Arthur M. Brown

Receptor-effector coupling by G proteins.

The primary structure of G proteins as deduced from purified proteins and cloned subunits is presented. When known, their functions are discussed, as are recent data on direct regulation of ionic channels by G proteins. Experiments on expression of alpha subunits, either in bacteria or by in vitro translation of mRNA synthesized from cDNA are presented as tools for definitive assignment of function to a given G protein. The dynamics of G protein-mediated signal transduction are discussed. Key points include the existence of two superimposed regulatory cycles in which upon activation by GTP, G proteins dissociate into alpha and beta gamma and their dissociated alpha subunits hydrolyze GTP. The action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP is emphasized, as is the role of subunit dissociation, without which receptors could not act as catalysts. To facilitate the reading of this review, we have presented the various subtopics of this rapidly expanding field in sections 1-1X, each of which is organized as a self-contained sub-chapter that can be read independently of the others.

Early outward current in rat single ventricular cells.

Voltage clamp experiments were conducted using single ventricular myocytes which had been dissociated enzymatically from adult rat hearts in order to examine further the membrane currents which contribute to the unusual plateau of the rat action potential. Membrane currents were recorded, using a single microelectrode (switching) voltage clamp circuit. From holding potentials near the resting potential (−80 to −90 mV), depolarizing clamp steps above −20 mV elicited an early outward current which overlapped in time with the slow inward current and displayed time-dependent inactivation. This is the first demonstration of a transient potassium current in an isolated ventricular myocyte. The early outward potential was voltage-inactivated at holding potentials of −50 to −40 mV and was blocked by 4-aminopyridine. The current was not dependent on Ca± or Ic and was blocked by Ba,. Double pulse experiments revealed that the time course for the recovery of the early outward current at −80 mV was rapid, and had a ± of 25 msec. The possible functional significance of this current is discussed.

Effects of unilateral cardiac sympathetic denervation on the ventricular fibrillation threshold

A train of gated stimuli scanning the entire vulnerable period was delivered to the right anterior or left posterior ventricular surface to study the ventricular fibrillation threshold in anesthetized and vagotomized dogs. Heart rate was held constant by atrial pacing. Measurements were obtained in control conditions and after surgical removal of one stellate ganglion. To avoid the shortcomings associated with an irreversible procedure like stellectomy, control fibrillation threshold measurements were also alternated with determinations during reversible blockade by cooling of one stellate ganglion. The results were similar with both techniques. In nine animals, ablation or cooling of the left stellate ganglion increased ventricular fibrillation threshold by 72 +/- 35 (mean +/- standard deviation) percent compared with control values (P less than 0.001). By contrast, in 11 animals, ablation or cooling of the right stellate ganglion lowered the threshold by 48 +/- 14 percent compared with control values (P less than 0.001). Electrode location did not influence the results. The observed changes depended solely upon unilateral removal of cardiac sympathetic activity and were not demonstrable if such activity was low. These results suggest that right and left cardiac sympathetic nerves may have different and specific effects on cardiac excitability. They also contribute to the understanding of the pathogenesis of the long Q-T syndrome (characterized by episodes of ventricular fibrillation associated with increased sympathetic activity) and increase the rationale for left stellectomy as the specific treatment for this illness. Left stellectomy, by raising the ventricular fibrillation threshold, may also represent an alternative measure in patients at high risk of sudden death from ventricular arrhythmias resistant to medical therapy.

Alteration and restoration of K+ channel function by deletions at the N- and C-termini

Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.

The action potential and comparative pharmacology of stem cell-derived human cardiomyocytes

INTRODUCTION The cardiac action potential (CAP) of stem cell-derived human cardiomyocytes (SC-hCMs) is potentially the most powerful preclinical biomarker for cardiac safety and efficacy in humans. Our experiments tested this hypothesis by examining the CAP and relevant pharmacology of these cells. METHODS The electrophysiological and pharmacological profiles of SC-hCMs were compared to rabbit and canine Purkinje fibers (PFs). Ventricular SC-hCMs provided the dominant electrophysiological phenotype (approximately 82%) in a population of ventricular, atrial and nodal cardiomyocytes (CMs). The effects of reference compounds were measured in SC-hCMs using perforated patch, current clamp recording. Selective inhibitors of I(Kr), I(Ks), I(Ca,L), and I(Na), and norepinephrine (NE), were tested on SC-hCM action potentials (APs). RESULTS AP prolongation was observed upon exposure to hERG channel blockers (terfenadine, quinidine, cisapride, sotalol, E-4031 and verapamil), with significantly shorter latencies than in PF assays. For the torsadogenic compounds, terfenadine and quinidine, SC-hCM AP prolongation occurred at significantly lower concentrations than in canine or rabbit PF APs. Moreover, the I(Ks) blocker chromanol 293B prolonged APs from SC-hCMs, whereas both rabbit and canine PF assays are insensitive to I(Ks) blockers in the absence of adrenergic preconditioning. Early afterdepolarizations (EADs) were induced by 100 nM E-4031 and 100 nM cisapride in the SC-hCM assay, but not in the canine or rabbit PF assay. Selective inhibition of I(Na) and I(Ca,L) slowed V(max) and shortened AP duration, respectively. NE prolonged the AP duration of SC-hCMs. DISCUSSION The CAP of SC-hCMs has been validated as a powerful preclinical biomarker for cardiac safety and efficacy. In addition to its human nature, the SC-hCM AP assay removes diffusion delays, reduces test compound consumption, demonstrates an overall pharmacological sensitivity that is greater than conventional rabbit or canine PF assays, and accurately predicts cardiac risk of known torsadogenic compounds.

MICE Models: Superior to the HERG Model in Predicting Torsade de Pointes

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.

Effects of unilateral stellate ganglion blockade on the arrhythmias associated with coronary occlusion

In anesthetized dogs the circumflex and/or the anterior descending coronary artery were briefly occluded (10 to 90 seconds) and ectopic beats occurring during the occlusion or for 60 seconds following release were counted. Control occlusions were alternated with occlusions performed during complete, reversible, unilateral blockade of either the right or the left stellate ganglion. This was achieved with thermodes through which coolant was circulated. In this way the shortcomings associated with stellectomy, which is irreversible, are avoided. Blockade of the right stellate ganglion increased the number of ectopic beats associated with coronary occlusion. The occurrence of episodes of ventricular tachycardia and fibrillation was also greater. By contrast, blockade of the left stellate ganglion reduced or abolished occlusion-induced arrhythmias. These effects are independent of changes in heart rate or vegal activity; they depend solely upon unilateral alteration in sympathetic tone, and are not demonstrable when such tone is low. We suggest that the right and left cardiac sympathetic nerves have a different influence upon cardiac excitability.

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 damp 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 μm 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.

Fast and slow gating of sodium channels encoded by a single mRNA

We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein.

Calcium current‐dependent and voltage‐dependent inactivation of calcium channels in Helix aspersa

1. Inactivation of the Ca channels has been examined in isolated nerve cell bodies of Helix aspersa using the suction pipette method for voltage clamp and internal perfusion.