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Background: Bifenthrin is usually considered a Type I pyrethroid, because it lacks an α-CN group present in Type II pyrethroids, but some previous studies suggest a mixed Type I/II mode-of-action. Results are presented for bifenthrin in a rat developmental neurotoxicity (DNT) study along with effects on Na currents in human VGSC subtypes. Molecular modeling comparisons were also made for bifenthrin and other pyrethroids., Results: In a rat DNT study, bifenthrin produced tremors and clonic convulsions in dams and pups and slightly reduced acoustic startle response amplitude, and increased Tmax, at PND20 in females. Similar blood levels of bifenthrin were measured in dams and pups at each dose level i.e. no concentration in pups. In human VGSC experiments, using the Nav1.8 subtype, bifenthrin's effects on inactivation were slight, as for Type II pyrethroids, but without large prolongation of the tail current (deactivation) seen with Type II. Molecular modeling of bifenthrin indicates that the o-Me group may occupy a similar space to the α-CN group of cypermethrin and fenpropathrin., Conclusion: In a DNT study and on human Nav1.8 tail currents bifenthrin showed Type I and II effects, similar to some published studies. Overall, bifenthrin acts as a mixed Type I/II pyrethroid. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3 V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration-approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3 V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.

The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity.

Voltage-gated Ca2+ channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity.

Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.

Methadone, a synthetic opioid for treatment of chronic pain and withdrawal from opioid dependence, has been linked to QT prolongation, potentially fatal torsades de pointes, and sudden cardiac death. Concomitant use of diazepam or other benzodiazepines in methadone maintenance treatment can increase the risk of sudden death. Therefore, we determined the effects of methadone and diazepam singly and in combination on cardiac action potentials (APs) and on the major ion channels responsible for cardiac repolarization. Using patch clamp recording in human stem cell-derived cardiomyocytes and stably transfected mammalian cells, we found that methadone produced concentration-dependent AP prolongation and ion channel block at low micromolar concentrations: hERG (IC50 = 1.7 μM), hNav1.5 (11.2 μM tonic block; 5.5 μM phasic block), and hCav1.2 (26.7 μM tonic block; 7.7 μM phasic block). Methadone was less potent in hKv4.3/hKChIP2.2 (IC50 = 39.0 μM) and hKvLQT1/hminK (53.3 μM). In contrast, diazepam blocked channels only at much higher concentrations and had no effect on AP duration at 1 μM. However, coadministration of 1-μM diazepam with methadone caused a statistically significant increase in AP duration and a 4-fold attenuation of hNav1.5 block (IC50 values were 44.2 μM and 26.6 μM, respectively, for tonic and phasic block), with no significant effect on methadone-induced block of hERG, hCav1.2, hKv4.3/hKChIP2.2, and hKvLQT1/hminK channels. Thus, although diazepam alone does not prolong the QT interval, the relief of methadone-induced Na channel block may leave hERG K channel block uncompensated, thereby increasing cardiac risk.

Introduction: There remains an unmet need for safe and effective antiarrhythmic drugs, especially for the treatment of atrial fibrillation. Vanoxerine is a drug that is free of adverse cardiac events in normal volunteers, yet is a potent blocker of the hERG (hK(v)11.1) cardiac potassium channel. Consequently,we hypothesized that vanoxerine might also be a potent blocker of cardiac calcium (Ca) and sodium (Na) currents, and would not affect transmural dispersion of repolarization., Methods: The whole cell patch clamp technique was used to measure currents from cloned ion channels overexpressed in stable cell lines and single ventricular myocytes. We measured intracellular action potentials from canine ventricular wedges and Purkinje fibers using sharp microelectrode technique., Results: We found that vanoxerine was a potent hK(v)11.1 blocker, and at submicromolar concentrations, it blocked Ca and Na currents in a strongly frequency-dependent manner. In the canine ventricular wedge preparation vanoxerine did not significantly affect transmural action potential waveforms, QT interval or transmural dispersion of repolarization., Conclusions: Vanoxerine (1) is a potent blocker of cardiac hERG, Na and Ca channels; (2) block is strongly frequency-dependent especially for Na and Ca channels; and (3) transmural dispersion of ventricular repolarization is unaffected. The multichannel block and repolarization uniformity resemble the effects of amiodarone, the exemplar atrial fibrillation drug. Vanoxerine is a completely different chemical and has none of amiodarone's toxic effects. Vanoxerine has characteristics of a potentially effective and safe antiarrhythmic.

Ion channels represent the third largest class of targets in drug discovery after G-protein coupled receptors and kinases. In spite of this ranking, ion channels continue to be under exploited as drug targets compared with the other two groups for several reasons. First, with 400 ion channel genes and an even greater number of functional channels due to mixing and matching of individual subunits, a systematic collection of ion channel-expressing cell lines for drug discovery and safety screening has not been available. Second, the lack of high-throughput functional assays for ion channels has limited their use as drug targets. Now that automated electrophysiology has come of age and provided the technology to assay ion channels at medium to high throughput, we have addressed the need for a library of ion channel cell lines by constructing the Ion Channel Panel (ChanTest Corp., Cleveland, OH). From 400 ion channel genes, a collection of 82 of the most relevant human ion channels for drug discovery, safety, and human disease has been assembled.Each channel has been stably overexpressed in human embryonic kidney 293 or Chinese hamster ovary cells. Cell lines have been selected and validated on automated electrophysiology systems to facilitate cost-effective screening for safe and selective compounds at earlier stages in the drug development process. The screening and validation processes as well as the relative advantages of different screening platforms are discussed.

The United States Food and Drug Administration (FDA) uses alfuzosin as an example of a drug having QT risk in humans that was not detected in nonclinical studies. FDA approval required a thorough clinical QT study (TCQS) that was weakly positive at high doses. The FDA has used the clinical/nonclinical discordance as a basis for mandatory TCQS, and this requirement has serious consequences for drug development. For this reason, we re-examined whether nonclinical signals of QT risk for alfuzosin were truly absent. Alfuzosin significantly prolonged action potential duration (APD)(60) in rabbit Purkinje fibers (p < 0.05) and QT in isolated rabbit hearts (p < 0.05) at the clinically relevant concentration of 300 nM. In man, the QT interval corrected with Fridericia's formula increased 7.7 ms, which exceeds the 5.0-ms threshold for a positive TCQS. Effects on hK(v)11.1, hK(v)4.3, and hK(v)7.1/hKCNE1 potassium currents and calcium current were not involved. At 300 nM, approximately 30x C(max), alfuzosin significantly increased whole-cell peak sodium (hNa(v)1.5) current (p < 0.05), increased the probability of late hNa(v)1.5 single-channel openings, and significantly shortened the slow time constant for recovery from inactivation. Alfuzosin also increased hNa(v)1.5 burst duration and number of openings per burst between 2- and 3-fold. Alfuzosin is a rare example of a non-antiarrhythmic drug that delays cardiac repolarization not by blocking hK(v)11.1 potassium current, but by increasing sodium current. Nonclinical studies clearly show that alfuzosin increases plateau potential and prolongs APD and QT, consistent with QT prolongation in man. The results challenge the FDA grounds for the absolute primacy of TCQS based on the claim of a false-negative, nonclinical study on alfuzosin.

Antimonial agents are a mainstay for the treatment of leishmaniasis, a group of protozoal diseases that includes visceral leishmaniasis, or Kala Azar. Chemotherapy with trivalent potassium antimony tartrate (PAT) and, more importantly, pentavalent antimony-carbohydrate complexes, such as sodium stibogluconate (SSG), has been reported to prolong the QT interval and produce life-threatening arrhythmias. PAT is chemically related to As2O3, which alters cardiac excitability by inhibition of human ether a-go-go related gene (hERG) trafficking and an increase of cardiac calcium currents. In this study, we report that PAT does not block hERG currents on short-term exposure but reduces current density on long-term exposure (IC50, 11.8 microM) and inhibits hERG maturation on Western blots (IC50, 62 microM). Therapeutic concentrations of 0.3 microM PAT increase cardiac calcium currents from -4.8 +/- 0.7 to -7.3 +/- 0.5 pA/pF at 10 mV. In marked contrast, pentavalent SSG, the drug of choice for the treatment of leishmaniasis, did not affect hERG/IKr or any other cardiac potassium current at therapeutic concentrations. However, both cardiac sodium and calcium currents were significantly increased on long-term exposure to 30 microM SSG in isolated guinea pig ventricular myocytes. We propose that the increase in calcium currents from -3.2 +/- 0.3 to -5.1 +/- 0.3 pA/pF at 10 mV prolongs APD90 from 464 +/- 35 to 892 +/- 64 ms. Our data suggest that conversion of Sb(V) into active Sb(III) in patients produces a common mode of action for antimonial drugs, which define a novel compound class that increases cardiac risk not by a reduction of hERG/IKr currents but-for the first time-by an increase in cardiac calcium currents.

Introduction: Direct block of I(Kr) by non-antiarrhythmic drugs (NARDs) is a major cause of QT prolongation and torsades de pointes (TdP), and has made the hERG potassium channel a major target of drug safety programs in cardiotoxicity. Block of hERG currents is not the only way that drugs can adversely impact the repolarizing current I(Kr), however. We have shown recently that two drugs in clinical use do not block hERG but produce long QT syndrome (LQTS) and TdP by inhibiting trafficking of hERG to the cell surface. To address the need for an inexpensive, rapid, and comprehensive assay to predict both types of hERG risk early in the drug development process, we have developed a novel antibody-based chemiluminescent assay called HERG-Lite., Methods: HERG-Lite monitors the expression of hERG at the cell surface in two different stable mammalian cell lines. One cell line acts as a biosensor for drugs that inhibit hERG trafficking, while the other predicts hERG blockers based on their ability to act as pharmacological chaperones. In this study, we have validated the HERG-Lite assay using a panel of 100 drugs: 50 hERG blockers and 50 nonblockers., Results: HERG-Lite correctly predicted hERG risk for all 100 test compounds with no false positives or negatives. All 50 hERG blockers were detected as drugs with hERG risk in the HERG-Lite assay, and fell into two classes: B (for blocker) and C (for complex; block and trafficking inhibition)., Discussion: HERG-Lite is the most comprehensive assay available for predicting drug-induced hERG risk. It accurately predicts both channel blockers and trafficking inhibitors in a rapid, cost-effective manner and is a valuable non-clinical assay for drug safety testing.

The diamidine pentamidine is used to treat leishmaniasis, trypanosomiasis, and Pneumocystis carinii pneumonia. Treatment may be accompanied by prolongation of the QT interval of the electrocardiogram and torsades de pointes tachycardias. Up to now, it has been thought that therapeutic compounds causing QT prolongation are associated with direct block of the cardiac potassium channel human ether a-go-go-related gene (hERG), which encodes the alpha subunit of cardiac I(Kr) currents. We show that pentamidine has no acute effects on currents produced by hERG, KvLQT1/mink, Kv4.3, or SCNA5. Cardiac calcium currents and the guinea pig cardiac action potential were also not affected. After overnight exposure, however, pentamidine reduced hERG currents and inhibited trafficking and maturation of hERG with IC(50) values of 5 to 8 microM similar to therapeutic concentrations. Surface expression determined in a chemiluminescence assay was reduced on exposure to 10, 30, and 100 microM pentamidine by about 30, 40, and 70%, respectively. These effects were specific for hERG since expression of hKv1.5, KvLQT1/minK, and Kv4.3 was not altered. In isolated guinea pig ventricular myocytes, 10 microM pentamidine prolonged action potential duration APD(90) from 374.3 +/- 57.1 to 893.9 +/- 86.2 ms on overnight incubation. I(Kr) tail current density was reduced from 0.61 +/- 0.09 to 0.39 +/- 0.04 pA/pF. We conclude that pentamidine prolongs the cardiac action potential by block of hERG trafficking and reduction of the number of functional hERG channels at the cell surface. We propose that pentamidine, like arsenic trioxide, produces QT prolongation and torsades de pointes in patients by inhibition of hERG trafficking.

Objective: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study., Methods: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology., Results: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation., Conclusion: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

Arsenic trioxide (As(2)O(3)) produces dramatic remissions in patients with relapsed or refractory acute promyelocytic leukemia. Its clinical use is burdened by QT prolongation, torsade de pointes, and sudden cardiac death. In the present study, we analyzed the molecular mechanisms leading to As(2)O(3)-induced abnormalities of cardiac electrophysiology. Using biochemical and electrophysiological methods, we show that long-term exposure to As(2)O(3) increases cardiac calcium currents and reduces surface expression of the cardiac potassium channel human ether-a-go-go-related gene (HERG) at clinically relevant concentrations of 0.1 to 1.5 microM. In ventricular myocytes, As(2)O(3) increases action potential duration measured at 30 and 90% of repolarization. As(2)O(3) interferes with hERG trafficking by inhibition of hERG-chaperone complexes and increases calcium currents by a faster cellular process. We propose that an increase in cardiac calcium current and reduced trafficking of hERG channels to the cell surface cause QT prolongation and torsade de pointes in patients treated with As(2)O(3). Our results suggest that calcium-channel antagonists will be useful in normalizing QT prolongation during As(2)O(3) therapy. As(2)O(3) is the first example of a drug that produces hERG liability by inhibition of ion-channel trafficking. Other drugs that interfere with proteins in the processing pathway of cardiac ion channels may be proarrhythmic for similar reasons.

Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG potassium channels are blocked by prazosin, doxazosin, and terazosin. These data may provide a hypothetical molecular explanation for the apoptotic effect of quinazoline-derived alpha1-adrenoceptor antagonists.

To evaluate the effects of the iron chelator deferoxamine on the functional and structural manifestations of iron-induced cardiac dysfunction, we measured cardiac power, left ventricular systolic, and diastolic function as (dP/dt)max and (dP/dt)min, respectively, and left ventricular and septal wall thickness in isolated heart preparations derived from the Mongolian gerbil model of iron overload. We induced iron overload with weekly subcutaneous injections of iron dextran (800 mg/kg/wk); deferoxamine (DFO; 100 mg/kg) was administered twice daily by subcutaneous injection, 5 of 7 days each week; and control animals received weekly subcutaneous injections of dextran alone. Animals administered iron alone initially exhibited, at 5 weeks, increased cardiac power but by 12 to 20 weeks, cardiac power was severely diminished, with impairment of both systolic and diastolic function of the left ventricle and marked cardiac hypertrophy (P<.001 for all vs control animals). Administration of DFO with iron did not interfere with the initial augmentation of cardiac power at 5 weeks but prevented the subsequent deterioration in cardiac performance. After 12 to 20 weeks, gerbils given DFO with iron had mean values of cardiac power indistinguishable from those of control animals; both systolic and diastolic function were significantly enhanced not only in comparison with those of animals treated with iron alone but also with respect to controls. In addition, DFO prevented cardiac hypertrophy; mean ventricular and septal wall thickness in gerbils given DFO and iron were not significantly different from those in controls. In the gerbil model of iron overload, concurrent administration of DFO with iron prevents both the development of cardiac hypertrophy and the progressive deterioration in cardiac performance that are produced by chronic iron accumulation.

Cardiac disease with arrhythmia or heart failure is the leading cause of death in patients with thalassemia major and a major complication of other forms of iron overload. Current antiarrhythmic treatment does not appear to alter the clinical course. Using a gerbil model of iron-overload cardiomyopathy, we previously observed a reduction in the fast inward sodium current in isolated cardiomyocytes. Electrocardiograms (ECGs) in the same gerbil model indicate PR-interval prolongation, QRS-interval widening, and arrhythmias. We hypothesize that such changes in the ECG in this model are the result of abnormal action-potential conduction at the level of the whole heart. To test this hypothesis, we took ECGs and recorded action potentials using high-resolution optical mapping from the anterior surface of 9 iron-overloaded and 9 age-matched control ventricular-paced, Langendorff-perfused gerbil hearts. The iron-overloaded gerbils received weekly iron-dextran injections of 800 mg/kg for 14 to 18 weeks. ECGs showed QRS- and PR-interval prolongation in iron-treated gerbils compared with that in controls. In addition, atrioventricular block was observed in 2 of 6 iron-treated gerbils but not in controls. Conduction velocity was significantly slower in iron-treated gerbils than in controls. At normal pacing rates, abnormal activation patterns caused by stable regions of conduction block were observed in iron-overloaded gerbils (33%) but not in controls. Such abnormal impulse conduction may be a mechanism of increased arrhythmia vulnerability in iron-overload cardiomyopathy.

We investigated the time course of electrocardiographic (ECG) changes in the Mongolian gerbil model of iron overload and the effects of the iron chelator deferoxamine (DFO) on these changes. Iron overload was produced with weekly subcutaneous injections of low doses (200 mg/kg/wk) or high doses (800 mg/kg/wk) of iron-dextran. DFO was administered subcutaneously at a dose of 200 mg/kg/day to high-dose animals. Our results show that (1) survival of iron-overloaded gerbils is dose-dependent, with median survival times of 68 and 14 weeks for low- and high-dose animals, respectively; (2) both low and high doses produce prolongation of the PR interval and bradycardia in early stages and prolongation of the QT interval, premature ventricular contractions, variable degrees of atrioventricular block, changes in the ST segment, and T-wave inversion at later stages coinciding with the development of heart failure; (3) DFO prevented death during 20 weeks of high-dose iron-dextran; (4) DFO prevented ECG changes, although delayed prolongation of PR intervals and QRS complexes occurred; and (5) despite marked prolongation of survival and prevention of ECG changes, DFO had modest effects on total cardiac iron content. We speculate that DFO chelates a small iron pool located within the cytoplasm of iron-overloaded cardiomyocytes.

Iron-overload cardiomyopathy is the most common cause of death in patients with thalassemia major, yet the associated changes in cardiac function have not been quantified. We studied the effects of iron overload on cardiac function in Mongolian gerbils, a species that responds to iron overload in the same manner as human beings. We injected iron-dextran or dextran alone at low subcutaneous doses (200 mg/kg/wk) for 20 to 60 weeks and at high doses (800 mg/kg/wk) for 6 to 20 weeks. At shorter durations for either dose, the mean values of cardiac work, coronary flow, left ventricular (dP/dt)(max) and left ventricular (dP/dt)(min) in isolated perfused hearts were significantly greater than control values; at longer durations, these values were significantly less than control values. Echocardiography in intact animals showed eccentric cardiac hypertrophy, increased cardiac output, and normal exercise tolerance at shorter durations of dosage. At longer durations, concentric cardiac hypertrophy developed, and cardiac output and exercise capacity were impaired. The response to iron overload in Mongolian gerbils progresses from an initial state of high cardiac output to a subsequent state of low-output failure similar to the course of cardiomyopathy that has been inferred in patients with transfusional iron overload.

K(+) channel-associated protein/protein inhibitor of activated STAT (KChAP/PIAS3beta) is a potassium (K(+)) channel modulatory protein that boosts protein expression of a subset of K(+) channels and increases currents without affecting gating. Since increased K(+) efflux is an early event in apoptosis, we speculated that KChAP might induce apoptosis through its up-regulation of K(+) channel expression. KChAP belongs to the protein inhibitor of activated STAT family, members of which also interact with a variety of transcription factors including the proapoptotic protein, p53. Here we report that KChAP induces apoptosis in the prostate cancer cell line, LNCaP, which expresses both K(+) currents and wild-type p53. Infection with a recombinant adenovirus encoding KChAP (Ad/KChAP) increases K(+) efflux and reduces cell size as expected for an apoptotic volume decrease. The apoptosis inducer, staurosporine, increases endogenous KChAP levels, and LNCaP cells, 2 days after Ad/KChAP infection, show increased sensitivity to staurosporine. KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15. Consistent with activation of p53 as a transcription factor, p21 levels are increased in infected cells. Wild-type p53 is not essential for induction of apoptosis by KChAP, however, since KChAP also induces apoptosis in DU145 cells, a prostate cancer cell line with mutant p53. Consistent with its proapoptotic properties, KChAP prevents growth of DU145 and LNCaP tumor xenografts in nude mice, indicating that infection with Ad/KChAP might represent a novel method of cancer treatment.

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.

Background: Patients with transfusional iron overload may develop a life-limiting cardiomyopathy. The sensitivity of lipid-metabolizing enzymes to peroxidative injury, as well as the reported effects of arachidonic acid (AA) and metabolites on cardiac rhythm, led us to hypothesize that iron-overloaded cardiomyocytes display alterations in the release of AA and prostaglandins., Methods and Results: Neonatal rat ventricular myocytes (NRVMs) cultured for 72 hours in the presence of 80 microgram/mL ferric ammonium citrate displayed an increased rate of AA release, both under resting conditions and after stimulation with agonists such as [Sar(1)]Ang II. Although iron treatment did not affect overall incorporation of [(3)H]AA into NRVM phospholipids, it caused a 2-fold increase in the distribution of precursor in phosphatidylcholine species, with a proportional decrease in phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine. Increased release of AA in iron-overloaded NRVMs was reduced by the diacylglycerol lipase inhibitor RHC80267 but was largely insensitive to inhibitors of phospholipases A(2) and C. Iron-overloaded cardiomyocytes also displayed increased production of eicosanoids and induction of cyclooxygenase-2 after stimulation with interleukin-1alpha., Conclusions: Iron overload enhances AA release and incorporation of AA into phosphatidylcholine, as well as cyclooxygenase-2 induction and eicosanoid production, in NRVMS: The effects of AA and metabolites on cardiomyocyte rhythmicity suggest a causal connection between these signals and electromechanical alterations in iron-overload-induced cardiomyopathy.

Administration of the 5-hydroxytryptamine 3 receptor class of antiemetic agents has been associated with prolongation in the QRS, JT, and QT intervals of the ECG. To explore the mechanisms underlying these findings, we examined the effects of granisetron, ondansetron, dolasetron, and the active metabolite of dolasetron MDL 74,156 on the cloned human cardiac Na(+) channel hH1 and the human cardiac K(+) channel HERG and the slow delayed rectifier K(+) channel KvLQT1/minK. Using patch-clamp electrophysiology we found that all of the drugs blocked Na(+) channels in a frequency-dependent manner. At a frequency of 3 Hz, the IC(50) values for block of Na(+) current measured 2.6, 88.5, 38.0, and 8.5 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Block was relieved by strong hyperpolarizing potentials, suggesting a possible interaction with an inactivated channel state. Recovery from inactivation was impaired at -80 mV compared with -100 mV, and the fractional recovery was impaired by drug in a concentration-dependent manner. IC(50) values for block of the HERG cardiac K(+) channel measured 3.73, 0.81, 5.95, and 12.1 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Ondansetron (3 microM) also slowed decay of HERG tail currents. In contrast, none of these drugs (10 microM) produced greater than 30% block of the slow delayed rectifier K(+) channel KvLQT1/minK. We concluded that the antiemetic agents tested in this study block human cardiac Na(+) channels probably by interacting with the inactivated state. This may lead to clinically relevant Na(+) channel blockade, especially when high heart rates or depolarized/ischemic tissue is present. The submicromolar affinity of ondansetron for the HERG K(+) channel likely underlies the prolongation of cardiac repolarization reported for this drug.

The concept of chaperones for K(+) channels is new. Recently, we discovered a novel molecular chaperone, KChAP, which increased total Kv2.1 protein and functional channels in Xenopus oocytes through a transient interaction with the Kv2.1 amino terminus. Here we report that KChAP is a chaperone for Kv1.3 and Kv4.3. KChAP increased the amplitude of Kv1.3 and Kv4.3 currents without affecting kinetics or voltage dependence, but had no such effect on Kv1.1, 1.2, 1.4, 1.5, 1.6, and 3.1 or Kir2.2, HERG, or KvLQT1. Although KChAP belongs to a family of proteins that interact with transcription factors, upregulation of channel currents was not blocked by the transcription inhibitor actinomycin D. A 98-amino acid fragment of KChAP binds to the channel and is indistinguishable from KChAP in its enhancement of Kv4.3 current and protein levels. Using a KChAP antibody, we have coimmunoprecipitated KChAP with Kv2.1 and Kv4.3 from heart. We propose that KChAP is a chaperone for specific Kv channels and may have this function in cardiomyocytes where Kv4.3 produces the transient outward current, I(to).

Background: Patients with chronic iron overload may develop a cardiomyopathy manifested by ventricular arrhythmias and heart failure. We hypothesized that iron-loaded cardiomyocytes may have abnormal excitability., Methods and Results: We examined a new model of human iron overload, the Mongolian gerbil given repeated injections of iron dextran. In ventricular myocytes, we measured iron concentration and distribution, action potential, sodium and potassium currents, and sodium channel protein. We showed for the first time that (1) the iron content of gerbil ventricular cardiomyocytes was increased to amounts similar to those of patients with iron-induced cardiomyopathy; (2) the overshoot and duration of the cardiac action potential decreased; (3) sodium current was reduced, steady-state inactivation was enhanced, and single-channel currents were unchanged; and (4) transient outward potassium current was increased, but inwardly rectifying potassium current was unchanged. Neonatal rat cardiomyocytes incubated with iron for 1 to 3 days showed similar changes, and levels of cardiac sodium channel proteins were unchanged., Conclusions: Abnormal excitability and heterogeneous cardiac iron deposition may cause the arrhythmogenesis of human siderotic heart disease.

1. The Kvbeta subunits of voltage-gated K+ channels alter the functional expression and gating of non- or slowly inactivating Kvalpha1 subunits via two separate domains. To determine how Kvbeta subunits modulate a rapidly inactivating Kvalpha1 subunit, we did two-microelectrode voltage clamp experiments on human Kv1.4 voltage-gated K+ channels expressed heterologously in Xenopus oocytes. In addition we tested a slowly inactivating mutant of Kv1.4 lacking amino acids 2-146 of the N-terminal alpha-ball domain (Kv1. 4DeltaN2-146). Kv1.4 or Kv1.4DeltaN2-146 were co-expressed with either rat Kvbeta2 or human Kvbeta1.2. To separate domain effects, we also used a mutant of Kvbeta1.2 lacking the unique 79 amino acid N-terminal beta-ball domain (Kvbeta1-C). 2. For the mutant Kv1.4DeltaN2-146 we found that Kvbeta1-C or Kvbeta2 increased current amplitude without altering activation or inactivation. By contrast Kvbeta1.2 produced rapid inactivation and slowed deactivation due to block produced by the beta-ball. The beta-ball also increased the rate of C-type inactivation in 5 mM, but not 50 mM, external K+ consistent with an effect of blockade on K+ efflux. 3. For Kv1.4, Kvbeta1-C produced a voltage-independent increase in the rate of inactivation and shifted the inactivation curve to more hyperpolarized potentials, but had no effect on deactivation. Kvbeta1-C, Kvbeta2 and Kvbeta1.2 slowed recovery from inactivation similarly, thereby excluding involvement of the beta-ball. Kvbeta1.2 produced an additional more rapid, voltage-dependent component of inactivation, significantly reduced peak outward current and shifted steady-state inactivation towards hyperpolarized potentials. 4. Yeast two-hybrid studies showed that alpha-beta interaction was restricted to the N-terminus of Kv1.4 and the C-terminus of Kvbeta1. 2 or Kvbeta2. Direct interaction with the alpha-ball did not occur. Our interpretation is that Kvbeta1-C and Kvbeta2 enhanced N-type inactivation produced by the Kv1.4 alpha-ball allosterically. 5. We propose that Kvbeta1.2 has three effects on Kv1.4, the first two of which it shares with Kvbeta2. First, Kvbeta1-C and Kvbeta2 have a current-enhancing effect. Second, Kvbeta1-C and Kvbeta2 increase block by the alpha-ball allosterically. Third, the beta-ball of Kbeta1.2 directly blocks both Kv1.4 and Kv1.4DeltaN2-146. When both alpha- and beta-balls are present, competition for their respective binding sites slows the block produced by either ball.

Voltage-gated K+ (Kv) channels are important in the physiology of both excitable and nonexcitable cells. The diversity in Kv currents is reflected in multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes. Given the fundamental importance and diversity of Kv channels, surprisingly little is known regarding the cellular mechanisms regulating their synthesis, assembly, and metabolism. To begin to dissect these processes, we have used the yeast two-hybrid system to identify cytoplasmic regulatory molecules that interact with Kv channel proteins. Here we report the cloning of a novel gene encoding a Kv channel binding protein (KChAP, for K+ channel-associated protein), which modulates the expression of Kv2 channels in heterologous expression system assays. KChAP interacts with the N termini of Kvalpha2 subunits, as well as the N termini of Kvalpha1 and the C termini of Kvbeta subunits. Kv2.1 and KChAP were coimmunoprecipitated from in vitro translation reactions supporting a direct interaction between the two proteins. The amplitudes of Kv2. 1 and Kv2.2 currents are enhanced dramatically in Xenopus oocytes coexpressing KChAP, but channel kinetics and gating are unaffected. Although KChAP binds to Kv1.5, it has no effect on Kv1.5 currents. We suggest that KChAP may act as a novel type of chaperone protein to facilitate the cell surface expression of Kv2 channels.

1. The perforated-patch-clamp technique was used to identify an inwardly rectifying K+ current (IK(IR)) in cultured rat anterior pituitary cells highly enriched in corticotropes. IK(IR) was rapidly activating and highly selective for K+. The K+ conductance was approximately proportional to the square root of the extracellular K+ concentration. 2. IK(IR) was blocked in a voltage-dependent manner by external Ba2+ and Cs+, slightly attenuated by 5 mM 4-aminopyridine (15% inhibition) and insensitive to 10 mM tetraethylammonium, 2 mM Ca2+, 1 mM Cd2+ and 50 microM La3+. 3. In physiological saline, 100 microM Ba2+, which inhibits 86% of IK(IR) at the cell resting potential, depolarized cells by 6.1 +/- 0.7 mV from a mean resting potential of -59.6 +/- 0.8 mV. 4. Corticotropin releasing hormone (CRH), which activates adenylyl cyclase and stimulates adrenocorticotropic hormone (ACTH) secretion from corticotropes, inhibited IK(IR) by 25% and depolarized the cells by 10.2 +/- 1.0 mV. Dibutyryl cAMP ((Bu)2cAMP) mimicked these effects. 5. The membrane depolarization evoked by Ba2+ or CRH increased the cell firing frequency. Comparison of cells exhibiting a membrane potential of approximately -50 mV revealed that spike frequency in the presence of CRH (109 +/- 7 spikes (5 min)-1) was greater than in control (60 +/- 5 spikes (5 min)-1) or Ba(2+)-treated (77 +/- 15 spikes (5 min)-1) corticotropes. 6. The data suggest that IK(IR) contributes to maintenance of the resting membrane potential of rat corticotropes. Inhibition of IK(IR) plays a role in, but does not account for all of, the membrane depolarization and enhancement of firing frequency evoked by CRH.

Corticotropin-releasing hormone (CRH) stimulates ACTH secretion from anterior pituitary corticotropes, largely, but possibly not exclusively, via activation of the adenylyl cyclase cascade. CRH stimulates secretion by increasing Ca(2+) influx and by Ca(2+)-independent mechanisms. As Ca(2+) influx is largely regulated by membrane electrical properties, we review the effects of CRH on membrane excitability and changes in cytosolic Ca(2+). We also speculate on possible pathways for CRH modulation of exocytosis by Ca(2+) independent mechanisms.

CRH induces corticotrope membrane depolarization and facilitates action potential firing. The increase in electrical excitability causes large oscillatory increases in cytosolic Ca2+ levels. In this study on highly enriched populations of cultured rat corticotropes, inhibitors were used to determine the contribution of the Na+ channel and Ca2+ channel subtypes to membrane excitability and cytosolic Ca2+ levels. Tetrodotoxin, an inhibitor of the voltage-dependent Na+ channel, inhibited a rapid initial component of the action potential, but generally did not influence spontaneous or CRH-induced firing frequency. Tetrodotoxin also had no effect on spontaneous or CRH-induced cytosolic Ca2+ levels. The L-type Ca2+ channel inhibitor nifedipine abolished spontaneous and CRH-induced action potentials and cytosolic Ca2+ transients, but did not eliminate the CRH-induced membrane depolarization or completely restore cytosolic Ca2+ to basal levels. Inhibition of P-type Ca2+ channels with omega-agatoxin-IVA decreased action potential firing frequency and reduced the CRH-induced increase in cytosolic Ca2+. The combination of nifedipine and omega-agatoxin-IVA abolished the CRH-induced rise in Ca2+, but did not abolish the membrane depolarization. Thus, cytosolic Ca2+ is mainly increased by CRH-induced action potentials that are completely dependent on L-type Ca2+ channels and partially regulated by P-type Ca2+ channels. CRH-induced Ca2+ entry also occurs independently of action potentials and is due to P-type, and possibly L-type, Ca2+ channels activated by the CRH-induced membrane depolarization.

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTP gamma S) affects ATP-activated Ca(2+)-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mM K+ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na+ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mM) and Na+ (145 mM) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca2+:Ba2+:Mn2+:Sr2+: Na+:K+ = 68:30:26:10:3.5:1. Slope conductances (in pS) for permeant ions rank as follows: Ca2+:Sr2+: Na+:Mn2+:Ba2+ = 19:18:14:12:10. Unitary Ca2+ currents display a tendency to saturate with the Ca2+ concentration increase with apparent dissociation constant (Kd) of 10 mM. No block of Na+ permeation by extracellular Ca2+ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptor-operated or voltage-gated Ca(2+)-permeable channels on the other cell types.

The modulation of membrane excitability and cytosolic Ca2+ levels by corticotropin-releasing hormone (CRH), (Bu)2cAMP (dBcAMP), and forskolin was examined in enriched populations of cultured rat anterior pituitary corticotropes. CRH (2 or 20 nM), dBcAMP (1 and 5 mM), and forskolin (10 microM) caused a long lasting membrane depolarization accompanied by the onset of cell firing in quiescent cells or by increased firing frequency in spontaneously active cells. All three substances also increased cytosolic Ca2+ levels by increasing the frequency and amplitude of cytosolic Ca2+ transients. These results are consistent with a previous report on human corticotrope tumor cells demonstrating that CRH-induced action potentials lead to enhancement of Ca2+ uptake through voltage-dependent Ca2+ channels. Preincubation with (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), an inhibitor of cAMP-dependent protein kinase A, did not inhibit the CRH-induced depolarization, but attenuated the CRH-induced increase in action potential frequency. H-89 inhibited CRH-induced changes in cytosolic Ca2+ by 69% in spontaneously active cells and by 83% in quiescent cells. In contrast, H-89 completely abolished the effects of dBcAMP and forskolin on membrane potential and cytosolic Ca2+ levels. It is concluded that activation of protein kinase A mediates all of the response to dBcAMP and forskolin, but only a portion of the response to CRH. The portion of the response to CRH that is resistant to H-89 is mediated by a cAMP-independent mechanism.

In this study on highly enriched populations of cultured rat corticotropes, Ca2+ channel inhibitors were used to identify subtypes of the high threshold Ca2+ channel current under voltage clamp conditions. From a holding potential (-50 mV) that eliminated the low threshold T-type current, 52 +/- 4% of the total current in 10 mM Ba2+ was mediated by dihydropyridine-sensitive L-type Ca2+ channels. Blockade of this current was half-maximal at a nifedipine concentration of 187 nM. omega-Agatoxin-IVA (20 nM) maximally inhibited 28 +/- 3% of the total current. This high sensitivity to omega-agatoxin-IVA indicates that this noninactivating current is mediated by P-type Ca2+ channels. A very high threshold, noninactivating current (23 +/- 4% of the total Ba2+ current) remained after maximal inhibition of L- and P-type Ca2+ channels. This current was also resistant to toxins that inhibit N (omega-conotoxin-GVIA)- and Q (omega-conotoxin-MVIIC)-type Ca2+ channels. Because this current had slow activation kinetics and voltage dependence very different from those of the L- and P-type currents in these cells, it was probably mediated by a third unclassified Ca2+ channel subtype (or subtypes). It is concluded that the high threshold current in corticotropes is due to the presence of at least three different Ca2+ channel subtypes.

1. To elucidate the possible involvement of a G protein in ATP-evoked Ca(2+)-permeable channel activity, membrane currents of rat peritoneal macrophages were recorded using inside-out and cell-attached configurations of the patch clamp technique. 2. In inside-out experiments with a pipette solution containing 105 mM Ba2+, application of 100 microM GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches investigated (n = 69). The mean value of NPo (N, number of open channels; Po, channel open probability) was equal to 0.49 +/- 0.27 (mean +/- S.E.M.; n = 16). The delay in the activity development was 21 +/- 8 s (n = 18) with 200 microM ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtained with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S-evoked channels were identical to those of channels activated by extracellular application of ATP. The channels exhibited at least four conductance sublevels, the 4th one being the least frequent. With 105 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 and 17 pS. Extrapolated reversal potential (Er) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channels with similar characteristics could be induced by the extracellular application of fluoride in cell-attached experiments without any agonist in the pipette solution.(ABSTRACT TRUNCATED AT 250 WORDS)

1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4- concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3.5-4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.

Aluminium fluoride (AlF4-), a G protein activator, was used to study a possible role of G protein in the control of the pathways for Ca2+ influx through plasma membrane of human carcinoma A431 cells. Fluorimetric measurements with the Ca2+ indicator Indo-1 have shown that addition of fluoride induces an increase in concentration of cytosolic free calcium ([Ca2+]in) due to both release of Ca2+ from intracellular stores and Ca2+ influx from the extracellular medium. The cells stimulated by fluoride became unresponsive to subsequent addition of epidermal growth factor (EGF), histamine and bradykinin. The Ca2+ signal induced by fluoride as well as one induced by EGF was inhibited by the pretreatment of cells with protein kinase C activator, phorbol myristate acetate (PMA). The pretreatment of the cells with pertussis toxin produced no effect on EGF-induced calcium response. In contrast, the pretreatment with cholera toxin (CTX) increased the basal level of [Ca2+]in and abolished the effect of EGF. The effects of CTX could not be reproduced by treating the cells with forskolin or IBMX, agents known to elevate cAMP content in the cell. Patch clamp experiments have shown that fluoride increases the activity of Ca(2+)-permeable channels identical to those activated by EGF from the extracellular side of the membrane [Mozhayeva et al. (1991) J. Membr. Biol. 124, 113-126]. The results obtained suggest the involvement of GTP-binding protein in signal transduction from the EGF receptor to Ca(2+)-permeable channel of plasma membrane in A431 cells.

Single Ca(2+)-permeable channels were studied in membrane patches from A431 carcinoma cells. Amplitudes of channel openings fell into three major groups with mean unitary conductances of 1.3, 2.4 and 5.1 pS (105 mM Ca2+ in the pipette as charge carrier). All three groups of events were activated with epidermal growth factor (EGF) from the outside and by GTP non-hydrolyzable analogues from the inside of the patch membrane. As a rule, channel openings were uniform in amplitude in each individual patch but sometimes transitions between openings of different conductance levels were seen. It is concluded that the plasma membrane of A431 cells contains a single type of EGF- and GTP-dependent Ca(2+)-permeable channel (or channel complex) that can display, at least, three conductance levels.

The patch-clamp technique was used to study mechanisms of ATP-induced Ca2+ influx in rat peritoneal macrophages. The experiments on whole-cell and patch membranes have shown that extracellular ATP activates channels permeable to di- and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca2+, Sr2+, Mn2+, Ba2+ and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non-hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP-binding protein is involved in receptor-to-channel coupling.

Ca2(+)-permeable channels in human carcinoma A431 cells were studied using the patch clamp technique. We have found two types of Ca2(+)-permeable channels which are activated by inositol 1,4,5-trisphosphate (IP3) applied to the intracellular side of the plasma membrane. Unitary conductances of these channels are 3.7 and 13 pS (105 mM Ca2+ in recording pipette, 30-33 degrees C). From the extracellular side of the membrane the channels are activated by EGF. It is assumed that extracellular agonists open both channel types by stimulating the release of IP3 from the membrane.

Patch clamp experiments on human carcinoma A431 cells have revealed two types of Ca2(+)-permeable channels, the activity of which can be increased by the application of non-hydrolyzable analogues of GTP to the intracellular side of the membrane. With 105 mM Ca2+ in recording pipette at 30-33 degrees C their unitary conductances (in pS) are 1.3 (SG-channels) and 2.4 (G-channels). G- and, possibly, SG-channels are activated from the extracellular side of the membrane with epidermal growth factor (EGF). The data are consistent with the hypothesis that both channels are activated via guanine nucleotide binding (G) proteins.