagonist drugs will be antiarrhythmic in this situation; rather there is no good evidence that these agents are antiarrhythmic unless they have a protective effect in the ischemic period. The antiarrhythmic role of alpha 1-adrenergic blocking drugs remains controversial; in isolated hearts they work in high concentrations, not through specific receptor antagonism. Beta-blocking drugs have no established place in the therapy of reperfusion arrhythmias. The role of lidocaine and other sodium channel blockers is also controversial. In isolated preparations, lidocaine can be antiarrhythmic and can inhibit DADs. Mexiletine, another sodium channel blocker, can inhibit reoxygenation and reperfusion arrhythmias as well as DADs, all in therapeutic concentrations (10 microM). Such drugs may indirectly inhibit sodium-calcium exchange, which is one of the mechanisms underlying the formation of DADs and, hence, a potential site of pharmacologic inhibition of reperfusion arrhythmias.

Effects of components of ischemia and metabolic inhibition on delayed afterdepolarizations in guinea pig papillary muscle.

Delayed afterdepolarizations (DADs) may develop into triggered automaticity and ventricular arrhythmias. However, the potential role of DADs in the genesis of ischemic arrhythmias is not clear. We studied the effects of different components of severe ischemia (acidosis, hypoxia, lactate, increased potassium, and the absence of glucose) on DADs. DADs were evoked using trains of 30-60 externally applied pulses at a rate of 4-5 Hz in the presence of isoproterenol (10(-7) M) or dibutyryl cyclic 3', 5' adenosine monophosphate (dB-cAMP, 10(-3) M). Acidosis, caused by the addition of protons (pH = 6.8), increased the amplitude of DADs from 3.2 +/- 0.4 to 5.9 +/- 0.5 mV (n = 8, p less than 0.001). DADs were abolished by hypoxia (pO2 less than 35 mm Hg, n = 7, p less than 0.001) from control values of 3.4 +/- 0.3 mV. DADs were also abolished by neutral lactate (20 mM, n = 7, p less than 0.001) in the absence of glucose. Acidotic lactate (20 mM, pH0 = 6.8), however, was unable to abolish DADs. Increasing the extracellular potassium concentration to 16.2 mM decreased DAD amplitude from 3.6 +/- 0.27 mV to 1.3 +/- 0.1 mV (n = 5, p less than 0.002) with an associated reduction of membrane potential from -86.2 +/- 0.9 to -58.6 +/- 0.9 mV. The overall effect of simulated ischemia (all components tested together) was to abolish DADs (n = 8, p less than 0.001), with hypoxia as the most important factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of oxygen free radicals on isolated cardiac myocytes from guinea-pig ventricle: electrophysiological studies.

Free oxygen radicals are formed during early reperfusion and are thought to contribute to some types of reperfusion abnormalities, including arrhythmias and myocardial stunning. The purpose of this study was to investigate electrophysiological effects of oxygen free radicals using voltage clamped single ventricular myocytes from guinea-pig hearts. Oxygen free radicals were produced enzymatically by the direct addition of xanthine oxidase (XOD, 0.04 U/ml) in the experimental chamber to a solution containing hypoxanthine (0.96 mM). The generation of oxygen radicals was confirmed by the formation of adrenochrome from adrenaline. Oxygen radicals caused automaticity of isolated myocytes within 20-30 min, followed by later hypercontracture. The percentage of rod-shaped cells declined sigmoidally as a function of time, with a half maximal value at 40.9 +/- 1.6 min, and a Hill slope of -0.10 +/- 0.01 (n = 26). These effects were prevented by a combination of superoxide dismutase (10(5) U/L) plus catalase (10(6) U/L). The rate at which cells underwent morphological shape changes was unchanged by ryanodine (0.5 microM) which is thought to act on the sarcoplasmic reticulum or by the Ca2+ channel blockers nisoldipine (1 microM) or Cd2+ (30 microM). Cellular automaticity and hypercontracture were delayed by variable degrees, and sometimes completely prevented, by zero (1 mM EGTA) extracellular Ca2+, MnCl2 (2 mM) and LaCl3 (50 microM), and amiloride (1 mM). On the other hand, in the presence of a low extracellular Na+ (30 mM) or caffeine (10 mM), hypercontracture occurred at a faster time scale. Whole cell voltage clamping revealed a decrease of the inward rectifying K+ current (IK1), and a decrease of the peak of the L-type Ca2+ current (ICa,L). The total ICa,L during the clamp step was increased, mainly because of an increased time constant of inactivation (47.6 +/- 4.7 ms to 72.7 +/- 15.5 ms after 30 min, n = 4, P less than 0.05). We conclude that oxygen radicals cause automaticity and hypercontracture of isolated myocytes, that these effects may be due to an increased intracellular Ca2+ concentration ([Ca2+]i), and despite an increased ICa,L, that the enhanced Ca2+ influx may occur predominantly via the Na/Ca exchange.

Proposed role of energy supply in the genesis of delayed afterdepolarizations--implications for ischemic or reperfusion arrhythmias.

Delayed afterdepolarizations (DADs) are Ca++-dependent electrophysiological abnormalities, which are evoked by a variety of conditions that induce intracellular Ca++ overload, including fast pacing, isoproterenol, dibutyryl cyclic AMP, and intracellular injection of Ca++. Since Ca++ overload is suspected of playing a role in both ischemic and reperfusion cellular damage, a reasonable hypothesis would be that DADs could play a role in ischemic or reperfusion arrhythmias. No direct proof has, however, been obtained for such a role for DADs. We propose that DADs could be associated with arrhythmias in which there is Ca++ overload of sufficient magnitude to cause an increased oscillatory release of Ca++ from the sarcoplasmic reticulum (SR), provided energy is available in the form of ATP. A sustained increase of Ca++ is likely to reflect energy depletion and therefore exclude a significant contribution of DADs to arrhythmia development. Thus, DADs are more likely to play a role in: (i) reperfusion arrhythmias and (ii) arrhythmias arising in moderately ischemic tissue, than in severe ischemia with marked energy depletion.

Inhibition by simulated ischemia or hypoxia of delayed afterdepolarizations provoked by cyclic AMP: significance for ischemic and reperfusion arrhythmias.

Controversy exists about the role of an increased level of tissue cyclic adenosine 3'-5' monophosphate (cAMP) in the genesis of early ischemic ventricular arrhythmias. Evidence for an arrhythmogenic role for cAMP was proposed by Podzuweit et al. (1978) and Opie et al. (1979) who argued that ischemic ventricular fibrillation was associated with increased levels of tissue cAMP in the ischemic zone. Lubbe et al. (1978) found that infusion of dibutyryl (dBcAMP), or the beta-adrenergic stimulant epinephrine, or the phosphodiesterase inhibitor theophylline, all produced a marked fall in the ventricular fibrillation threshold and an increase in the duration of the vulnerable period of the isolated perfused rat heart. In contrast, Muller et al. (1986) recently showed that prevention of ventricular fibrillation by beta-adrenergic blockade is not directly associated with decreased levels of cAMP, while Manning et al. (1985) used forskolin to stimulate adenylate cyclase and found that the markedly elevated tissue cAMP levels in the rat heart did not promote ischemic or reperfusion arrhythmias. Some of these contradictions could be resolved if the electrophysiological mechanisms by which increased levels of cAMP might predispose to arrhythmias were better understood. It is known that intracellular injection of cAMP into cardiac myocytes can enhance delayed afterdepolarizations (DADs; Matsuda et al. 1982) and that DADs may explain certain arrhythmias such as those evoked by digitalis toxicity (Ferrier, 1977) or reperfusion (Ferrier et al. 1985).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thiol-modifying agents on KATP channels in guinea pig ventricular cells.

ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)