Biomedical subjects

E Marban

Publications and source records attributed to E Marban.

At least 55 records · Page 3Linked to original sources

PubMed1996

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Allosteric Regulation↗

PubMed1996

The equine periodic paralysis Na+ channel mutation alters molecular transitions between the open and inactivated states.

1. The Na+ channel mutation associated with equine hyperkalaemic periodic paralysis (HPP) affects a highly conserved phenylalanine residue in an unexplored region of the alpha-subunit. This mutation was introduced into the rat skeletal muscle Na+ channel gene at the corresponding location (i.e. F1412L) for functional expression and characterization in Xenopus oocytes. 2. In comparison with wild-type (WT) channels, equine HPP channels showed clear evidence for disruption of inactivation: increased time-to-peak current, slowed rates of whole-cell current decay, significant increases in sustained current, rightward shifts in the steady-state inactivation curve by 9.5 mV, a 6-fold acceleration in the rate of recovery from inactivation at -80 mV, decreased number of blank single-channel sweeps, repetitive opening of single channels throughout depolarizing steps, increased open probability per sweep, and an increased mean open time. 3. The observed disruption of inactivation in HPP occurred without measurable changes in steady-state activation and first latency kinetics of channel opening. 4. Kinetic modelling demonstrates that the equine HPP phenotype can be simulated by altering the rate constants for transitions entering and leaving the inactivated states resulting from an energetic destabilization of the inactivated state. 5. These results suggest that the highly conserved cytoplasmic end of the third transmembrane segment (S3) in the fourth internal repeat domain (domain IV) plays a critical role in Na+ channel inactivation.

Animals↗

PubMed1996

Selective effects of oxygen free radicals on excitation-contraction coupling in ventricular muscle. Implications for the mechanism of stunned myocardium.

BACKGROUND: Oxygen free radicals (OFRs) have been implicated in the pathogenesis of myocardial stunning, but the precise mechanism by which OFRs foster stunning remains unclear. We investigated the pathophysiology of the contractile dysfunction that occurs after direct exposure of OFRs to cardiac muscle and compared the results with the pathophysiology of stunned myocardium. METHODS AND RESULTS: Trabeculae from the right ventricles of rat hearts were loaded iontophoretically with fura-2 to determine [Ca2+]i. Steady-state force-[Ca2+]i relations were obtained by rapid electrical stimulation in the presence of ryanodine. Two exogenous OFR-generating systems were used: H2O2 + Fe(3+)-nitrilotriacetic acid (H2O2 + Fe3+) to produce hydroxyl radical, and xanthine oxidase+purine (XO + P) to produce superoxide. In muscles exposed to H2O2 + Fe3+ for 10 minutes, both twitch force and Ca2+ transients were decreased (eg, in 1.5 mmol/L external [Ca2+], force decreased from 41 +/- 7 to 23 +/- 4 mN/mm2, P < .05, and Ca2+ transient amplitude from 0.96 +/- 0.09 to 0.70 +/- 0.05 mumol/L, P < .05). Maximal Ca(2+)-activated force (Fmax) decreased slightly, from 103 +/- 5 to 80 +/- 12 mN/mm2 (P = NS). Neither the [Ca2+]i required to achieve 50% of Fmax (Ca50) nor the Hill coefficient was changed. In muscles exposed to XO + P for 20 minutes, twitch force was reduced (in 1.5 mmol/L external [Ca2+]) from 50 +/- 9 to 39 +/- 8 mN/mm2 (P < .05). Ca2+ transients, on the other hand, were not affected. Fmax decreased insignificantly from 100 +/- 16 to 81 +/- 14 mN/mm2. Ca50 increased from 0.71 +/- 0.06 to 1.07 +/- 0.07 mumol/L (P < .05), with no change in the Hill coefficient. CONCLUSIONS: These results indicate that exposure to the H2O2 + Fe3+ free radical-generating system reduces activator Ca2+ availability, whereas XO + P decreases the Ca2+ sensitivity of the myofilaments. Exogenously generated OFRs, particularly those produced by XO + P, mimic the effects of myocardial stunning on cardiac excitation-contraction coupling.

Animals↗

PubMed1996

Coupling between fast and slow inactivation revealed by analysis of a point mutation (F1304Q) in mu 1 rat skeletal muscle sodium channels.

1. We sought to elucidate the mechanism of the defective inactivation that characterizes sodium channels containing mutations in the cytoplasmic loop between the third and fourth domains (the III-IV linker). Specifically, we measured whole-cell and single-channel currents through wild-type and F1304Q mutant mu 1 rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes. 2. In wild-type channels, inactivation is complete and the faster of two decay components predominates. In F1304Q, inactivation is incomplete; the slow decay component is larger in amplitude and slower than in wild-type. The fraction of non-inactivating current is substantial (37 +/- 2% of peak current at -20 mV) in F1304Q. 3. Cell-attached patch recordings confirmed the profound kinetic differences and indicated that permeation was not altered by the F1304Q mutation. The F1304Q phenotype must be conferred entirely by changes in gating properties and is not remedied by coexpression with the beta 1-subunit. 4. Recovery from inactivation of F1304Q channels is faster than for wild-type channels and three exponentials are required to describe recovery adequately following long (5 s) depolarizations. Thus, there are three inactivated states even in 'inactivation-deficient' F1304Q channels. 5. The steady-state voltage dependence of F1304Q inactivation is right-shifted by 26 +/- 2 mV. 6. A gating model incorporating three inactivated states, all directly accessible from multiple closed states or the open state, was constrained to fit wild-type and F1304Q inactivation (h infinitive) data and repriming data simultaneously. While it was necessary to alter the rate constants entering and exiting all three inactivated states, the model accounted for the F1304Q-induced rightward shift in steady-state inactivation without imposing voltage dependence on the inactivation rate constants. 7. We conclude that the F1304Q mutation in mu 1 sodium channels modifies several inactivation processes simultaneously. The fact that a single amino acid substitution profoundly alters both fast and slow inactivation indicates that these processes share physical determinants in Na+ channels.

Animals↗

PubMed1996

External pore residue mediates slow inactivation in mu 1 rat skeletal muscle sodium channels.

1. Upon depolarization, voltage-gated sodium channels assume non-conducting inactivated states which may be characterized as "fast' or "slow' depending on the length of the repolarization period needed for recovery. Skeletal muscle Na+ channel alpha-subunits expressed in Xenopus laevis oocytes display anomalous gating behaviour, with substantial slow inactivation after brief depolarizations. We exploited this kinetic behaviour to examine the structural basis for slow inactivation. 2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusion of the pore by III-IV linker residues, the structural features of slow inactivation are unknown. Since external pore-lining residues modulate C-type inactivation in potassium channels, we performed serial cysteine mutagenesis in the permeation loop (P-loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residues are involved in Na+ channel slow inactivation. 3. Wild-type and mutant alpha-subunits were heterologously expressed in Xenopus oocytes, and Na+ currents were recorded using a two-electrode voltage clamp. Slow inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptophan residues in domains II, III and IV did not alter slow inactivation. 4. Analogous to the W402C mutation, coexpression of the wild-type alpha-subunit with rat brain Na+ channel beta 1-subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta 1-subunit coexpression did not. We propose that the W402C mutation and the beta 1-subunit modulate gating through distinct mechanisms. 5. Removal of fast inactivation in wild-type alpha-subunits with the III-IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed the development of slow inactivation. We propose that slow inactivation in Na+ channels involves conformational changes in the external pore. Mutations that affect fast and slow inactivation appear to interact despite their remote positions in the channel.

Amino Acid Sequence↗

PubMed1996

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Amino Acid Sequence↗

PubMed1996

Enhancement of ionic current and charge movement by coexpression of calcium channel beta 1A subunit with alpha 1C subunit in a human embryonic kidney cell line.

1. Coexpression of the beta subunit with the alpha 1C subunit of the cardiac L-type Ca2+ channel has been shown to increase ionic current. To examine the mechanism of this increase, ionic and gating currents were measured in transiently transfected HEK293 cells. 2. Beta 1A subunit coexpression increased the maximal whole-cell conductance (Gmax) measured in 10 mM Ba2+ from 91 +/- 11 to 833 +/- 107 pS pF-1 without a change in the voltage dependence of activation (V1/2: -6.1 +/- 1.1 and -6.6 +/- 0.9 mV, respectively). 3. Gating currents were smaller in cells expressing only the alpha 1C subunit (only four out of eleven cells exhibited gating currents above the limits of detection, whereas eight out of eight beta 1A coexpressing cells had measurable gating currents). The gating currents were integrated to measure the intramembrane charge movement (Q). The ON charge movement (Qon) could be described by a Boltzmann distribution reaching a maximal value of Qon,max. 4. The mean ratio of Gmax: Qon,max increased from 99 +/- 6 to 243 +/- 30 pS fC-1 with beta 1A coexpression, demonstrating that the beta 1A subunit changes the gating of alpha 1C channels to favour the opening of the channels. However, this 2.5-fold change in the Gmax: Qon,max ratio explains less than half of the 9.2-fold increase in Gmax with beta 1A subunit coexpression. The major effect is due to a 3.7-fold increase in Qon,max, demonstrating that beta 1A subunit coexpression increases the number of functional surface membrane channels.

Action Potentials↗

PubMed1996

Control of ion flux and selectivity by negatively charged residues in the outer mouth of rat sodium channels.

1. The sodium channel has a ring of negatively charged amino acids on its external face. This common structural feature of cation-selective channels has been proposed to optimize conduction by electrostatic attraction of permeant cations into the channel mouth. We tested this idea by mutagenesis of mu1 rat skeletal sodium channels expressed in Xenopus oocytes. 2. Replacement of the external glutamate residue in domain II by cysteine reduces sodium current by decreasing single-channel conductance. While this effect can be reversed by the negatively charged sulfhydryl modifying reagent methanethiosulphonate ethylsulphonate (MTSES), the flux saturation behaviour cannot be rationalized simply by changes in the surface charge. 3. The analogous mutations in domains I, III and IV affect not only conductance but also selectivity. These changes in selectivity are only partially reversed by exposure to MTSES. 4. Our findings necessitate revision of prevailing concepts regarding the role of superficial negatively charged residues in the process of ion permeation. These residues do not act solely by electrostatic attraction of permeant ions, but instead may help to form ion-specific binding sites within the pore.

Amino Acids↗

PubMed1996

Structure of the sodium channel pore revealed by serial cysteine mutagenesis.

The pores of voltage-gated cation channels are formed by four intramembrane segments that impart selectivity and conductance. Remarkably little is known about the higher order structure of these critical pore-lining or P segments. Serial cysteine mutagenesis reveals a pattern of side-chain accessibility that contradicts currently favored structural models based on alpha-helices or beta-strands. Like the active sites of many enzymes of known structure, the sodium channel pore consists of irregular loop regions.

Amino Acid Sequence↗

PubMed1996

Single-channel analysis of inactivation-defective rat skeletal muscle sodium channels containing the F1304Q mutation.

The intracellular linker between domains III and IV of the voltage-gated Na channel mediates fast inactivation. Targeted alteration of one or more of a triplet of hydrophobic amino acids within this linker region results in a marked slowing in the decay of ionic current. The mechanism of this defective inactivation was explored in rat skeletal muscle sodium channels (mu 1) containing the F1304Q mutation in Xenopus laevis oocytes with and without coexpression of the rat brain beta 1 subunit. Cell-attached single-channel patch-clamp recordings revealed that the mu 1-F1304Q channel reopens multiple times with open times that are prolonged compared with those of the wild-type channel. Coexpression of the beta 1 subunit stabilized a dominant nonbursting gating mode and accelerated the activation kinetics of mu 1-F1304Q but did not modify mean open time or fast-inactivation kinetics. A Markov gating model incorporating separate fast- and slow-inactivation particles reproduced the results by assuming that the F1304Q mutation specifically influences transitions to and from fast-inactivated states. These effects are independent of interactions of the mutant channel with the beta 1 subunit and do not result from a change in modal gating behavior. These results indicate that F1304Q mutant channels can still enter the inactivated state but do so reversibly and with altered kinetics.

Animals↗

PubMed1996

Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis.

We used serial cysteine mutagenesis to study the structure of the outer vestibule and selectivity region of the voltage-gated Na channel. The voltage dependence of Cd(2+) block enabled us to determine the locations within the electrical field of cysteine-substituted mutants in the P segments of all four domains. The fractional electrical distances of the substituted cysteines were compared with the differential sensitivity to modification by sulfhydryl-specific modifying reagents. These experiments indicate that the P segment of domain II is external, while the domain IV P segment is displaced internally, compared with the first and third domain P segments. Sulfhydryls with a steep voltage dependence for Cd(2+) block produced changes in monovalent cation selectivity; these included substitutions at the presumed selectivity filter, as well as residues in the domain IV P segment not previously recognized as determinants of selectivity. A new structural model is presented in which each of the P segments contribute unique loops that penetrate the membrane to varying depths to form the channel pore.

Amino Acid Sequence↗

PubMed1996

Functional consequences of lidocaine binding to slow-inactivated sodium channels.

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.

Anesthetics, Local↗

PubMed1996

Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure.

Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097 +/- 73 milliseconds [failing hearts, n = 30] versus 842 +/- 56 milliseconds [control hearts, n = 25]; P < .05), and the prominent repolarizing notch in phase 1 was dramatically attenuated. Basal L-type Ca2+ current and whole-cell Na+ current did not differ in cells from failing and from control hearts, but significant differences in K+ currents were observed. The density of the inward rectifier K+ current (IKl) was reduced in cells from failing hearts at test potentials below -90 mV (at -150 mV, -19.1 +/- 2.2 pA/pF [failing hearts, n = 18] versus -32.2 +/- 5.1 pA/pF [control hearts, n = 15]; P < .05). The small outward current component of IKl was also reduced in cells from failing hearts (at -60 mV, 1.7 +/- 0.2 pA/pF [failing hearts] versus 2.5 +/- 0.2 pA/pF [control hearts]; P < .05). The peak of the Ca(2+)-independent transient outward current (Ito) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0 +/- 0.9 pA/pF [failing hearts, n = 20] versus 20.4 +/- 3.2 pA/pF [control hearts, n = 15]; P < .001), while the steady state component was unchanged. There were no significant differences in Ito kinetics or single-channel conductance. A reduction in the number of functional Ito channels was demonstrated by nonstationary fluctuation analysis (0.4 +/- 0.03 channels per square micrometer [failing hearts, n = 5] versus 1.2 +/- 0.1 channels per square micrometer [control hearts, n = 3]; P < .001). Pharmacological reduction of Ito by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp-release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of Ito in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.

Action Potentials↗

PubMed1996

Synergistic modulation of ATP-sensitive K+ currents by protein kinase C and adenosine. Implications for ischemic preconditioning.

Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K+ (K ATP) channels. We investigated the effects of PKC activation and adenosine on K(ATP) current (I KATP) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 micromol/L), an opener of K(ATP) channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). I(KATP) measured at 0 mV was increased by PMA pretreatment from 0.55 +/- 0.32 to 3.25 +/- 0.47 nA (n=6, P < .01). We next determined whether PKC activation abbreviates the time required to turn on I(KATP) developed after an average of 15.1 +/- 2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9 +/- 2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 micromol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop I(KATP) (12.3 +/- 1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5 +/- 1.6 minutes (n=8;P < .02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline. The opening of K(ATP) channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 micromol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the I(KATP) Induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of I(KATP) by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.

Adenosine↗

PubMed1996

Intrinsic myofilament alterations underlying the decreased contractility of stunned myocardium. A consequence of Ca2+-dependent proteolysis?

We investigated the mechanism of the decreased myofilament Ca2+ responsiveness in stunned myocardium. The steady state force-[Ca2+] relationship was measured before and after skinning in thin ventricular trabeculae from control or stunned (20 minutes of ischemia, 20 minutes of reperfusion) rat hearts.[Ca2+]i was determined using microinjected fura 2 salt in intact muscles, whereas the myofilaments of chemically skinned trabeculae were activated directly with solutions of varied [Ca2+]. Maximal Ca2+- activated force (F max) before and after skinning was identical within either the control or stunned groups but was markedly depressed in both groups of stunned trabeculae (P < .001)). After ischemia and reperfusion, the [Ca2+] required for 50% of maximal activation (Ca50) was increased in both intact (control, 0.60 +/- 0.09 micromol/L; stunned, 0.85 +/- 0.09 micromol/L;P < .001) and skinned (control, 1.13 +/- 0.24 micromol/L; stunned 1.39 +/- 0.21 micromol/L; P = .0025) trabeculae. These data indicate that the decreased Ca2+ responsiveness of stunned myocardium is due to intrinsic alterations of the myofilaments. Therefore, we tested the hypothesis that activation of proteases by reperfusion-induced Ca2+ overload decreases the Ca2+ responsiveness of the cardiac myofilaments. Force-[Ca2+] relations were compared before and 5 to 30 minutes after direct exposure of skinned trabeculae to calpain I (18 microgram/mL, 20 minutes at [Ca2+]=10.8 micromol/L), a Ca2+-activated protease that is present in myocardium. Calpain I reduced F max from 94.3 +/- 8.3 to 56 +/- 8.5 mN/mm2 while increasing Ca50 from 0.94 +/- 0.11 to 1.36 +/- 0.21 micromol/L (P < .01). Calpastatin, a specific calpain inhibitor prevented the effects of calpain I on skinned trabeculae. The results show that the reduced Ca2+ responsiveness of stunned myocardium reflects alteration of the myofilaments themselves, not of soluble cytosolic factors, which can be faithfully reproduced by exposure to Ca2+-dependent protease.

Actin Cytoskeleton↗

PubMed1996

Reversal of potassium channel deficiency in cells from failing hearts by adenoviral gene transfer: a prototype for gene therapy for disorders of cardiac excitability and contractility.

Heart failure is a common, often lethal disorder in which conventional pharmacologic strategies have achieved limited success. Failing hearts exhibit a delay of electrical repolarization which predisposes to fatal arrhythmias. To explore the feasibility of gene therapy for this condition, we isolated myocytes from normal and failing dog hearts and quantified electrophysiologic and contractile parameters in primary culture. Action potentials were prolonged in failing cells as a result of diminished potassium currents. Exposure to AdShK, an adenovirus that overexpresses potassium channels, reversed the action potential prolongation of failing cells. The precise phenotype varied as a function of the density of expressed channels; modest increases in potassium current sufficed to mimic the non-diseased state most faithfully, while more robust expression of the transgene excessively abbreviated excitation and contraction. Our results demonstrate that viral gene transfer can modify the electrical properties of adult mammalian heart cells in a manner appropriate to reverse a fundamental disorder of excitability. Realistic application of this form of therapy will need to include a sensitive mechanism for control of the level and distribution of transgene expression.

Action Potentials↗

PubMed1995

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1995

Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels.

Electrical activity initiates a program of selective gene expression in excitable cells. Although such transcriptional activation is commonly attributed to depolarization-induced changes in intracellular Ca2+, zinc represents a viable alternative given its prominent role as a cofactor in DNA-binding proteins coupled with evidence that Zn2+ can enter excitable cells in a voltage-dependent manner. Here it is shown that Zn2+ entry into heart cells depends upon electrical stimulation and occurs via dihydropyridine-sensitive Ca2+ channels. The addition of extracellular Zn2+ to spontaneously depolarizing GH3 pituitary tumor cells induced the expression of a reporter gene driven by the metallothionein promoter, an effect that was prevented by exposure to dihydropyridine Ca2+ channel blockers. Thus, Zn2+ influx through L-type Ca2+ channels can mediate voltage-dependent gene expression.

Animals↗