I. INTRODUCTION

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Under aerobic conditions, NADH and FADH₂, formed during glycolysis and in the citric acid cycle, transfer their electrons to O₂ through the electron transport chain. This provides the energy to build up the chemiosmotic gradient that drives the synthesis of ATP. Oxidative phosphorylation is coupled to the demands of the cell. A feedback is generated by the breakdown products of ATP (772) and the rise in mitochondrial Ca²⁺ that modulates the activity of the mitochondrial dehydrogenases (692) and of the ATP synthase (199).

The turnover rate of high-energy phosphates is 30-40 mol · g wet wt¹ · min¹, while the storage is only 15 mol/g wet wt. This means that the time limit for exhaustion is short and only 15-30 s (339). When oxygen falls below a critical level in the cytoplasm, the electron transport and the process of H⁺ ejection in the mitochondrion stops. The energy stored in the proton electrochemical gradient becomes insufficient to synthesize ATP in appropriate quantity. Some of the energy in ATP may be used to maintain the mitochodrion membrane potential (E_m) and to inhibit irreversible reactions leading to cell death (772). As a consequence, [ATP] may be expected to fall (Fig. 1) and [ADP] to increase. The cell, however, has efficient means to maintain the ATP level: 1) energy demand falls very rapidly during the first 30 s of ischemia as a consequence of contraction failure, 2) an important amount of phosphocreatine (PCr) is continuously used to restore ATP from ADP with concomitant increase in P_i, and 3) anaerobic glycolysis starts and intensifies.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Metabolic consequences of ischemia on intramitochondrial levels of ATP, Pi, and phosphocreatine (PCr) as percent of control [ATP] obtained from 31P-NMR spectra in perfused ferret hearts. [From Marban et al. (640). Copyright 1990 American Heart Association.]

The fall in the ATP/ADP stimulates the glycolytic pathway by activation of three important enzymes: 1) hexokinase responsible for the formation of glucose-6-phosphate; 2) phosphofructokinase, responsible for the transformation of fructose-6-phosphate to fructose-1,6-biphosphate; and 3) pyruvate kinase for the formation of pyruvate. The reactions leading to pyruvate are stimulated but glucose metabolism stops at the pyruvate-lactate stage, due to inhibition of pyruvate dehydrogenase. Pyruvate is transformed in L-lactate under simultaneous oxidation of NADH to NAD⁺. At the same time, phosphorylase a is transformed in its active form, facilitated by accompanying increases in AMP, P_i, Ca²⁺, and cAMP and increases the breakdown of glycogen. High glycogen content of cells postpones the development of contracture during ischemia (190). Finally, glucose transport through the cell membrane is stimulated, secondary to a translocation of glucose transporters from an intracellular pool to the plasma membrane (1155) and increases the flow down to pyruvate. Glycolytic enzymes are especially dense in the subsarcolemmal space; stimulation of anaerobic glycolysis at this level is important for the regulation of intracellular ion concentrations and channels (KATP) (1082).

Stimulation of the glycolytic pathway explains why [ATP] stays remarkably constant during the initial 10-15 min of ischemia. However, the free energy change upon hydrolysis of ATP falls immediately because of the rise in [ADP] (281). This fall has important consequences for a number of ATP-driven transporters such the Na⁺-K⁺ATPase and the Ca²⁺-ATPase. The anaerobic glycolytic process is self-inhibiting however. When acidosis becomes too pronounced, glycolysis in turn is inhibited, and ATP synthesis is further reduced.

Whenever the free energy of ATP hydrolysis exceeds the energy stored in the proton electrochemical gradient, net ATP hydrolysis may occur; the ATP synthase then acts as an ATP hydrolase, and the energy is used to update the electrical gradient in the mitochondrion (224) (Fig. 2). Although the arrest of electron flow is expected to cause depolarization of the mitochondrial membrane during anoxia, such depolarization only occurs after a delay (244). The delay corresponds to the time needed to block glycolysis to cause serious ATP deficiency and rise in [Ca²⁺]_i. Blocking ATP hydrolysis by oligomycin slows the fall in [ATP] (808) and delays activation of the mitochondrial mega-channel. When [ATP] drops to levels <1 mM and at the same time the concentration of H⁺, [Ca²⁺]_i (>1 µM), PO₄³ (>10 mM), and long-chain acylcarnitines (LCAC) increase (772), the mitochondrial membrane becomes abnormally leaky through activation of the mega-channel or transition pore in the inner membrane of the mitochondrion. Efflux of ATP through the plasma membrane is an additional reason for a fall of intracellular ATP. In the extracellular medium, this ATP is further broken down to adenosine and may activate purinergic P₁ and P₂ receptors.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Ionic channels and carriers in outer and inner mitochondrial membranes. Under aerobic conditions, electron-carrying chain ejects protons and generates an important electrical gradient over inner membrane. Proton inflow through synthase provides necessary energy for ATP synthesis. In anaerobic conditions, mitochondrion should depolarize; synthase however may now act as an ATPase and guarantee persistence of electrical as well as proton gradient. Ca2+ homeostasis in mitochondrion is dependent on this electrical and proton gradient. MCC, multiple conductance channel, mega-channel, or permeability transition pore; mCS, mitochondrial cento/picosiemens channel; KATP, ATP-sensitive K+ channel; ANC, adenine nucleotide carrier; VDAC, voltage-dependent anion channel.

Block of oxidative metabolism and fall in ATP/ADP will have consequences on lipid metabolism, the generation of oxidative stress, the release of catecholamines, and ion concentrations.

Upon reperfusion after 5-30 min of ischemia, oxygen consumption rapidly recovers. The NADH/NAD⁺ quickly decreases, although the level may remain higher than the control for some time. After brief (5-10 min) ischemic bouts, PCr concentration quickly returns to normal, but recovery of ATP is slow. After longer periods of ischemia, PCr concentration still recovers within 5 min, but ATP, which may have dropped to 50%, stays at this low level for 30 min or more (641).

The preischemic pattern of substrate utilization is restored, i.e., oxidation of fatty acids (FA), is the main contributor to ATP synthesis; the level of glycolysis, however, remains elevated in the early period of reperfusion and is important for ATP generation. This situation may explain why block of glycolysis at this time is disadvantageous and results in aggravation of Ca²⁺ overload and release of intracellular enzymes. Such a block occurs when FA are present in high concentration (621). A high rate of FA oxidation generates NADH which inhibits pyruvate dehydrogenase. Glycolysis which stops at the pyruvate level is accompanied by an overproduction of protons and may negatively affect the recovery of [Na⁺]_i and [Ca²⁺]_i (612). High levels of lactate are deleterious for recovery.

The high level of oxygen consumption early after reperfusion stays in contrast to the continuing deficiency of the contractile machinery or "stunning." This uncoupling between oxygen consumption and contractility is not due to impairment of the respiratory chain flux and insufficient ATP synthesis. Energy is sufficiently present but apparently not used. As a possible explanation for the high rate of oxygen consumption, the existence of futile cycles has been proposed, such as the cycling of Ca²⁺ between the cytoplasm and mitochondria (189). The absorption of Ca²⁺ by the mitochondria as well as its removal via the Na⁺/Ca²⁺ exchanger depends on the energy flux in the electron chain. The ejection of protons generates the negative matrix potential that drives the absorption of Ca²⁺ and creates the proton gradient that is required for removing Na⁺ from the mitochondrial matrix via the Na⁺/H⁺ exchanger and subsequently Ca²⁺ via the Na⁺/Ca²⁺ exchanger. Adenosine 5'-triphosphate, which for its synthesis is also dependent on the proton gradient, is used by the plasma membrane Na⁺-K⁺ pump to keep cytosolic [Na⁺] low (Fig. 2). Under normal conditions, this cycle only requires little energy, but in the case of Ca²⁺ overload, the expenditure may become excessive. The situation is aggravated by an eventual intermittent opening of the mega-channel activated by elevated matrix [Ca²⁺] (91). The leak created causes breakdown of the mitochondrial electrical gradient. Activation of the mega-channel may propagate from mitochondrion to mitochondrion, creating a Ca²⁺ wave in the cytosol (449).

Reperfusion after more than 30 min leads still to an important but only transient recovery of oxygen consumption, while contractile function remains severely and persistently depressed. Intracellular enzymes and other substances are lost. On the microscopic level, cells appear necrotic, and later important fibrosis develops.

	II. ION CHANNELS AND TRANSPORTERS

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A.  Ion Channels and Transporters in the Plasma Membrane

1.  Na⁺ channels

In most excitable cells, the Na⁺ current is responsible for the upstroke and the conduction of the action potential. In heart, the density of the channel is low in sinoatrial node (SAN) and atrioventricular node (AVN) cells and highest in Purkinje cells. The Na⁺ channel is voltage operated and shows activation and inactivation. It is specifically blocked by tetrodotoxin (TTX) (for a general review, see Ref. 291); modulation of channels is described in section III.

A) ACTIVATION AND INACTIVATION. When a cardiac cell is depolarized, an inward current is generated that rises rapidly and decreases afterward on a much slower time course. The rise is sigmoidal (567) or exponential (685). The time to peak shortens with depolarization (634). The decline in macroscopic current or inactivation is best described by a sum of two exponentials. The time constant of the first component becomes shorter with depolarization.

Peak current-voltage relation can be translated into a conductance-voltage relation from which an activation curve can be constructed. Peak current is voltage dependent and increases for more negative holding potentials. The relation has been explained as a change in availability of the channel to become activated. Both relations, activation and inactivation, can be described by a Boltzmann distribution, with slopes of ~6 mV. Midpoints of voltages in multicellular preparations are 30 mV for the activation process and 85 mV for the inactivation process.

The time course and the steady-state properties of the current have been explained in terms of two voltage-dependent processes, activation and inactivation independent from each other (425). The opening and closing of the channel depends on the movement of charged gates, and the hypothesis thus predicts the existence of a gating current. In heart cells, such a gating current has been measured for the activation process (50, 350, 372, 486) but not for the inactivation process, as if inactivation is voltage independent. The activation gating current, however, becomes smaller during the development of inactivation. The gating charge becomes immobilized, suggesting some coupling between the two processes (372, 486). Depending on the type of cell, coupling between activation and inactivation is variable. Coupling is strong in neuroblastoma cells (9) in which activation shows a variable onset and inactivation follows rapidly. In cardiac cells, the coupling is weak; openings always occur at the beginning of the depolarizing pulse (activation is fast) and their duration is variable (inactivation is slower and variable) (69, 90, 567, 587, 856).

Recovery from inactivation is normally very fast (1-10 ms), with rates increasing upon hyperpolarization. After long depolarizations, recovery can become very slow (order of seconds). The process has been called slow inactivation (125, 160, 825, 876). The existence of this phenomenon may be important to understand changes occurring during and after ischemia, where the cells are subjected to long depolarizations.

Is there more than one type of Na⁺ current? In heart cells, decay of the Na⁺ current is normally very rapid. In a limited range of potentials, where activation and inactivation overlap, a small noninactivating current or window current can be recorded, which is due to the cycling of channels between the rested, activated, and inactivated state (27, 325, 755). This is not due to any abnormal behavior of the channel, but in pathological conditions, the overlapping may increase, resulting in an enhancement of the current.

A slowly inactivating Na⁺ current, which represents a small percentage of the total Na⁺ current, can be recorded in rabbit (125) and canine (325) Purkinje fibers, in rat ventricular cells (826), and in expressed human Na⁺ channels (801) over a broad range of potentials. In the rat, it becomes more pronounced during hypoxia (489) (Fig. 3). Part of it does not inactivate completely. Compared with the fast Na⁺ current component, activation is shifted in the hyperpolarized direction. This may cause more pronounced overlapping of activation and inactivation, generating in this way a constant steady-state component. The current deactivates immediately upon hyperpolarization. At the single-channel level, it is characterized by a bursting behavior, i.e., clusters of repetitive short openings (5 ms) sometimes alternating with long openings (order of 200 ms) (1187). An activity characterized by short openings (<0.5 ms), becoming shorter with depolarization, has been described as "background" current because it carries current at the resting potential (1187). The current undergoes inactivation, however, and is very selective for Na⁺. According to Böhle and Benndorf (90), one and the same channel can show many modes of gating behavior; this channel may thus not be different from the slowly inactivating one. The slowly inactivating current has been described as being more sensitive to block by TTX (174), a finding which has been advanced in favor of the existence of a different isoform; the result, however, can also be explained by the slow kinetics of the TTX block (126).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Hypoxia increases persistent Na+ channel activity. Voltage steps from 120 to 50 mV were applied to a cell-attached patch from a rat ventricular myocyte. A: control. B: during hypoxia, different cells. Bottom trace in each series is average current of 50 traces. [Adapted from Ju et al. (489).]

After exposure of membrane patches to lysophosphatidylcholine (108, 1002), a persistent current can be recorded over a voltage range of 150 to 0 mV. It is probably due to a modulation of the fast component. The current does not deactivate upon hyperpolarization and is substantial at the resting potential. It may play an important role as an inward leak current, causing K⁺ loss during ischemia.

A Na⁺-dependent, TTX-insensitive background current is induced by high concentrations of ACh in guinea pig ventricular myocytes (659). The current reverses at 25 mV in normal Tyrode solution, and its single-channel conductance has been estimated by noise analysis to be ~2 pS (897). Because of its different pharmacological and single-channel characteristics, it can be regarded as a different isoform.

B) ION PERMEATION. In the presence of 150 mM [Na⁺]_o, single-channel conductance is 20-25 pS; it is dependent on the concentration of external Na⁺ with a dissociation constant (K_d) of 300-400 mM (885) and shows different substates (554, 727, 856). The channel is permeable to Na⁺ and Li⁺ and much less to K⁺ (9.5%) and Cs⁺ (2%) or tetramethylammonium (<1%) (885). A small Ca²⁺ permeability exists (3) and may increase in the presence of ouabain and upon -receptor activation (840). The current-voltage relation is linear in the absence of bivalent ions; deviation from this linear behavior in normal bathing solutions is due to a voltage-dependent block by [Ca²⁺]_o and [Mg²⁺]_o; electrical distance is 0.3-0.4 (884). The block by [Ca²⁺]_o and [Mg²⁺]_o is characterized by a reduction in apparent single-channel conductance, suggesting a very fast block. Other bivalent ions efficiently but more slowly block the current: Cd²⁺ > Mn²⁺ > Co²⁺ > Ca²⁺ > Mg²⁺ > Ba²⁺. Of importance to note is the highly sensitive block by Zn²⁺ and Cd²⁺ (297, 1042); these ions are much less effective in neuronal and skeletal muscle cells, whereas the opposite sensitivity exists for TTX. Internal [Mg²⁺] blocks outward current through the channel (786). The block is slightly voltage dependent with an electrical distance of 0.18 from inside.

At the molecular level, the channel consists of an -subunit (260 kDa) and two -subunits (36 and 33 kDa each) (291). The -subunit, which is sufficient for channel activity, is composed of six transmembrane segments repeated four times in a tetrameric structure. The voltage sensor for activation has been related to the S4 segment. The short intracellular segment between domains III and IV has been identified as the fast inactivation gate. Spontaneous deletion of the KPQ segment in the junctional part gives rise to the LQT3 syndrome, characterized by a slowing of the inactivation process and generation of long action potentials. The absence or weak voltage dependency of the inactivation process is consistent with the location of this inactivation gate outside the electrical field of the membrane. Slow inactivation involves conformational changes in the external pore (C-type inactivation) (1058). The presence of sialic acid at the external surface shifts the activation and inactivation in the negative direction (64). Interaction with the cytoskeleton modulates the inactivation and activation voltage dependence; F-actin disruption and microtubule stabilization accelerate the shift in the negative direction that occurs during whole cell recording (636). The two -subunits exert a modulatory role, speed up inactivation, and decrease block by local anesthetics (633).

The permeation process is dependent on the hydrophilic part of the -subunit between transmembrane segments 5 and 6. The selectivity for Na⁺ is due to specific amino acids in this region, and mutation of only two critical amino acid residues is sufficient to confer permeability properties similar to the Ca²⁺ channel. A cysteine in domain I is responsible for the high affinity of the channel for Zn²⁺ and Cd²⁺ and low sensitivity to TTX of the cardiac isoform.

Different types of Ca²⁺-permeable channels have been described in the plasma membrane of heart cells: the L- and T-type channels, both voltage activated, and a background channel (see Ref. 673). They can be differentiated on the basis of their electrophysiological and pharmacological characteristics. The density of the T- and L-type channels differs in different sections of the heart. The ratio of T-type over L-type channel is highest in Purkinje and sinoatrial cells where it approaches the value of 0.2-0.6, and it is less in atrial and ventricular cells where the ratio only attains 0.015-0.025 (48).

A) BACKGROUND CA²⁺ CHANNELS. Calcium-permeable channels are seen following incorporation of plasma membrane protein fractions in bilayers (806). No voltage steps are required for activation, and spontaneous single-channel activity with long openings of >100 ms can be recorded at negative E_m values of 90 mV. The conductance is 7 pS in isotonic Ba²⁺; the channels are not blocked by dihydropyridines (DHP) and show no rundown. Their selectivity is not pronounced with a permeability ratio P_Ba/P_Cs of 10.

A similar channel but with higher conductance (22, 45, and 78 pS in Ba²⁺) has been described in neonatal rat hearts (187). It is not blocked by Cd²⁺ or nifedipine; instead, activity is rather increased in the presence of DHP but suppressed by protamine. In rat ventricular myocytes, the channel is induced by exposure of the inside-out patches to phenothiazines (593). Activity is also increased by exposure to oxygen free radicals and metabolic inhibition (1056). The channel may be responsible for the Na⁺-independent Ca²⁺ entry pathway described for rat trabeculae (583).

B) L-TYPE CA²⁺ CHANNEL. Calcium influx through the L-type Ca²⁺ channel is responsible for the upstroke of the action potential in the SAN and AVN and plays an important role in determining the plateau and eventual spike-dome appearance of the action potential in other cardiac cells. It is further responsible for the coupling between excitation and contraction, induces release of Ca²⁺ from the sarcoplasmic reticulum, and regulates intracellular Ca²⁺ load. In this way it determines activity of a number of mitochondrial and cytoplasmatic Ca²⁺-sensitive enzymes.

I) Kinetics. A) Activation and inactivation. Threshold for activation is around 25 mV, and half-maximum activation is attained at about 15 mV for most cells (see Ref. 673) and at more positive potentials (3 mV) in the AV node (373). The rise in current follows a sigmoidal time course, suggesting a multistep process as the underlying mechanism. Activation is preceded by a gating current (50, 352, 486, 898). The density of the channels derived from the gating current is much larger than the ionic current density, suggesting that some of the channels although gating are not carrying current.

At the single-channel level, three modes of activity have been distinguished (137, 403). In mode 1, the channel shows repetitive short (<1 ms) openings and closures (0.2 and 2 ms), forming a burst of activity separated from other bursts by longer closures. A number of consecutive bursts may be grouped in a cluster. A variable waiting time precedes the openings; it decreases at more depolarized levels and corresponds to the faster activation and shorter time to peak values of the Ca²⁺ current. Mode 2 occurs in the presence of DHP agonists (403) or after -receptor stimulation (137, 767) and is characterized by much longer open times. Mode 3 is characterized by the complete absence of openings or presence of only rare short openings. The frequency of this latter mode increases with preceding depolarizations and corresponds to the occurrence of steady-state inactivation.

The L-type Ca²⁺ current inactivates in two ways: a voltage-dependent and a current-dependent way. The existence of two types of inactivation explains the complex time course of current decay and the presence of a dip in the inactivation curve. Half-maximum steady-state inactivation occurs at 20 to 30 mV. The curve shows a minimum at ~0 mV and increases again at more positive potentials. When intracellular Ca²⁺ is well buffered, this turning up is absent and the decay of the current during a pulse is much slower. These observations have led to the conclusion that inactivation is dependent on voltage as well as on Ca²⁺ influx. The latter or Ca²⁺-induced inactivation is the faster process, whereas the voltage-induced inactivation is rather slow. Activation and inactivation show a remarkable overlapping (window current) (414, 900).

B) Intracellular Ca²⁺ or current-dependent inactivation. Evidence for intracellular Ca²⁺-dependent inactivation at the whole cell level is based on the change in time course of current decay with changes in [Ca²⁺]_i (see Ref. 673) (Fig. 4, A and B). The decay is faster the larger the Ca²⁺ current, and it is slowed in the presence of intracellular Ca²⁺ buffers. At the single-channel level (cell-attached patches), Ca²⁺ permeation through the channel reduces the open probability of subsequent reopenings of the channel and shifts the gating mode toward a mode with long-lived closed states (454). In excised patches, with Ba²⁺ as the charge carrier, steady-state elevation of Ca²⁺ in the range of micromolar concentration or flash photolysis of Ca²⁺ reduces the open probability of the Ca²⁺ channels. It is especially the Ca²⁺ originating from the sarcoplasmic reticulum (SR) that is responsible for the inactivation process during the first 50 ms of depolarization (949, 1154); at later times also Ca²⁺ permeating through the Ca²⁺ channel contributes to the inactivation (949). The increase in cytosolic Ca²⁺ by the release from the SR is indeed 10-fold greater than the Ca²⁺ entering the cell via the L-type Ca²⁺ channel (915). In favor of this explanation is the observation that inactivation is much slower after depletion of the SR by caffeine or in the presence of ryanodine. It is important to note that [Ca²⁺] seen by the channel may importantly deviate from the cytosolic [Ca²⁺] as measured by fluorescence techniques. Especially during the first 50 ms of a depolarizing pulse the concentration seen by the channel may be much higher. Such a difference may explain why the relative inhibition of the current estimated from the Ca²⁺ transient is greater during this initial period than later (611).

View larger version (31K): [in this window] [in a new window]   Fig. 4. A: existence of facilitation during recovery of Ca2+ current from previous inactivation in dog ventricular myocytes. Paired depolarizing pulses of 100-ms duration were applied from a holding potential of 80 mV to 10 mV with a delay variable between 50 and 3,000 ms. Ca2+ current recovers rapidly within 30 ms to its control value; afterward, current transiently becomes larger. Facilitatory effect requires presence of Ca2+ influx and is absent when external solution contains Ba2+. [Adapted from Tseng (991).] B: Ca2+-induced inactivation. Ca2+ currents (top traces) and Ca2+ transients (bottom traces) in guinea pig ventricular myocytes in presence of 2 different external Ca2+ concentrations (Cao). Superimposed tracings for control (a) and after Ca2+ loading (b) are shown. Note marked, rapid Ca2+-induced inactivation in presence of Ca2+ overload, but also partial recovery during pulse. I, current; [Ca2+]i, intracellular Ca2+ concentration. [From Sipido et al. (911). Copyright 1995 American Heart Association.]

The existence of an intact cytoskeleton is important in determining the local [Ca²⁺]. The cytoskeleton normally keeps the channels separated from each other and limits the rise in local [Ca²⁺]. In ischemia, the cytoskeleton may become disturbed. Disruption of the cytoskeleton structure by colchicine or cytochalasin, with consequent clustering of the channels, favors inactivation, whereas substances such as taxol and phalloidin that stabilize the skeleton remove inactivation and improve reopening (taxol and colchicine act on microtubules; phalloidin on F-actin) (311).

Different mechanisms have been proposed to explain [Ca²⁺]_i-dependent inactivation (719). 1) A fall in driving force is improbable. In cell-attached patches, single-channel conductance does not change, whereas open probability is markedly reduced. 2) Dephosphorylation by phosphatase (e.g., calcineurin) and proteolysis by Ca²⁺-stimulated proteases (calpain) (351) are mechanisms that may be responsible for long-term changes in Ca²⁺ channel behavior but are too slow to explain the time course of changes during a single depolarization. 3) Calcium binds to the channel protein and induces a change in configuration. In the cloned channel, a Ca²⁺ binding motif (an EF hand) exists at the COOH terminal of the _1C-subunit; deletion eliminates Ca²⁺-induced inactivation. The site is located near the inner mouth but outside the electrical field (205, 719). The reduction of Ca²⁺ current by [Mg²⁺]_i also has been explained by a direct binding to this site, reducing the number of functional channels (1132). Because Ca²⁺ binding is a fast process, it provides an explanation for the time course of inactivation and the observation that Ca²⁺ oscillations are accompanied by equivalent changes in current (911). Photolysis of Ca²⁺ results in a rapid inactivation, within 20 ms (351) to 75 ms (46); the process is not accompanied by a change in gating current, confirming that intracellular Ca²⁺-induced inactivation and voltage-dependent inactivation are two distinct phenomena (273).

C) Voltage-dependent inactivation. The evidence for voltage-dependent inactivation is based on the following observations (see Ref. 673). 1) Under conditions where intracellular Ca²⁺-dependent inactivation is excluded (e.g., current carried by monovalent cations or current carried by Ba²⁺ or Sr²⁺), the current decays with time, and the rate of decay is faster the higher the depolarization. 2) Inactivation develops when prepulses are applied that do not result in Ca²⁺ inward current. 3) Inactivation is present in channels incorporated in lipid bilayers with Ca²⁺ buffered (806). 4) Inactivation is slowed by trypsin treatment, but the intracellular Ca²⁺-dependent inactivation is not affected (862). No charge movement occurs on inactivation, but as inactivation of the current proceeds, the charge movement that accompanies activation becomes smaller (351, 898).

D) Recovery from inactivation: Ca²⁺-induced facilitation. Upon hyperpolarization, the Ca²⁺ current recovers from inactivation induced by a previous depolarization. Because inactivation is voltage and intracellular Ca²⁺ dependent, it is logical to expect repriming also to depend on these two parameters. For the voltage-induced inactivation, the rate and the degree of recovery is greater the more hyperpolarized the membrane, with time constants in the order of 300 ms at 50 mV and 100 ms or shorter at 80 mV. A much slower component (seconds) is present after long depolarizations, indicating the occurrence of slow inactivation (94, 868). This slow inactivation may play a role in overdrive suppression (1074). Recovery from Ca²⁺-induced inactivation as such is voltage independent (911, 912) but indirectly it is modulated by voltage, since the fall in [Ca²⁺]_i is dependent in part on the Na⁺/Ca²⁺ exchange that is faster the more negative the E_m.

At negative holding potentials, recovery from inactivation may show an overshoot, i.e., the Ca²⁺ current transiently becomes larger than in steady state. The original finding of an overshoot was made in Purkinje fibers treated with digitalis (505) but can also be observed without Ca²⁺ overload (see references in Refs. 606, 770). The potentiated Ca²⁺ current is characterized by a larger peak and a slower time course of decay (991, 1159, 1193) (Fig. 4).

At the single-channel level, facilitation is characterized by an increase in open probability (P_o) with a larger proportion of long openings (413) (mode 2) and an increase in number of functional channels (1132). In all these approaches, it is clear that a moderate increase in Ca²⁺ is required (991, 1159, 1193); the overshoot in recovery is inhibited by rising intracellular Ca²⁺ buffering, or by ryanodine or using Ba²⁺ as the current carrier. Excessive rises in Ca²⁺ lead to inhibition by Ca²⁺-induced inactivation (351).

How elevated Ca²⁺ causes facilitation remains a matter of debate. Phosphorylation of the channel protein is a possibility. Flash photolysis induces facilitation with a delay, and the effect is counteracted by inhibitors of protein kinases (PK) (18, 1159). Other groups, however, did not find an effect of PK inhibitors (46, 1132), and facilitation still occurred with nonhydrolyzable ATP analogs (1132) or even improved (46). The conclusion of these authors is that a Ca²⁺-nucleotide complex directly potentiates Ca²⁺ current (I_Ca) through a phosphorylation-independent mechanism. The existence of two phases in the recovery (decrease followed by an overshoot) has practical consequences in determining down- or upregulation of I_Ca as a function of frequency and diastolic E_m.

II) Permeation and selectivity. The channel is 500-1,000 times more permeable to bivalent ions such as Ca²⁺ and Ba²⁺ than to monovalent ions. The exclusion of monovalent ions depends on the presence of a minimum concentration of bivalent ions. In the absence of bivalent ions, the channel becomes highly permeable to monovalent ions (651). Although restricted, the permeability for K⁺ is responsible for a substantial current during the action potential, the reason being that the concentration of intracellular K⁺ is quite high compared with the nanomolar free Ca²⁺ concentration. The K⁺ contribution also explains why the reversal potential (E_rev) of the Ca²⁺ current is much less positive than expected for the equilibrium potential for Ca²⁺. The bivalent ion current through the channel increases with the concentration and shows saturation. Compared with the single-channel conductance in isotonic Ba²⁺ (8-10 pS and 15-25 pS, see Ref. 673), the single-channel conductance at the physiological concentration of 1 mMCa²⁺ was found to be surprisingly high at 7 pS (1162). The presence of negative charges at the pore mouth of the channel, which attract bivalent ions and increase their local concentration (331), is probably the reason for this behavior. In the presence of both Ca²⁺ and Ba²⁺, the current across the channel is smaller than in the presence of either Ca²⁺ or Ba²⁺ alone (997). This kind of behavior has been called anomalous mole fraction behavior and suggests a multi-ion channel. Different ions thus interact in such a way that the flux of one species is hampered by the presence of the other species. Multi-ion occupancy also explains the high conductance and at the same time the high selectivity. High selectivity is conditioned by high affinity; high conductance by the presence of more than one ion in the channel and repulsion of one ion by the other. The multi-ion nature of the channel also explains the flickery block behavior of the channel in the presence of elevated proton concentration (784).

The channel can be blocked by a number of extracellular bi- and trivalent ions such as Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cd²⁺, Zn²⁺, and La³⁺ (997). The ions Cd²⁺, Zn²⁺, and La³⁺ block the channel in a voltage-dependent way with an apparent electrical distance of 0.15 for Cd²⁺ and Zn²⁺ and 0.60 for La³⁺. Intracellular Mg²⁺ is needed to activate enzymes that phosphorylate the channel but may, on the other hand, reduce the current that has been augmented by isoproterenol or BAY K 8644 by a direct blocking action and by activating phosphatases (1090, 1132). The cAMP-dependent phosphorylation reduces the sensitivity to [Mg²⁺]_i block (1131).

The cardiac Ca²⁺ channel molecular structure consists of four subunits: two -subunits, ₁ and ₂, a -subunit, and a -subunit (a -subunit is exclusively expressed in skeletal muscle). The -subunit is sufficient to express channel activity. It resembles the Na⁺ channel with four times six transmembrane segments, a highly charged S4 segment that probably acts as the voltage sensor for activation, and an intracellular link between domain II and III responsible for inactivation. Trypsin treatment removes voltage-dependent inactivation but not internal Ca²⁺-dependent inactivation (862). Binding of Ca²⁺ to the COOH terminal is a possible mechanism for Ca²⁺-induced inactivation (205).

The highly conserved glutamate residues located in the pore region of all four repeats are involved in high-affinity bivalent binding (1146). A mutation from E to Q in domain III has shown that this group is the strongest determinant of Ca²⁺ binding (331).

The function of the -subunit is markedly modulated by the -subunit. Coexpression of the two results in a fourfold increase of peak current, which is not due to a change in single-channel conductance (331) but to a marked increase in density of functional channels (488, 718, 759). The gating current remains unchanged, but coupling between conductance and gating is improved (759).

The L-type Ca²⁺ channels is highly regulated; this aspect is analyzed in section III.

C) T-TYPE CA²⁺ CHANNEL. A Ca²⁺ current of short duration is activated at potentials more negative than the threshold for the L-type Ca²⁺ current (48, 726; see review in Ref. 1026). The current is well represented in SAN cells, atrial cells, Purkinje cells, and nodal cells. In the embryonic chick ventricle, it is the major Ca²⁺ current. The current is not found in human atrium (258, 606) or human ventricle (76). The channel has been proposed to play a role in pacemaking. It may also interfere with steroidgenesis, cell proliferation, and cardiac growth (400).

I) Activation, inactivation, and repriming. Threshold for activation is around 70 to 50 mV and maximum activation is seen at 30 to 10 mV (see Ref. 1026). Inactivation is rapid and complete, with time constants of 30 ms at 50 mV, becoming shorter at more depolarized levels. This behavior is opposite to the L-type channel, where inactivation is decelerated at positive potentials. Steady-state inactivation extends from 85 to 40 mV with half maximum around 60 mV and slope of 5.5 mV (14, 36, 412). An increase in [Ca²⁺]_i does not induce inactivation but rather facilitates the T-type current (13, 994). At the single-channel level, this increase in current is characterized by a shift to long openings (mode 2 behavior).

Repriming is voltage dependent and becomes faster with hyperpolarization: 250 ms at 70 mV and 100 ms at 90 mV (145). It is slower the longer the preceding depolarization, suggesting the existence of slow inactivation (412).

II) Permeation. In 100 mM [Ca²⁺], the single-channel conductance is 8 pS, compared with 20 pS for the L type. Contrary to the behavior of the L-type channel, permeability for Ca²⁺ and Ba²⁺ ion is the same (48, 900). The channel is permeable to Sr²⁺, blocked by Ni²⁺, but much less sensitive to Cd²⁺. Extracellular protons inhibit the channel with greater efficiency than the L-type current, whereas intracellular protons have no effect (1000). Extracellular Mg²⁺ reduce the current and shift the activation and inactivation curves in the positive direction (1113).

Recently two isoforms, the G and H isoform of the -subunit, have been cloned (760). Sialic acid probably forms an important component of the extracellular part of the channel (1150).

3.  K⁺ channels

Because the equilibrium potential of K⁺ is rather negative, all cardiac K⁺ channels when activated will carry outward current, repolarize the membrane during the action potential, or stabilize the membrane at a hyperpolarized level. Among the many K⁺ currents, distinction can be made between voltage-activated currents (I_to, I_Kur, I_Kss, I_Kr, and I_Ks), ligand-activated currents (I_KACh, I_KATP, I_KNa, and I_KAA), and a current (the inward rectifier I_K1) that apparently does not gate and can be called a background current. Under physiological circumstances the voltage-activated K⁺ currents, I_KACh among the ligand-activated and I_K1, play an important role in shaping the normal action potential. Under ischemic conditions, ligand-activated currents, especially I_KATP and I_KAA, become primordial, whereas some of the "physiological" currents are inhibited. Voltage-activated K⁺ currents show activation and inactivation upon depolarization; the rates of these two processes can vary from fast to ultra-slow. Ligands can bind to receptors, which then activate the channel via a G protein, or can interact directly with an intracellular site of the channel.

The amino acid composition of most K⁺ channels is known (201), and recently, the molecular structure of the pore has been elucidated from X-ray analysis of the crystallized molecule (234). The channels show a remarkable homology with the Na⁺ and Ca²⁺ channels. However, whereas Na⁺ and Ca²⁺ channels consist of tandemly linked four domains of six transmembrane segments that are connected in one long polypeptide, only one domain with six transmembrane segments is found for the K⁺ channel. Segment 4 shows a high density of positive charges and acts as potential sensor. Two types of inactivation have been described in expressed channels (794): N-type inactivation in which the negatively charged NH₂ terminal acts as a ball and blocks the open channel and C-type inactivation (COOH terminal) in which conformational changes on the extracellular side close to the pore result in some kind of constriction. C-type inactivation is sensitive to drug binding and extracellular K⁺. The ligand-activated and background channels have a simpler structure and contain only two transmembrane segments. Recently, a new family of K⁺ channels with two pore segments in tandem and four transmembrane segments have been expressed. They act as background channels (530); some of them are activated by arachidonic acid and polyunsaturated fatty acids (280). A tetrameric structure for all K⁺ channels is highly likely. The pore in K⁺ channels is formed by a stretch of 19 amino acids in the link between S5 and S6 in the four repeats. The motif GYG (or of GFG) in the P-region is the signature of K⁺ selectivity, but other residues also participate in determining K⁺ selectivity.

A) K⁺ OUTWARD CHANNELS WITH FAST ACTIVATION. Upon depolarization, three different K⁺ currents are rapidly activated. They can be distinguished by their rate of inactivation, which is relatively fast for I_to, slow to ultraslow for I_Kur, and nonexistent for I_Kss, also called background current. On the latter current, no detailed information is available at the present time.

I) The fast transient outward current. The fast transient outward current (I_to) is a transient outward K⁺ current that is rapidly activated and inactivated and blocked by millimolar concentrations of 4-aminopyridine (4-AP). The criterion of 4-AP sensitivity is not exclusive. In some species, another K⁺ current, the I_Kur, is also blocked even by micromolar concentrations of the drug (see below), and in the dog ventricle part of the I_to is insensitive to millimolar concentrations (607). It should be distinguished from another transient outward current carried by Cl and activated by [Ca²⁺]_i; this current is also called I_to2. In this review it will be indicated as I_ClCa. I_to is partly responsible for the initial fast repolarization or phase 1 during the action potential. The density of I_to varies among species and in a particular species it varies in different parts of the heart (see Ref. 117). It is more expressed in the atrium and Purkinje fibers and in the ventricle more in the epicardial than endocardial fibers. The density of I_to in the heart increases after birth (259, 483, 639), although its presence is variable (191, 342), an observation which is possibly related to pathological downregulation.

A) Activation and inactivation. The current is activated upon depolarization. Values for time course and steady-state vary among species and experimental conditions. In the rabbit, time course is fast and monoexponential (269); in the ferret it is sigmoidal (118). Midpoint voltage values for steady-state activation vary between 10 mV in the rabbit (269) and +20 mV in the ferret (118).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Inactivation and frequency dependence of Ito and IKur in human atrial myocytes. A: identification of 2 outward currents based on their different time dependence of inactivation. For prepulses of 400 ms between 80 and 20 mV, peak currents () inactivated while late currents () were constant. For longer prepulses (2,500 ms) also, late current partially inactivated (). [From Firek and Giles (284). Copyright 1995 Elsevier Science.] B: rate dependence of Ito and IKur. Atrial myocyte was stimulated at 0.1 and 4 Hz (top traces); trace marked by solid circle is difference current and demonstrates that current affected is IKur. [From Fermini et al. (272).]

After activation, the current decays. The time course of inactivation has been described as monoexponential or biexponential. Time constants again vary but are in the order of 25-75 ms and are voltage independent. Steady-state inactivation shows half-maximum potentials between 50 and 15 mV (see Ref. 117) (Fig. 5A). Recovery from inactivation is very sensitive to voltage, being faster the more hyperpolarized the membrane. It is also facilitated by increasing [K⁺]_o (284); this supports the hypothesis that inactivation is of the N type (750). For C-type inactivation, the process is slowed by extracellular K⁺ acting as a "foot in the door."

Actual time constants for recovery vary with species. In most species, including humans (15, 284, 342), recovery is fast with time constants in the order of 20-60 ms at 80 mV; frequency dependence is small (Fig. 5B). In rabbit atrium and ventricle, in sheep and dog Purkinje fibers (see Ref. 117), and in human subendocardial fibers (708), recovery is slow to very slow (time constants of 1-6 s). In these latter preparations, the current is markedly reduced (272) and shortening of the action potential markedly less at elevated frequencies (512).

B) Permeation. On the basis of measurements of E_rev (25, 118, 158, 416, 707, 708, 1088), the I_to current is considered to be mainly carried by K⁺, although it seems less selective than other K⁺ currents, such as I_K1. At the single-channel level, the current-voltage relation is linear and single-channel conductance in 145 mM [K⁺]_o is on the order of 10-30 pS (63, 158, 717) with the exception of a much lower value of 3-4 pS for ferret ventricle (118). The conductance increases at elevated [K⁺]_o, with a K_d of 200 µM (284).

As molecular substrates for I_to, Kv4.2 (922) and Kv4.3 (229) have been proposed. On the basis of differences in voltage dependence, kinetics of inactivation and recovery, and block by 4-AP, Kv4.2 is the better candidate for I_to in the human, rat, and ferret, and Kv4.3 in the canine and human subendocardium. Kv1.4 may be the better choice for the sheep and the rabbit. In the rat, expression of the protein shifts from Kv1.4 to Kv4.3 after birth and during thyroid treatment (1094).

II) I_Kur. A rapidly activated K⁺ current, with no or very slow inactivation is present in different heart preparations. The voltage dependency of the slow inactivation process and the sensitivity to 4-AP is species variable, and on this basis, it can be concluded that the current does not correspond to a unique channel. On the basis of the sensitivity to 4-AP, the currents can be subdivided into two groups. In the human atrium (15, 192, 388, 1065), dog atrium (1164), cultured rat neonatal ventricle (347), and mouse ventricle (287, 1182), I_Kur is exceptionally sensitive to 4-AP and completely blocked by concentrations of 50 µM or less. In rat atrium (97) and ventricle (25) and human ventricle (707), the current is insensitive to 4-AP. In many publications it is described as a noninactivating component of I_to.

A) Activation and inactivation. On depolarization to levels positive to 40 mV, an outward current remains after subtraction of a rapidly inactivating I_to in many species: rat atrium (97, 1021), rat ventricle (25, 347, 1083), human atrium (890, 1065) and human ventricle (707, 1087), rabbit ventricle (272), guinea pig ventricle (1161), and dog atrium (1164). It is rapidly activated and shows no or only very slow decay. The current inactivates however. Inactivation has been determined using long (tens of seconds) conditioning pulses. Midpoint inactivation potential is variable: 70 mV (25) and 90 mV (1083) in the rat and much less negative values of 9 and 20 mV in the human atrium (284, 890, 1065) (Fig. 5A). In accord with the existence of slow recovery from inactivation, the current is markedly reduced at elevated frequencies in the rat ventricle (25), rabbit ventricle (272)), and human atrium (272, 284, 890) (Fig. 5B). In these preparations, a rest current (I_Kss) remains, which seems different from I_Kur. It is reduced by -receptor stimulation in rat atrium (1021) and by -receptor stimulation in rat ventricle (849).

B) Permeation. I_Kur is assumed to be carried by K⁺, but direct demonstration is mostly lacking. In human atrium, tail currents reverse at negative potentials, suggesting a predominant (388, 1065) but not exclusive (191) permeability to K⁺. In favor of the K⁺ nature is the observation of sensitivity to tetraethylammonium (TEA) in rat ventricle (25) and dog atrium (1164) and block by Ba²⁺ in guinea pig ventricle (33, 1161) (in humans the current is not sensitive to Ba²⁺). Single-channel conductance in 5.4 mM [K⁺]_o is in the order of 14 pS for the guinea pig ventricle (1161) and 20 pS for the dog atrium (1164) and is sensitive to [K⁺]_o. Fully activated current-voltage relations show outward rectification (388, 1065).

The Kv.1.5 protein is a possible molecular candidate for the I_Kur current in the human atrium (271, 923, 1065). It shows a high sensitivity to 4-AP (1067) and a limited, partial inactivation at positive potentials but is insensitive to TEA. The single-channel conductance is 17 pS. The protein is present in the rat and the human atrium and ventricle, as determined by immunolocalization; it is highly concentrated in the intercalated disks (669). In the dog, Kv3.1 has been proposed as a molecular candidate (1164).

B) K⁺ CURRENTS WITH DELAYED ACTIVATION: DELAYED K⁺ CURRENTS. On the basis of kinetics, rectification, sensitivity to blockers, and modulation by intracellular messengers, two delayed K⁺ currents, I_Kr and I_Ks, can be distinguished (153, 838); I_Kr shows activation and inactivation, I_Ks only activation. Both are present in the human atrium (1066), human ventricle (77, 556, 605, 1033), guinea pig ventricle (838), guinea pig atrium (433), dog ventricle (324, 613) and atrium (1165), rabbit atrium (700) and ventricle (832), mouse neonatal ventricle (1055), and rat ventricle (141, 1115). Only I_Kr has been clearly described for the cat ventricle (290), the ferret ventricle (617), and rabbit SAN (1032) (466). I_Ks seems the only delayed current in the guinea pig SAN (22). Density of the two currents varies in different layers of the myocardial wall. In midmyocardium of the dog ventricle, expression of I_Ks is small (613); this explains the longer action potential in these cells. In the ferret ventricle, the ERG protein (Kr) is most abundant in the epicardial layers (99).

I) Rapid delayed K⁺ current, I_Kr. A) Kinetics. I_Kr activates rapidly for depolarizations positive to 40 mV, with a midpoint voltage between 20 and 5 mV; this value is [K⁺]_o independent (889). Time constants of activation vary among species; in the guinea pig, they are in the order of 175 ms at 30 mV and shorten at more positive or more negative potentials to ~50 ms (838). In the rabbit ventricular cell, time constants for activation are longer, 500 ms at 40 mV to less than 100 ms at 0 mV (128). Compared with I_Ks, these time constants are shorter, a finding on which the distinction of the two currents has been based. Deactivation does not follow the same pattern. It is fast in the guinea pig but much slower in the rabbit (128) and in the dog (324); in these preparations, the time course is composed of at least two exponentials, of which the first one is on the order of 0.5 s and the second on the order of 5-10 s at 40 mV (129, 324).

At the single-channel level, the mean open time is on the order of 3 ms. Closed time distribution is biexponential with values of 0.6 and 22 ms (at 100 mV and 100 [K⁺]_o) (889).

In whole cell recordings, the time course of the macroscopic current shows saturation with no indication of a secondary decrease. The tail currents on hyperpolarization, however, are preceded by a "hook," and the current temporarily increases before it declines in an exponential way (838, 889). The initial increase in outward current has been interpreted (889) as due to recovery from inactivation, a process supposed to be faster than the deactivation process. The hypothesis implies that the current during depolarization very rapidly undergoes inactivation, before there is any substantial activation (Fig. 6). It implies that steady-state inactivation extends over a voltage range that is quite positive. The consequence is that the current rectifies in the inward direction. Inactivation preceding activation has also been demonstrated in the expressed HERG channel (836, 932, 1141), which has been shown to be responsible for the I_Kr current. Fast recovery from inactivation is the reason for an increase in the number of openings of the Kr channel upon repolarization during early diastole in nodal cells (466, 1033).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Rapid inactivation determines inward rectification. Experiments were performed on HERG channels expressed in AT1 cells. In A, a depolarizing pulse to +40 mV reveals a small activating current, followed by a prominent tail at 40 mV. , Time constant. In B, protocol was identical except that a brief 20-ms hyperpolarizing pulse to 120 mV was interpolated in pulse to +40 mV. This hyperpolarizing pulse was followed by a large outward current that decayed rapidly. Result is interpreted as being due to rapid removal of inactivativation by hyperpolarizing pulse, followed by fast inactivation at +40 mV. In C, data from 8 experiments are displayed. Activating current (circles) (see also A) display inward rectification. Instantaneous currents, following a short hyperpolarizing pulse (see B), show a quasilinear, slightly outward going current-voltage relation (squares). [From Yang et al. (1141). Copyright 1997 American Heart Association.]

Inactivation of the I_Kr channel is of the C type (836). The evidence is based on the following observations: 1) truncation of the NH₂ terminal has no effect on the phenomenon; 2) intracellular TEA has no effect, but external TEA reduces the current and slows inactivation; and 3) an increase of [K⁺]_o slows inactivation in expressed channels and in AT-1 cells (1141). It is further of interest to mention that increases in [Mg²]_i or [Ca²⁺]_i, which generate inward rectification in other K⁺ channels, do not change rectification of I_Kr (836).

B) Permeation. Although preferentially permeable to K⁺, the channel's K⁺ selectivity is less pronounced than that of I_K1. Especially at lower [K⁺]_o, the E_rev is quite positive to the equilibrium potential for K⁺ (E_K). The conductance falls at lower [K⁺]_o (850, 889), a phenomenon explained as block by external Na⁺ or more pronounced inward rectification. C-type inactivation and thus inward rectification is enhanced at low [K⁺]_o (1141).

In 150 mM [K⁺]_o, the single-channel conductance is ~10 pS in SAN (466), AVN cells of the rabbit (889), human ventricular cells (1033), and guinea pig atrial cells (433). A value of <2 pS can be extrapolated for normal Tyrode solution.

External bivalent and trivalent cations block the channel. Especially sensitive is the block by Co²⁺ and La³⁺ (10 µM) (265, 837). External Cd²⁺ causes a positive shift of the activation curve (290) and a reduction in inward rectification, in this way increasing the current during a depolarizing pulse (749). The block by Ca²⁺ and Mg²⁺ is reduced by elevating [K⁺]_o but does not change inward rectification (421, 422). The HERG gene is responsible for the I_Kr protein expression (836, 932).

II) The slowly activated I_Ks current. A) Activation. The I_Ks current only shows activation and no inactivation. Activation occurs over a broad range of depolarizing potentials. In many experimental conditions it is difficult to obtain a clear-cut saturation. Half-maximum values vary considerably from 13 mV (666) to 26 mV (38). Kinetics are slow; the time course of the rise in current is sigmoidal, whereas the decay of the tails is monoexponential at voltages negative to 50 mV but biexponential at more positive potentials (605, 666). Deactivation is slow in the guinea pig but relatively fast in the dog and the rabbit (128, 324, 613). At the single-channel level, kinetics are complex with many open and closed times (38, 242).

B) Permeation. The channel is less selective than I_Kr. The E_rev is more positive than that for I_K1 and changes only by 49 mV for a 10-fold change in [K⁺]_o (666). The fully activated current-voltage relation approaches linearity, except for the current in frog atrial cells where inward rectification is present (243).

Single-channel conductance is relatively low with estimations of 5.4 pS in guinea pig ventricle (38), 3 pS in guinea pig atrium (433), and a greater value of 20 pS in frog atrial cells (242).

Extracellular K⁺ has no direct effect on the conductance but affects the current through changes in the chemical gradient; thus in zero [K⁺]_o, the current is greatly increased, especially in Na⁺-free conditions (850). Cobalt (1 mM) (265) and La³⁺ (at 100 µM or higher) (38, 837) block the current. A rise in [Na⁺]_i or [Ca²⁺]_i enhances I_Ks (978, 728).

C) Molecular structure. Coexpression of the minK and the Kv.LQT1 generates a current with the characteristics of the cardiac I_Ks (39, 835). The KvLQT protein has the classical constitution of voltage-activated K⁺ channels. The minK protein consists of only 129 or 130 amino acids and a single putative transmembrane domain, with the NH₂ terminal turned to the external side of the membrane (956, 1023). It plays an essential role in the function of Kv.LQT1 and can be considered a regulator protein.

C) THE INWARD RECTIFIER. The inward rectifier current (I_K1) is the current responsible for maintaining the negative resting potential in cardiac cells; it also plays an important role during the final rapid repolarization during an action potential (894). The density of the I_K1 is highest in the Purkinje and ventricular system (445), less in atrium (396); in the SAN, the I_K1 current is absent (459). A substantial increase of the current occurs during development from the neonatal to the adult stage (487, 1049).

I) Activation-deactivation: inward rectification. Is I_K1 a background or voltage-activated current? The I_K1 current, the first K⁺ current to be characterized in cardiac cells, was considered initially to be a time-independent background current. Its pronounced inward rectification provided an explanation for the existence of a long plateau in the cardiac action potential (see Ref. 133). With the improvement of recording techniques, it became clear that I_K1 showed time-dependent changes, which were analyzed as activation, deactivation, and inactivation (569). Upon hyperpolarization from a holding potential of 50 mV to 100 mV, a quasi-instantaneous current jump is followed by an exponential increase in inward current to a steady state (385, 569, 982). On depolarization, the reverse sequence is seen. This led to the hypothesis that the I_K1 channel opens and closes by an intrinsic gating process not different from other voltage-operated channels. More recently, the time-dependent changes in the current or gating have been recognized to be generated by a time-dependent block-unblock by Mg²⁺ (656, 1009) and polyamines (622, 1093). Magnesium and putrescine ions are responsible for the very rapid phase, spermidine and spermine ions for the slower phase (622, 723) (Fig. 7). The difference in rate corresponds to the difference in positive charge (263, 1139). The block is voltage dependent, with an electrical distance of 0.3 for [Mg²⁺]_i; polyamines seem to penetrate deeper in the pore (622, 723). With time at the depolarized level, the block shifts from fast Mg²⁺ to slow polyamine block. On hyperpolarization, this is seen as an increase in the slower phase of activation (463). This new concept of activation being an unblocking is in accord with the information obtained on the molecular structure of the inward rectifier family, in which a voltage sensor or S4 segment is lacking. The I_K1 molecule consists of only two transmembrane segments with a H5 or pore sequence in between (563, 954). The I_K1 channel can thus be considered a background channel, and a distinction between gating and permeation becomes less obvious. Whether on top of block-unblock there still exists an intrinsic gating mechanism is not fully resolved, and recent experiments on IRK1 channels expressed in oocytes have been explained in this way (893).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Block by intracellular positvely charged substances determines inward rectification. Experiments performed on HRK1 channels expressed in Xenopus oocytes. A: current-voltage relations for 20-ms test pulses to different potentials, after patch isolation () and in presence of 25 µM () and 1 µM spermidine (). B: normalized currents as a function of membrane potential in 500 µM spermine (spe), spermidinine (spd), and putrescine (put). Curves are fitted by Boltzmann functions with electrical distances of 2.64 for spe, 2.21 for spd, 1.69 for put, and 1.12 for Mg2+. [From Lopatin et al. (622). Copyright 1994 Macmillan Magazines Ltd.]

At the single-channel level, activation at hyperpolarized levels is correlated with a change from a lower to a higher conductance substate eventually to the fully open state and a prolongation of the open time: 10 ms around E_K and 100 ms at 60 mV negative to the E_rev (569, 652, 756, 990).

Activation or unblocking from Mg²⁺ or polyamines depends on [K⁺]_o, [K⁺]_i, and the time spent in the depolarized state. In K⁺-free medium, no I_K1 current can be recorded (see references in Ref. 123); the channel seems to remain blocked. The process has been called "K⁺ activation" (123, 155).

Also, intracellular K⁺ interferes with activation: the higher [K⁺]_i, the faster the current rise during activation (735). The K⁺ gradient or E_K furthermore determines the position of the apparent activation curve on the voltage axis (167).

At hyperpolarized levels, the current after being "activated" frequently undergoes a secondary decrease or inactivation, which is due to block by external ions such as Na⁺, Mg²⁺, and Ca²⁺ (86, 386). At the single-channel level, the inactivation corresponds to a fall in open probability (831).