Strains of [PSI(+)] are distinguished by their efficiencies of prion-mediated conformational conversion.

Yeast prions are protein-based genetic elements that produce phenotypes through self-perpetuating changes in protein conformation. For the prion [PSI(+)] this protein is Sup35, which is comprised of a prion-determining region (NM) fused to a translational termination region. [PSI(+)] strains (variants) with different heritable translational termination defects (weak or strong) can exist in the same genetic background. [PSI(+)] variants are reminiscent of mammalian prion strains, which can be passaged in the same mouse strain yet have different disease latencies and brain pathologies. We found that [PSI(+)] variants contain different ratios of Sup35 in the prion and non-prion state that correlate with different translation termination efficiencies. Indeed, the partially purified prion form of Sup35 from a strong [PSI(+)] variant converted purified NM much more efficiently than that of several weak variants. However, this difference was lost in a second round of conversion in vitro. Thus, [PSI(+)] variants result from differences in the efficiency of prion-mediated conversion, and the maintenance of [PSI(+)] variants involves more than nucleated conformational conversion (templating) to NM alone.

Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast.

Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.

Nucleated conformational conversion and the replication of conformational information by a prion determinant.

Prion proteins can serve as genetic elements by adopting distinct physical and functional states that are self-perpetuating and heritable. The critical region of one prion protein, Sup35, is initially unstructured in solution and then forms self-seeded amyloid fibers. We examined in vitro the mechanism by which this state is attained and replicated. Structurally fluid oligomeric complexes appear to be crucial intermediates in de novo amyloid nucleus formation. Rapid assembly ensues when these complexes conformationally convert upon association with nuclei. This model for replicating protein-based genetic information, nucleated conformational conversion, may be applicable to other protein assembly processes.

Flecainide and the electrophysiologic matrix: the effects of flecainide acetate on the determinants of cardiac excitability in sheep Purkinje fibers.

Flecainide was associated with excess mortality distributed virtually equally throughout the period of the Cardiac Arrhythmia Suppression Trial, suggesting the intersection of two events, drug effect and perhaps ischemia. Flecainide's effect on active properties has been studied extensively, but nothing is known of its effects on passive properties or on the balance among active and passive cellular properties that determines cardiac excitability. The multiple microelectrode method of intracellular current application and transmembrane voltage recording was used in sheep Purkinje fibers to determines strength- and charge-duration as well as constant current-voltage relationships and to estimate active properties, liminal length, and cable properties at a normal [K+]o and in a setting of hyperkalemia analogous to that of ischemia. A computer tracked in time the alterations in the active and passive properties relevant to excitability. Flecainide slightly decreased excitability at a normal [K+]o, primarily by depressing the sodium system with some contributory effect of passive properties. At high [K+]o, flecainide caused a frequency-dependent decrease in excitability and conduction, the latter best interpreted as a failure of the fiber to attain the liminal length requirements to produce a local action potential due primarily to an effect on sodium conductance. Together, the observations suggest that the action potential is the local phenomenon and that the propagated event is the sequential fulfillment of liminal length requirements. The data were interpreted in terms of the electrophysiologic matrix first proposed in detail in this Journal, which indicated that the electrophysiologic universe moved as a system in response to the drug and a change in [K+]o, the presumed antiarrhythmic and proarrhythmic electrophysiologic matrices for flecainide were quite similar, and the matrical configuration shared characteristics with the matrices of other drugs with known proarrhythmic potential.

Effects of ethmozin on excitability in sheep Purkinje fibers: the balance among active and passive cellular properties which comprise the electrophysiologic matrix.

Ethmozin is a phenothiazine derivative that is effective against supraventricular and ventricular arrhythmias. Studies to date have examined ethmozin's effects on active cellular properties and automaticity, but nothing is known of its effects on passive properties or on the interrelationships among the several active and passive properties that are of particular relevance to cardiac excitability. The hypothesis tested in this study was that ethmozin, in concentrations equivalent to clinically effective antiarrhythmic levels, would simultaneously affect passive and active cellular properties so as to produce a net decrease in cardiac excitability. The multiple micro-electrode method of intracellular constant current application and trans-membrane voltage recording was used in sheep Purkinje fibers to determine strength-duration and constant current-voltage relationships as well as cable properties. A rapid, on-line computerized data analysis system tracked in time the alterations in the active and passive properties relevant to excitability. Ethmozin, at concentrations of 1.1 and 2.2 microM (0.5 and 1.0 mg/l), decreased cardiac excitability as manifested by an increase in the current required to attain threshold and/or an upward shift in strength- and charge-duration relationships, by depressing the sodium system (decreased maximal rate of rise of phase 0 of the action potential, voltage threshold and overshoot), by decreasing slope resistance and altering nonlinearities of the current-voltage relationships in the subthreshold potential range, by decreasing membrane resistance and by affecting other properties dependent on membrane resistance which would depress excitability. The data for ethmozin and other antiarrhythmic drugs are interpreted in terms of the recently proposed electrophysiologic matrix which we believe has important advantages over traditional hierarchical classifications.

Effects of quinidine sulfate on the balance among active and passive cellular properties that comprise the electrophysiologic matrix and determine excitability in sheep Purkinje fibers.

Quinidine is the most commonly used drug for the chronic treatment of ventricular arrhythmias, but it may be arrhythmogenic. Much information exists concerning quinidine's effects on active properties in cardiac tissues, but virtually nothing is known of its effects on passive properties. We studied the effects of quinidine, in a clinically relevant concentration, on the balance among active and passive cellular properties that comprise the electrophysiologic matrix that determines cardiac excitability. The multiple microelectrode method of intracellular-current application and transmembrane voltage recording was used in sheep Purkinje fibers to determine strength-duration and constant current-voltage relations as well as cable properties. A rapid, on-line computerized data analysis system tracked in time the alterations in the active and passive properties relevant to excitability. In normal fibers at [K+]o = 5.4 mM, quinidine increased cardiac excitability as manifested by a decrease in the current required to attain threshold and/or a downward shift in strength- and charge-duration relations by altering passive properties despite a depressed sodium system and a slowed conduction velocity. During washout, excitability and passive properties remained altered despite a return of descriptive action potential parameters such as the resting potential, the maximum rate of rise of phase 0, overshoot, and the action potential duration to or nearly to normal. At [K+]o = 8.0 mM, quinidine could either increase or decrease excitability; net excitability depends on the balance between altered passive properties and the depressed sodium system. The results explain, in part, the antiarrhythmic actions and arrhythmogenic potential of quinidine. The data for quinidine and other antiarrhythmic drugs are interpreted in terms of the electrophysiologic matrix, which we believe has important advantages over traditional hierarchical classifications.

[Effect of ethmozin on the electrophysiologic parameters of Purkinje cells in the sheep heart. The frequency-dependent blockade of sodium channels]. / Vliianie étmozina na élektrofiziologicheskie parametry kletok Purkin'e serdtsa ovtsy. Chastotno-zavisimaia blokada natrievykh kanalov.

The effect of ethmozin (E) on action potential (AP) duration (ADP90, APD50), AP upstroke velocity (Vmax) and cable properties (Ri, Rm, Rin, Cm, lambda m, tau m) was studied. Vmax was significantly reduced by E (0.2 to 5 mkM). Duration of AP plateau (APD50) was more sensitive to E than APD90. Thus, application of 0.5 mkM E resulted in 30% decrease of APD90 and 60% of APD50. This suggested that E not only inhibits INa, what is reflected by Vmax reduction, but also can affect other currents involved in plateau phase. Cable properties remained unchanged in the wide concentration range of E. Blockade of Vmax by E was use-dependent. Uptake rates of ethmozin by sodium channel for different stimulation frequencies were estimated and kinetic binding and unbinding constants (k = 40322 M-1 ms-1; 1 = 7.17 X 10(-5) ms-1) with unguarded receptor were calculated using novel drug-channel interaction model (Starmer and Grant, 1985).

Electrophysiologic actions and interactions between lysophosphatidylcholine and lidocaine in the nonsteady state: the match between multiphasic arrhythmogenic mechanisms and multiple drug effects in cardiac Purkinje fibers.

The effects and interactions between an arrhythmogenic intervention and an antiarrhythmic drug on active and passive cellular properties relevant to excitability were studied with multiple microelectrode methods and rapid online data analysis in cardiac Purkinje fibers. The arrhythmogenic intervention was superfusion with lysophosphatidylcholine (LPC), a metabolite that accumulates in the ischemic myocardium; and the antiarrhythmic drug was lidocaine. LPC (10-20 microM) initially increased excitability as manifested by a decreased threshold current and, when tested, by a downward shift in nonnormalized strength- and charge-duration curves. Normalized strength- and charge-duration curves suggested altered passive properties to be primarily responsible for increased excitability. Cable analysis showed LPC to increase significantly input resistance, membrane resistance, time constant and length constant; current-voltage relationships showed LPC to decrease chord and slope conductances over the subthreshold range. Lidocaine (4 micrograms/ml) decreased excitability both by depressing the sodium system and by directly countering the effects of LPC on conductance and related properties. LPC subsequently decreased excitability by depressing the sodium system and, in this phase, the further depressant effect of lidocaine on the sodium system predominated. Lidocaine could normalize action potentials that were prolonged or had two stable steady states after LPC, at times retarded LPC-induced inexcitability and could render the tissue inexcitable to intracellular point stimulation but not to extracellular stimulation. Interactions between the arrhythmogenic and pharmacologic interventions affected net excitability by altering the matrix of active and passive cellular properties in time. The results are relevant to the development of a rational matrical approach to understanding drug action and treating arrhythmias.

Effects of encainide on the determinants of cardiac excitability in sheep Purkinje fibers.

Encainide is a benzanilide derivative that is effective against ventricular arrhythmias but, at times, may be arrhythmogenic. The microelectrode technique of intracellular current application and transmembrane voltage recording was used to study the effects of encainide, in a concentration equivalent to a clinically effective antiarrhythmic plasma level, on the determinants of cardiac excitability in sheep Purkinje fibers. A rapid, on-line computerized data analysis system was used to track the alterations in the active and passive membrane properties relevant to excitability in time. Cardiac excitability was defined experimentally in terms of the current required to attain threshold and/or the shift in strength- or change-duration curves. Encainide produced multiphasic changes in cardiac excitability, the final state of excitability depended on the balance between altered passive and active membrane properties. Encainide could enhance excitability by increasing membrane and slope resistance without altering the nonlinearities of the current-voltage relationship despite an actual depression of the sodium system. Encainide could decrease excitability by 1) depressing the sodium system; 2) decreasing membrane resistance without altering the nonlinearities of the subthreshold current-voltage relationship; 3) altering the nonlinearities of the current-voltage relationship; and 4) by a combination of these actions. During washout, excitability could remain altered despite a return of descriptive parameters such as the maximal rate of rise of phase 0, overshoot and action potential duration to normal. The study demonstrated the importance of time-related changes of an antiarrhythmic drug at a constant concentration. The results explain, in part, the antiarrhythmic actions and arrhythmogenic potential of encainide.