Anthony Varghese

Kinetic analysis of open‐ and closed‐state inactivation transitions in human Kv4.2 A‐type potassium channels

1 We studied the gating kinetics of Kv4.2 channels, the molecular substrate of neuronal somatodendritic A‐type currents. For this purpose wild‐type and mutant channels were transiently expressed in the human embryonic kidney (HEK) 293 cell line and currents were measured in the whole‐cell patch‐clamp configuration. 2 Kv4.2 channels inactivated from pre‐open closed state(s) with a mean time constant of 959 ms at ‐50 mV. This closed‐state inactivation was not affected by a deletion of the Kv4.2 N‐terminus (Δ2‐40). 3 Kv4.2 currents at +40 mV inactivated with triple‐exponential kinetics. A fast component (τ= 11 ms) accounted for 73 %, an intermediate component (τ= 50 ms) for 23 % and a slow component (τ= 668 ms) for 4 % of the total decay. 4 Both the fast and the intermediate components of inactivation were slowed by a deletion of the Kv4.2 N‐terminus (τ= 35 and 111 ms) and accounted for 33 and 56 %, respectively, of the total decay. The slow component was moderately accelerated by the truncation (τ= 346 ms) and accounted for 11 % of the total Kv4.2 current inactivation. 5 Recovery from open‐state inactivation and recovery from closed‐state inactivation occurred with similar kinetics in a strongly voltage‐dependent manner. Neither recovery reaction was affected by the N‐terminal truncation. 6 Kv4.2 Δ2‐40 channels displayed slowed deactivation kinetics, suggesting that the N‐terminal truncation leads to a stabilization of the open state. 7 Simulations with an allosteric model of inactivation, supported by the experimental data, suggested that, in response to membrane depolarization, Kv4.2 channels accumulate in the closed‐inactivated state(s), from which they directly recover, bypassing the open state.

Gating currents associated with intramembrane charge displacement in HERG potassium channels

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K+ channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K+ current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K+ channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K+ channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

Simulating cardiac sinus and atrial network dynamics on the Connection Machine

Abstract Computational methods for simulating biophysically detailed, large-scale models of mammalian cardiac sinus and atrial networks on the massively parallel Connection Machine CM-2, and techniques for visualization of simulation data, are presented. Individual cells are modeled using the formulations of Noble et al. Models incorporate properties of voltage-dependent membrane currents, ion pumps and exchangers, and internal calcium sequestering and release mechanisms. Network models are used to investigate factors determining the site of generation and direction of propagation of the pacemaker potential. Models of the isolated sinus node are used to show that very few gap junction channels are required to support frequency entrainment. When cell membrane properties in the isolated sinus node models are modified to reproduce regional differences in oscillation properties, as described by the data of Kodama and Boyett, an excitatory wave is generated in the node periphery which propagates towards the node center. This agrees with activation patterns measured in the isolated sinus node by Kirchoff. When the model sinus node is surrounded by a region of atrial cells, the site of pacemaker potential generation is shifted away from the periphery towards the node center. This is in agreement with activation patterns measured by Kirchoff in the intact sinus node of the rabbit heart, and demonstrates the importance of sinus node boundary conditions on shaping the site of generation and direction of propagation of the pacemaker potential.

Effects of premature stimulation on HERG K+ channels

1 The unusual kinetics of human ether‐à‐go‐go‐related gene (HERG) K+ channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K+ channels, transfected in Chinese hamster ovary cells, at 37 °C. 2 HERG K+ channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). 3 HERG K+ channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90% repolarization (APD90) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD90– 20 ms) and atrial (APD90– 30 ms) action potentials. 4 The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. 5 A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre‐open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. 6 These properties of HERG K+ channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria.

Generation and Propagation of Ectopic Beats Induced by Spatially Localized Na--K Pump Inhibition in Atrial Network Models

A biophysically detailed two-dimensional network model of the cardiac atrium has been implemented on the Thinking Machines massively parallel CM-5 supercomputer. The model is used to study the effects of spatially localized inhibition of the Na–K pump. Na overloading produced by pump inhibition can induce spontaneous, propagating ectopic beats within the network. At a cell-to-cell coupling value yielding a realistic plane wave conduction velocity of 0.6 m s-1, pump inhibition in roughly 1000 cells can induce propagating ectopic beats in a 512 x 512 lattice of cells.

Role of Store-Operated Calcium Channels and Calcium Sensitization in Normoxic Contraction of the Ductus Arteriosus

Background— At birth, the increase in oxygen causes contraction of the ductus arteriosus, thus diverting blood flow to the lungs. Although this contraction is modulated by substances such as endothelin and dilator prostaglandins, normoxic contraction is an intrinsic property of ductus smooth muscle. Normoxic inhibition of potassium channels causes membrane depolarization and calcium entry through L-type calcium channels. However, the studies reported here show that after inhibition of this pathway there is still substantial normoxic contraction, indicating the involvement of additional mechanisms. Methods and Results— Using ductus ring experiments, calcium imaging, reverse-transcription polymerase chain reaction, Western blot, and cellular electrophysiology, we find that this depolarization-independent contraction is caused by release of calcium from the IP3-sensitive store in the sarcoplasmic reticulum, by subsequent calcium entry through store-operated channels, and by increased calcium sensitization of actin-myosin filaments, involving Rho-kinase. Conclusions— Much of the normoxic contraction of the ductus arteriosus at birth is related to calcium entry through store-operated channels, encoded by the transient receptor potential superfamily of genes, and to increased calcium sensitization. A clearer understanding of the mechanisms involved in normoxic contraction of the ductus will permit the development of better therapy to close the patent ductus arteriosus, which constitutes ≈10% of all congenital heart disease and is especially common in premature infants.

Not All hERG Pore Domain Mutations Have a Severe Phenotype: G584S Has an Inactivation Gating Defect with Mild Phenotype Compared to G572S, Which Has a Dominant Negative Trafficking Defect and a Severe Phenotype

Introduction: Mutations in the pore domain of the human ether‐a‐go‐go‐related gene (hERG) potassium channel are associated with higher risk of sudden death. However, in many kindreds clinical presentation is variable, making it hard to predict risk. We hypothesized that in vitro phenotyping of the intrinsic severity of individual mutations can assist with risk stratification.

Generation and propagation of normal and abnormal pacemaker activity in network models of cardiac sinus node and atrium

Effects of cell-to-cell coupling conductance on dynamics of sinus node cells are examined. Cell models are biophysically detailed, and are based on the kinetic equations developed by Noble et al. [Neuronal and Cellular Oscillators, edited by J. W. Jacklet, Marcel Deckker, New York (1989).] Resistively coupled cell pairs show five regimes of behavior as a function of coupling conductance: (1) independent oscillation for Gc < 1 pS; (2) primarily quasiperiodic oscillation for 1 ⩽ Gc < 116 pS; (3) windows of periodic behavior which undergo period doubling bifurcation to chaos for 116 ⩽ Gc < 212 pS; (4) frequency entrainment for Gc ⩾ 212 pS; (5) waveform entrainment for Gc ⩾ 50 nS. Thus, only 4–5 gap junction channels are required for frequency entrainment. This is shown to also be the case for large networks of sinus cells modeled on the Connection Machine CM-5. A biophysically detailed two-dimensional network model of the cardiac atrium has also been implemented on the CM-5 supercomputer. The model is used to study effects of spatially localized inhibition of the Na-K pump. Na overloading produced by pump inhibition can induce spontaneous, propagating ectopic beats within the network. At a cell-to-cell coupling value yielding a realistic plane wave conduction velocity of 60cms−1 pump inhibition in small regions of the network containing as few as 1000 cells can induce propagating ectopic beats.

Low potassium dextran lung preservation solution reduces reactive oxygen species production

BACKGROUND Low potassium dextran lung preservation solution has reduced primary graft failure in animal and human studies. Though the mechanism of reducing primary graft failure is unknown, low potassium dextran differs most significantly from solutions such as Euro-Collins (EC) and University of Wisconsin in its potassium concentration. The aim of this study was to investigate the impact that potassium concentration in lung preservation solutions had on pulmonary arterial smooth muscle cell depolarization and production of reactive oxygen species. METHODS Using isolated pulmonary artery smooth muscle cells from Sprague-Dawley rats, the patch-clamp technique was used to measure resting cellular membrane potential and whole cell potassium current. Measurements were recorded at base line and after exposure to low potassium dextran, EC, and University of Wisconsin solutions. Pulmonary arteries from rats were isolated from the main pulmonary artery to the fourth segmental branch. Arteries were placed into vials containing low potassium dextran, EC, low potassium EC, Celsior, and University of Wisconsin solutions with reactive oxygen species measured by lucigenin-enhanced chemiluminescence. RESULTS Pulmonary artery smooth muscle cell membrane potentials had a significant depolarization when placed in the University of Wisconsin or EC solutions, with changes probably related to inhibition of voltage-gated potassium channels. Low potassium dextran solution did not alter the membrane potential. Production of reactive oxygen species as measured by chemiluminescence was significantly higher when pulmonary arteries were exposed to University of Wisconsin or EC solutions (51,289 +/- 5,615 and 35,702 +/- 4353 counts/0.1 minute, respectively) compared with low potassium dextran, Celsior, and low potassium EC (12,537 +/- 3623, 13,717 +/- 3,844 and 15,187 +/- 3,792 counts/0.1 minute, respectively). CONCLUSIONS Preservation solutions with high potassium concentration are clearly able to depolarize the pulmonary artery smooth muscle cells and increase pulmonary artery reactive oxygen species production. Low potassium preservations solutions may limit reactive oxygen species production and thus reduce the incidence of primary graft failure in lung transplantation.

Dynamics of the calcium subsystem in cardiac Purkinje fibers

Abstract A minimal model of the dynamics of internal calcium concentration of the mammalian cardiac Purkinje fiber is examined in order to identify the cause of certain arrhythmias of the heart. The effect of inhibition of the sodium/potassium pump is modeled by an elevated value of internal sodium cocentration. Effects of pump inhibition are examined at different clamp voltages. Such conditions mimic those which have been examined experimentally and which are known to cause oscillatory calcium release [W.J. Lederer, PhD thesis, Yale University, New Haven, CT (1976), 168 pp.; R.S. Kass, W.J. Lederer, R.W. Tsien and R. Weingart, J. Physiol. (London) 281 (1978) 187–208]. System dynamics are investigated using numerical continuation methods. Results of these analyses predict the existence of stable periodic oscillations of internal calcium over a range of voltage-clamp values. The emergence of these oscillations depends on the intracellular sodium concentration.