Na+ channels as sites of action of the cardioactive agent DPI 201-106 with agonist and antagonist enantiomers.

This paper shows the interaction of the cardiotonic agent 4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy]-1H-indole- 2-carbonitrile (DPI 201-106) and its optic enantiomers R-DPI (205-429) and S-DPI (205-430) with the Na+ channel of a variety of excitable cells. Voltage-clamp experiments show that DPI 201-106 acts on neuroblastoma cells and rat cardiac cells. S-DPI (205-430) increases the peak Na+ current, slows down the kinetics of Na+ channel inactivation, and is cardiotonic on heart cells. Conversely, R-DPI (205-429) reduces the peak Na+ current and blocks Na+ channel activity and cardiac contractions. Binding experiments using radioactively labeled toxins indicate that DPI 201-106 and its enantiomers do not interact with sites already identified for tetrodotoxin or sea anemone and scorpion toxins. DPI 201-106 and its enantiomers inhibit binding of a 3H-labeled batrachotoxin derivative, [3H]batrachotoxinin A 20-alpha-benzoate, to brain membranes. The dissociation constant of the complex formed between the Na+ channel and both R-DPI and S-DPI is Kd congruent to 100 nM. 22Na+ uptake experiments using different cell types have shown that R and S enantiomers of DPI 201-106 are active on the different Na+ channel subtypes with similar IC50 values. These results are discussed in relation with the cardiotonic properties of DPI 201-106 that are not accompanied by cardiotoxic effects.     

Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C.

Cardiac rH1 Na+ channel alpha subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line expressing cardiac Na+ channels (SNa-rH1) was isolated. Mean Na+ currents of 2.2 +/- 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per micron2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 microM) and had voltage-dependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 microM) decreased this current approximately 33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8- to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 microM) or Ca(2+)-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microinjected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effect of OAG. Single-channel recordings from SNa-rH1 cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation of neuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 microM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.     

Mesoridazine: an open-channel blocker of human ether-a-go-go-related gene K+ channel

Mesoridazine, a phenothiazine antipsychotic agent, prolongs the QT interval of the cardiac electrocardiogram and is associated with Torsade de pointes-type arrhythmias. In this study, we examined the effects of mesoridazine on human ether-a-go-go-related gene (HERG) K+ currents. HERG channels were stably expressed in human embryonic kidney 293 cells and studied using standard whole-cell patch-clamp technique (37 degrees C). Mesoridazine blocked HERG currents in a concentration-dependent manner (IC50 550 nM at 0 mV); block increased significantly over the voltage range where HERG activates and saturated at voltages eliciting maximal HERG channel activation. Tonic block of HERG current by mesoridazine (1.8 microM) was minimal (< 2-4%). The rate of the onset of HERG channel block was rapid and dose dependent (tau = 54 +/- 7 ms at 0 mV and 1.8 microM mesoridazine), but not significantly affected by test potentials ranging from -30 to +30 mV. The V1/2 for steady-state activation was shifted from -31.2 +/- 1.0 to -39.2 +/- 0.5 mV (P < 0.01). The apparent rate of HERG channel deactivation was significantly reduced (fast tau = 153 +/- 8 vs. 102 +/- 6 ms at -50 mV, P < 0.01; slow tau = 1113 +/- 63 vs. 508 +/- 27 ms, P < 0.01). The inactivation kinetics and voltage dependence of steady-state inactivation of the HERG channel were not significantly altered by mesoridazine. These findings demonstrate that mesoridazine is a potent and rapid open-channel blocker of HERG channels. This block would explain the QT prolongation seen clinically at therapeutic concentrations (0.3-3.6 microM).     

Diphenylhydantoin blocks cardiac calcium channels and binds to the dihydropyridine receptor

Diphenylhydantoin was studied for its effects on Ca currents in single isolated guinea pig ventricular cells. The whole-cell patch-clamp technique was used, and Ca currents were studied after suppressing Na and K currents. At low frequencies (0.1 Hz) and negative holding potentials (-50 mV), diphenylhydantoin produced a concentration-dependent decrease in Ca currents without any significant change in the current-voltage relations. Half blocking effect occurred at 2 X 10(-4) M. The effects of diphenylhydantoin on Ca currents were dependent upon the holding potential. Inactivation curves for Ca currents were shifted to more negative potentials by the drug. The recovery of Ca currents from inactivation was prolonged by diphenylhydantoin, and the repriming of the current displayed an additional component, attributed to slow release of the drug from the channels. The voltage-dependent block was attributed to preferred binding by the inactivated channel state. Diphenylhydantoin also blocked specific [3H]-nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of [3H]-nitrendipine binding by diphenylhydantoin was competitive. Diphenylhydantoin also blocks cardiac Na channels in a voltage-dependent manner. We suggest that diphenylhydantoin binding sites exist on both Ca and Na channels.     

Heterogeneity of sodium current in atrial vs epicardial ventricular myocytes of adult guinea pig hearts

The different sodium channel currents (I(Na)) were reported in myocardium, neuron, and skeletal muscles. To study whether I(Na) is homogeneous within the heart, we applied whole-cell voltage clamp technique to evaluate fast voltage-gated I(Na) in atrial and ventricular myocytes isolated from guinea pig heart. It was found that the density of inward I(Na) was 50% greater at -35 mV in atrial (-42.6+/-2.9 pA/pF) than in ventricular (-27.5+/-1.8 pA/pF, P<0.01) myocytes. The half activation and inactivation voltages (V(0.5)) of I(Na) in atrial myocytes were shifted 4.5+/-0.2 and 9.6+/-0.3 mV negative to those of ventricular myocytes. Time constants for I(Na) activation (tau(m)) and inactivation (tau(h)) were twice as rapid in atrial as in ventricular myocytes. The tau(m) and tau(h) were 0.34+/-0.03 and 1.36+/-0.07 ms for atrial myocytes, and 0.69+/-0.05 and 3.27+/-0.23 ms for ventricular myocytes, respectively. Recovery of I(Na) from inactivation was slower in atrial than in ventricular myocytes, whereas the development of resting state inactivation was more rapid in atrial (tau=67.5+/-4.3 ms) than in ventricular (152.8+/-7.5 ms, P<0.01) myocytes. The results reveal marked heterogeneity of I(Na) in the density and biophysical properties in atrial and ventricular myocytes, and the study suggests the potential possibility of tissue specific cardiac sodium channel isoforms.     

Effects of lidocaine on single cardiac sodium channels

Lidocaine block of single cardiac sodium channels was studied in cell free inside-out patches of ventricular cells isolated from guinea-pig hearts. When applied to the inner surface of the membrane lidocaine depressed Na channel currents by decreasing the probability P of the channels to open measured from the peaks of the averaged currents. In parallel to the decrease in P the relative number of empty sweeps (nulls) was increased. Half maximum block of the activity of single Na channels was observed at 2.9 microM. Lidocaine affected the gating behaviour of Na channels by shortening of the mean open time tau 0 from 0.44 +/- 0.17 (control) to 0.19 +/- 0.13 (5 microM lidocaine, holding potential-120 mV, test potential -60 mV). Five micromolar lidocaine completely suppressed burst-like openings of Na channels and abolished the slow decaying phase of the averaged currents. A shift from - 120 towards - 160 mV exerted relief from the effect of both P and tau 0.     

A comparison of calcium currents in rat and guinea pig single ventricular cells

The slow inward calcium currents were compared in rat and guinea pig heart using enzymatically dissociated, single ventricular cells. A single electrode voltage clamp was used, in which current and voltage were sampled separately using a time-sharing method. Spatial homogeneity of membrane potential during peak slow inward calcium current was assessed by measuring the potential with two microelectrodes 50 micron apart; the potentials were within 3 mV of each other. Peak current-voltage relations for slow inward calcium currents were similar for the two species, but the individual currents showed a faster time course of inactivation and a slower time course of recovery from inactivation for rat, compared with guinea pig. The potassium current blockers 4-aminopyridine and tetraethylammonium chloride did not produce significant effects on the net membrane currents recorded at the holding potentials (-50 to -40 mV) used in this study. The underlying mechanism for the inactivation of the slow inward calcium currents was explored using a double pulse procedure. In both rat and guinea pig heart cells prepulses to very positive potentials were associated with a partial restoration of the slow inward calcium current in the following test pulse. In addition, internal ethylene glycol-bis N,N,N',N'-tetraacetic acid or substitution of barium for calcium slowed the rate of inactivation of the slow inward calcium current in rat heart cells. Calcium activation of nonspecific currents was thought less likely to have produced these results due to the lack of effect of depolarizing prepulses on hyperpolarizing test pulses. A calcium-dependent component of inactivation may be responsible for the differences observed in both the inactivation and the recovery time courses of the slow inward calcium current in these species.     

Diltiazem inhibits hKv1.5 and Kv4.3 currents at therapeutic concentrations

OBJECTIVE: In the present study we examined the effects of diltiazem, an L-type Ca(2+) channel blocker widely used for the control of the ventricular rate in patients with supraventricular arrhythmias, on hKv1.5 and Kv4.3 channels that generate the cardiac ultrarapid delayed rectifier (I(Kur)) and the 4-aminopyridine sensitive transient outward (I(to)) K(+) currents, respectively. METHODS: hKv1.5 and Kv4.3 channels were stably and transiently expressed in mouse fibroblast and Chinese hamster ovary cells, respectively. Currents were recorded using the whole-cell patch clamp. RESULTS: Diltiazem (0.01 nM-500 muM) blocked hKv1.5 channels, in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC(50)=4.8+/-1.5 nM and 42.3+/-3.6 muM). Diltiazem delayed the initial phase of the tail current decline and shifted the midpoint of the activation (Vh=-16.5+/-2.1 mV vs -20.4+/-2.6 mV, P<0.001) and inactivation (Vh=-22.4+/-0.7 mV vs. -28.2+/-1.9 mV, P<0.001) curves to more negative potentials. The analysis of the development of the diltiazem-induced block yielded apparent association (k) and dissociation (P) rate constants of (1.6+/-0.2) x 10(6) M(-1)s(-1) and 46.8+/-4.8 s(-1), respectively. Diltiazem (0.1 nM-100 muM) also blocked Kv4.3 channels in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC(50)=62.6+/-11.1 nM and 109.9+/-12.8 muM). Diltiazem decreased the peak current and, at concentrations > or =0.1 microM, accelerated the inactivation time course. The apparent association and dissociation rate constants resulted (1.7+/-0.2) x 10(6) M(-1)s(-1) and 258.6+/-38.1 s(-1), respectively. Diltiazem, 10 nM, shifted to more negative potentials the voltage-dependence of Kv4.3 channel inactivation (Vh=-33.1+/-2.3 mV vs -38.2+/-3.5 mV, n=6, Plt;0.05) the blockade increasing at potentials at which the amount of inactivated channels increased. CONCLUSION: The results demonstrated for the first time that diltiazem, at therapeutic concentrations, decreased hKv1.5 and Kv4.3 currents by binding to the open and the inactivated state of the channels.     

Characterization of the sodium current in single human atrial myocytes.

Patch-clamp recording techniques have permitted measurement of the fast Na+ current (INa) in isolated cardiac cells from a number of species in recent years. However, there is still only very little information concerning human cardiac INa. The purpose of this study was to describe the kinetics of INa in normal-appearing, Ca(2+)-tolerant, enzymatically isolated human atrial myocytes using whole-cell voltage-clamp techniques. Atrial specimens were obtained from 46 patients undergoing open heart surgery. Cs+ was substituted for K+ in both pipette and external solutions and F- was added to the former. The reversal potential of the rapid inward current varied approximately 57 mV at 17 +/- 1 degrees C with a 10-fold change in [Na+]o, and the current was completely blocked by 100 microM tetrodotoxin, findings typical of the fast cardiac Na+ current. The tetrodotoxin dose-response curve was best fitted by an equation describing binding to high- and low-affinity sites. INa was activated at a voltage threshold of -70 to -60 mV, and peak inward current was obtained at approximately -30 mV (holding potential, -140 mV). The inactivation time course was voltage dependent and was fitted best by the sum of two exponentials. The relation between voltage and steady-state availability (h infinity) was sigmoidal with the half-inactivation at -95.8 +/- 0.9 mV and a slope factor of 5.3 +/- 0.1 mV (n = 46), and we did not observe a significant difference with disease and age. The overlap of the h infinity and activation curves suggested the presence of a Na+ "window" current. Recovery from inactivation also was voltage dependent and best fitted by a model describing the sum of two exponentials. Recovery occurred after an initial delay at potentials positive to -140 mV, suggesting that inactivation of human atrial INa is a multistate process. We conclude that INa of normal-appearing, Ca(2+)-tolerant human atrial myocytes is similar to that of other mammalian cardiac cells with the possible exception of having two tetrodotoxin binding sites.     

Independent modulation of L-type Ca2+ channel in guinea pig ventricular cells by nitrendipine and isoproterenol

Dihydropyridine (DHP) Ca2+ channel blockers decrease L-type Ca2+ channel current (I(CaL)) by enhancing steady-state inactivation, whereas beta-adrenergic stimulation increases I(CaL) with small changes in the kinetics. We studied the effects of DHP Ca2+ channel blockers on cardiac I(CaL) augmented by beta-adrenergic stimulation. We recorded I(CaL) as Ba2+ currents (I(Ba)) from guinea pig ventricular myocytes using the whole-cell patch clamp technique. and compared the effects of nitrendipine (NIT) in the absence and presence of isoproterenol (1 microM, ISO) or forskolin (10 microM, FSK). Maximal I(Ba) elicited from a holding potential of -80 mV were diminished to 69.4+/-13.5% (mean and SE, n=5) of control by NIT (100 nM) and the diminished I(Ba) were increased to 180.3+/-23.2% of control by ISO in the presence of NIT, which was similar to the enhancement seen in the absence of NIT. NIT shifted the V(1/2) of the I(Ba) inactivation curve from -34.6+/-1.9 mV (n=5) to -48.7+/-1.2 mV, enhancing I(Ba) decay with shortening T(1/2) at -10 mV from 164.6+/-24.2 ms (n=7) to 105.4+/-15.2 ms. ISO elicited a small additional shift in the V(1/2) of I(Ba) inactivation in the same direction. ISO and FSK each slowed I(Ba) decay in the absence of NIT, but not in its presence. Thus, beta-adrenergic agonists increase and DHP Ca2+ channel blockers decrease the amplitude of cardiac I(CaL) independently and the kinetics of I(CaL) is determined mainly by the latter when these drugs coexist.     

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes.     

Functional expression of an inactivating potassium channel cloned from human heart.

Recently a putative K+ channel with homology to the Shaker family of potassium channels has been cloned from human ventricular myocardium. However, proof that the cDNA encodes a K+ channel requires appropriate translation and expression of a functional ion-selective channel. Therefore, expression of this putative human K+ channel DNA was attempted by cytoplasmic injections of in vitro transcribed cRNA into Xenopus laevis oocytes and screening by two-electrode voltage-clamp methods. This resulted in expression of voltage-gated channels that rapidly inactivated (time constant of inactivation, 47.6 +/- 3.6 msec; 0 mV; n = 10) and were at least 50 times more selective for K+ than Na+ (Na+/K+ permeability ratio of 0.02). The channels showed voltage-dependent activation (half-maximal voltage, -34 +/- 0.7 mV; n = 5), and 50% of the channels were inactivated within 2 seconds when the membrane potential was clamped near -60 mV (half-maximal voltage, -62 +/- 7 mV; n = 10). The expressed protein resulted in a K+ current that had many properties similar to the 4-aminopyridine-sensitive calcium-insensitive component of the cardiac transient outward current that is observed in native cardiac myocytes and thus may serve as one molecular substrate for this current.     

Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels.

Functional expression of the rat brain alpha 1A Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alpha 1A Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alpha 1A current was partially blocked (approximately 23%) by omega-agatoxin IVA (200 nM) and substantially blocked by omega-conotoxin MVIIC (5 microM blocked approximately 70%). Bay K 8644 (10 microM) or omega-conotoxin GVIA (1 microM) had no significant effect on the alpha 1A current. Coexpression with rat brain Ca channel beta subunits increased the alpha 1A whole-cell current and shifted the current-voltage relation to more negative values. While the beta 1b and beta 3 subunits caused a significant acceleration of the alpha 1A inactivation kinetics, the beta 2a subunit dramatically slowed the inactivation of the alpha 1A current to that seen typically for P-type Ca currents. In situ localization with antisense deoxyoligonucleotide and RNA probes showed that alpha 1A was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alpha 1A expression was detected in Purkinje cells with some labeling also in granule cells. Overall, the results show that alpha 1A channels are widely expressed and share some properties with both Q- and P-type channels.     

Evidence for voltage-dependent block of cardiac sodium channels by tetrodotoxin

The effects of tetrodotoxin (TTX) on cardiac sodium channels in guinea-pig ventricular muscle were investigated. Membrane potential was controlled using a single sucrose gap voltage clamp method, and the maximum upstroke velocity of the ventricular action potential (Vmax) was used as an indicator of drug-free sodium channels. Reduction of Vmax by TTX was found to be both voltage- and time-dependent, similar to the effects of many local anesthetic drugs, with the exception that TTX concentrations high enough to produce significant use-dependent block (e.g. 2 microM), also produced significant tonic block, even at potentials negative to -85 mV. The mechanism underlying use-dependent block was determined by defining the time course of block development at potentials between -40 and +20 mV, and the time course of recovery at -85 mV. In 2 microM TTX, the time course of block development at +20 mV contained two phases, a fast phase (tau less than 3 ms) having a mean amplitude of 8.1 +/- 3.2% of control Vmax, and a slow phase (tau = 429 +/- 43 ms) having an amplitude of 35 +/- 2% of control Vmax (n = 5). Recovery from use-dependent block at -85 mV occurred with a time constant of 324 +/- 58 ms (n = 5). The effects of TTX could be well-described by a modulated receptor model with an estimated 12 mV drug-induced shift of inactivation, and state-dependent dissociation constants of 10, 4 and 0.3 microM for rested, activated and inactivated channels. These same drug rate constants could also be used to adequately simulate the reported effects of TTX on plateau sodium currents in a variant model with slow inactivation kinetics.     

Kv1.4通道电流与大鼠心室肌细胞瞬间外向钾电流特性的比较

克隆的大鼠外向钾通道Ｋｖ１．４亚型表达于２９３细胞（ＲＣＫ４）。用膜片钳全细胞钳制法系统比较该克隆的大鼠瞬间外向钾电流（Ｉｔｏ）和天然大鼠心室肌细胞Ｉｔｏ的特点和动力学特性。两种通道电流形态相似,呈“Ａ”型电流,在＋４０ｍＶ时电流失活时间常数τ依次为３６．６±２ｍｓ和４１．０±２ｍｓ（Ｐ＞０．０５）。Ｋｖ１．４通道电流激活曲线用二相Ｂｏｌｔｚｍａｎｎ方程拟合,一相半数最大激活电位（Ｖ１／２,１）为－２１．０±３．９ｍＶ、二相半数最大激活电位（Ｖ１／２,２）为２７．０±３．９ｍＶ;天然大鼠心室肌细胞Ｉｔｏ激活曲线用单相Ｂｏｌｔｚｍａｎｎ方程拟合,半数最大激活电位为１０．８±１．１ｍＶ（Ｐ＜０．０５,ｖｓＫｖ１．４通道电流的Ｖ１／２,１）。ＲＣＫ４细胞通道电流半数最大灭活电位（Ｖ１／２）为－４９．８±１．８ｍＶ,斜率因子（ｋ）为３．８±０．２７;天然大鼠心室肌细胞Ｉｔｏ的Ｖ１／２为－３１．６±１．７ｍＶ,ｋ为５．４±０．２１。灭活后再激活的恢复时间比较,Ｋｖ１．４通道电流明显长于天然大鼠心室肌细胞Ｉｔｏ,分别为１．８９±０．２ｓ和３９．２±１．６ｍｓ（Ｐ＜０．０５）。研究表明克隆的大鼠Ｋｖ１．４通道电流与天然大?     

Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+ current

The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (I(K1)). In addition to differences in current density, atrial and ventricular I(K1) have differences in outward current profiles and in extracellular potassium ([K+]o) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular I(K1) in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than -30 mV (I approximately 0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, I(K1) rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, I(K1) did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1+/-0.1 pS in 15 cells, which corresponded with I(K1) in sheep atria (9.9+/-0.1 pS in 32 cells). Outward Kir2.1 currents were increased in 10 mmol/L [K+]o, whereas Kir2.3 currents did not increase. Correspondingly, guinea pig (but not sheep) atrial I(K1) showed an increase in outward currents in 10 mmol/L [K+]o. Although the ventricles of both species expressed Kir2.1 and Kir2.3, outward I(K1) currents rectified completely and increased in high [K+]o-displaying Kir2.1-like properties. Likewise, outward current properties of heterologously expressed Kir2.1-Kir2.3 complexes in normal and 10 mmol/L [K+]o were similar to Kir2.1 but not Kir2.3. Thus, unique properties of individual Kir2.x isoforms, as well as heteromeric Kir2.x complexes, determine regional and species differences of I(K1) in the heart.     

Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride.

Na+/Ca2+ exchange is inhibited in both guinea pig cardiac membrane vesicles and papillary muscles in a concentration-dependent fashion by several analogs of the pyrazine diuretic amiloride. Structure/activity studies based on transport measurements in vesicles prepared from guinea pig left ventricle indicate that hydrophobic substitutions at the terminal nitrogen atom of the guanidinium moiety of amiloride improved the inhibitory potency almost 100-fold over that of the parent compound. 3',4'- Dichlorobenzamil ( DCB ) is one of the most active inhibitors (IC50 = 17 microM). In electrically stimulated papillary muscles isolated from guinea pig heart, 10-40 microM DCB decreases contractile force. At 100 microM inhibitor, diastolic tension is significantly increased. The positive inotropic responses to veratridine and ouabain are inhibited by 20 and 40 microM DCB . Since the responses to these interventions were a consequence of increased intracellular Na+ concentration, these data indicate that DCB is an inhibitor of Na+-dependent Ca2+ influx in the intact tissue. Interpretation of mechanical responses elicited by paired pulses suggests that 40 microM but not 100 microM DCB decreases release of Ca2+ from the sarcoplasmic reticulum. The mechanical data obtained with concentrations of DCB that inhibited Na+/Ca2+ exchange in vesicles suggest that a significant amount of Ca2+ can enter the cardiac cell via Na+/Ca2+ exchange under normal conditions and that this transport system may be an important source of Ca2+ supplying the sarcoplasmic reticulum in guinea pig heart. Moreover, these amiloride analogs function as potent inhibitors of the positive inotropic effect caused by increased intracellular Na+ concentration.     

Molecular cloning of a putative tetrodotoxin-resistant rat heart Na+ channel isoform.

Voltage-gated Na+ channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na+ channels are blocked by 1-10 microM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.