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R S Kass

Publications and source records attributed to R S Kass.

At least 37 records · Page 2Linked to original sources
PubMed1998

MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled IsK channel.

IsK, a slowly activating delayed rectifier K+ current through channels formed by the assembly of two channel proteins KvLQT1 and MinK, modulates the repolarization of cardiac action potentials. Mutations that map to the KvLQT1 and minK genes account for more than 50% of an inherited cardiac disorder, the Long QT syndrome (Splawski, I., Tristani-Firouzi, M., Lehmann, M. H., Sanguinetti, M. C., and Keating, M. T. (1997) Nat. Genet. 17, 338-340). Despite the importance of these channels to human cardiac function, the molecular basis of their uniquely slow gating properties as well as the stoichiometry and interaction sites of these two subunits are still unclear. We have constructed several fusion channel proteins to begin investigating the stoichiometry of these two subunits and the role of voltage-dependent subunit assembly in channel gating. Functional properties of these constructs were measured using whole cell patch clamp recordings of transiently transfected Chinese hamster ovary cells. The constructs we tested are as follows: MK24 (C terminus of MinK linked to N terminus of KvLQT1); KK40 (a tandem homodimer of KvLQT1); and MKK44 (C terminus of MinK linked to N terminus of KK40). In control experiments (no DNA, control DNA, or only MinK), no time-dependent K+ current was observed. Expression of KvLQT1 or KK40 produced currents that activate and inactivate in a voltage-dependent manner as reported by others for KvLQT1. In contrast, expression of MK24 and MKK44 elicited current with activation kinetics and voltage dependence very similar to native IsK and identical to currents expressed by cells co-transfected with independent MinK and KvLQT1 cDNA. Expression of MK24 plus additional MinK significantly slows current kinetics. Our data raise the possibility 1) of multiple MinK/KvLQT1 stoichiometries and 2) indicate that uniquely slow kinetics of IsK channels is due to voltage-dependent conformational changes of the channel protein and not to assembly of channel subunits.

Animals↗
PubMed1998

Overexpression of nerve growth factor in the heart alters ion channel activity and beta-adrenergic signalling in an adult transgenic mouse.

1. The electrophysiological and pharmacological properties of cardiac myocytes from the hearts of adult transgenic mice engineered to overexpress nerve growth factor (NGF) in the heart were studied. 2. There was a 12% increase in the ventricular myocyte capacitance in NGF myocytes consistent with cardiac hypertrophy, and action potential duration at 90% repolarization (APD90) was prolonged by 142 % compared with wild-type (WT) myocytes. This was due, at least in part, to a decrease in the density of two K+ currents, Ito and IK(ur), which were significantly reduced in NGF mice with no change in their electrophysiological characteristics. We found no change in the current density or electrophysiological properties of the L-type Ca2+ current. 3. The effect on Ito and IK(ur) of TEA and 4-aminopyridine (4-AP) was not different in cells isolated from WT and NGF mice. The prolongation of APD observed in NGF cells was mimicked in WT cells by exposure to 1 mM 4-AP, which partially blocked Ito, completely blocked IK(ur) and increased APD90 by 157%. 4. The isoprenaline-induced increase in ICa was significantly smaller in NGF myocytes than in WT myocytes. This was not due to a decrease in beta-adrenergic receptor (beta-AR) density, as this was increased in NGF tissue by 55%. Analysis of beta-AR subtypes showed that this increase was entirely due to an increase in beta2-AR density with no change in beta1-ARs. 5. The response of the beta-AR-coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin was studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in WT myoyctes and this was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to guanylylimidodiphosphate (Gpp(NH)p) and forskolin were unaffected. 6. In conclusion, the overexpression of NGF in the mouse heart resulted in a decrease in the current density of two K+ channels, which contributed to the prolongation of the cardiac action potential. Despite an increase in beta2-AR density in the hearts of the NGF mice, the response to isoprenaline was diminished, and this was due to an uncoupling of the beta-ARs from the intracellular signalling cascade. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in cardiac hypertrophy and failure, and this mouse provides a novel model in which to study such changes.

Action Potentials↗
PubMed1998

Novel LQT-3 mutation affects Na+ channel activity through interactions between alpha- and beta1-subunits.

The congenital long-QT syndrome (LQT), an inherited cardiac arrhythmia characterized in part by prolonged ventricular repolarization, has been linked to 5 loci, 4 of which have been shown to harbor genes that encode ion channels. Previously studied LQT-3 mutations of SCN5A (or hH1), the gene that encodes the human Na+ channel alpha-subunit, have been shown to encode voltage-gated Na+ channels that reopen during prolonged depolarization and hence directly contribute to the disease phenotype: delayed repolarization. Here, we report the functional consequences of a novel SCN5A mutation discovered in an extended LQT family. The mutation, a single A-->G base substitution at nucleotide 5519 of the SCN5A cDNA, is expected to cause a nonconservative change from an aspartate to a glycine at position 1790 (D1790G) of the SCN5A gene product. We investigated ion channel activity in human embryonic kidney (HEK 293) cells transiently transfected with wild-type (hH1) or mutant (D1790G) cDNA alone or in combination with cDNA encoding the human Na+ channel beta1-subunit (hbeta1) using whole-cell patch-clamp procedures. Heteromeric channels formed by coexpression of alpha- and beta1-subunits are affected: steady-state inactivation is shifted by -16 mV, but there is no D1790G-induced sustained inward current. This effect is independent of the beta1-subunit isoform. We find no significant effect of D1790G on the biophysical properties of monomeric alpha- (hH1) channels. We conclude that the effects of the novel LQT-3 mutation on inactivation of heteromeric channels are due to D1790G-induced changes in alpha- and beta1-interactions.

Cell Line↗
PubMed1997

Molecular pharmacology of UK-118, 434-05, a permanently charged amlodipine analog.

We studied the effects of UK-118, 434-05, a permanently charged form of amlodipine, on recombinant smooth muscle and cardiac L-type calcium channels to determine the distinct modulatory properties of the ionized form of amlodipine. We found that the short distance between the permanent charge group and the active dihydropyridine (DHP) ring of UK-118, 434-05 reduces the potency of this compound as an inhibitor of smooth muscle (alpha(1c-b)) L-type channels, and is similar to the effects of other charged DHP derivatives on cardiac (alpha(1c-a)) L-type channels. However, we found surprisingly that the tonic block of cardiac (alpha(1c-a)) L-type channels was more pronounced than the tonic block of smooth muscle (alpha(1c-b)) L-type channels. This result contrasts with the previously reported subunit-specificity of neutral DHP compounds, and suggests that interactions between the amlodipine charge group and site(s) on the L-type channel alpha1 subunit distinguish the action of charged from neutral DHPs and may contribute to amlodipine's unique pharmacological profile.

Amlodipine↗
PubMed1997

Decreased density of Ito in left ventricular myocytes from German shepherd dogs with inherited arrhythmias.

INTRODUCTION: A colony of inbred German shepherd dogs with inherited ventricular arrhythmias has been established. METHODS AND RESULTS: The inward rectifier (IK1), the slow delayed rectifier (IKs), and the transient outward current (I(to)) were recorded from epicardial myocytes, and Ito was recorded from Purkinje myocytes isolated from the left ventricles of dogs mildly or severely affected with arrhythmias, and unaffected relatives. There were no differences between unaffected and severely affected dogs in the densities of either IK1 or IKs. Peak Ito density at +40 mV was reduced by 49% in epicardial myocytes from severely affected dogs. I(to) density was also reduced in a subset of Purkinje myocytes. Boltzmann analysis of steady-state inactivation showed no differences between groups in slope factor. V1/2, the half-inactivation voltage, was shifted by +6.2 mV in epicardial cells from severely affected versus unaffected dogs. In addition, the time constant for I(to) decay was reduced in mildly and severely affected dogs compared to unaffected dogs. CONCLUSION: Altered density and inactivation of I(to) are associated with the presence of severe ventricular arrhythmias in inbred dogs at risk for sudden death.

Action Potentials↗
PubMed1996

The utility of fluorescent in vivo reporter genes in molecular cardiology.

In vivo reporter genes can be used in different ways in molecular cardiology. In this paper studies are presented using the green fluorescent protein and one of its mutants, S65T-GFP, as in vivo reporter genes. With this new molecular tool we studied cell type specificity of the murine ventricular myosin light chain 2 promoter, positive cell identification prior to patch clamp procedures, and the use of fluorescence activated cell sorting of transiently transfected mammalian cells.

Animals↗
PubMed1996

Influence of L-type Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293 cells.

We have measured ionic and gating currents in human embryonic kidney (HEK 293) cells transiently transfected with cDNAs encoding subunits of the cardiac voltage-gated L-type Ca2+ channel. Robust recombinant ionic current and associated nonlinear charge movement could be measured over a broad voltage range without contamination by endogenous channel activity. Coexpression of the alpha 2/delta-subunit along with alpha 1- and beta 2-subunits speeded activation and deactivation kinetics and significantly increased the maximal conductance of ionic current. Charge movement was measured at voltages negative to the threshold for activation of ionic current, and gating charge could be immobilized at positive holding potentials that did not inactivate ionic current. The ratio of maximal ionic conductance to maximal charge moved remained the same in the absence or presence of the alpha 2/delta-subunit. However, the maximal amount of charge moved was increased about twofold in the presence of the alpha 2/delta-subunit. These results suggest that coexpression of the alpha 2/delta-subunit enhances the expression of functional L-type channels and, in addition, provide evidence that most of the L-type channel-associated nonlinear charge movement is caused by transitions between nonconducting states of the channel protein that precede the open and inactivated states.

Calcium Channels↗
PubMed1996

Developmental changes in ionic channel activity in the embryonic murine heart.

We have isolated murine embryonic atrial and ventricular cells derived from timed-pregnant females at different periods and used patch-clamp procedures to investigate age- and chamber-specific expression of ionic channels in the developing fetal mouse. Our data indicate that L-type Ca2+ channels play a dominant role in excitation during early murine cardiac embryogenesis and that Na+ channel expression increases dramatically just before birth. K+ channel expression is particularly sensitive to changes during development. Neither atrial nor ventricular cells express a slowly activating component of delayed rectification (IKs) until just before birth, and inwardly rectifying channel activity, associated with determination of cellular resting potential, is not markedly apparent until late stages of embryogenesis. Instead, we find robust expression of the ATP-regulated K+ channel at early and late states of embryonic development, which may indicate a novel functional role for this channel during morphogenesis of the heart. These results have important implications for the physiology and development of the murine cardiac conduction system and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in the myocardium of both wild-type and genetically modified mice.

Adenosine Triphosphate↗
PubMed1996

Developmental changes in beta-adrenergic modulation of L-type Ca2+ channels in embryonic mouse heart.

In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca2+ entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca2+ channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L- type Ca2+ channel currents and to assay functional activity of distinct steps in the beta-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that the L-type Ca2+ channels in early-stage (day-11 to -13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca2+ channels in late-stage (day-17 to -19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca2+ channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca2+ channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca2+ channel insensitivity to beta-A agonists in early stages of the developing murine heart.

Adrenergic beta-Agonists↗
PubMed1996

Lidocaine block of LQT-3 mutant human Na+ channels.

In transiently transfected mammalian cells we have identified pharmacological consequences of a naturally occurring deletion mutation, delta KPQ, of the human heart Na+ channel alpha subunit that previously has been linked to one form of the long QT syndrome, an inherited heart disease. Our results show that the Class IB antiarrhythmic agent lidocaine blocks maintained inward current through and slows recovery from inactivation of delta KPQ-encoded Na+ channels. Block is greater for maintained than for peak current. Because incomplete inactivation of mutant Na+ channels is now thought to underlie the prolonged ventricular action potential, which is the phenotype of this disease, and we find that the delta KPQ mutation speeds the recovery from inactivation of drug-free mutant channels, our results provide evidence, for the first time, that clinically relevant dysfunctional properties of an ion channel can be selectively targeted on the basis of the molecular properties conferred on the channel by an inherited genetic disorder.

Anti-Arrhythmia Agents↗
PubMed1996

Dual actions of the novel class III antiarrhythmic drug NE-10064 on delayed potassium channel currents in guinea pig ventricular and sinoatrial node cells.

We have investigated the concentration-dependent modulation, by the novel class III antiarrhythmic compound NE-10064, of the delayed potassium channel current Iks in isolated guinea pig sinoatrial nodal (SAN) and ventricular cells. At concentrations greater than 1 micron, the drug potently inhibited Iks in each of the cell types investigated. The concentration-dependent inhibition of Iks (IC50 = 700 nM) was the same in ventricular and SAN cells. At near-threshold drug concentrations, we also observed increases of Iks activity in both SAN and ventricular cells. The NE-10064-induced enhancement of Iks was more pronounced at voltages near the Iks activation threshold (0 mV), than at more positive voltages in both cell types. Furthermore, the agonistic effects of the drug were more prominent before steady-state effects of the compound were attained, which suggests parallel agonistic and antagonistic pathways. Our results demonstrate that Iks channels in cells of the sinoatrial node region of the guinea pig heart respond to NE-10064 in the same manner as cells of the ventricle and that this compound, although a potent inhibitor of Iks activity, possesses an interesting but as yet mechanistically unidentified agonistic action at low concentrations in both cell types.

Animals↗
PubMed1995

Dependence of the GABAA receptor gating kinetics on the alpha-subunit isoform: implications for structure-function relations and synaptic transmission.

1. To examine the dependence of gamma-aminobutyric acid (GABAA) receptor gating on the alpha-subunit isoform, we studied the kinetics of GABA-gated currents (IGABA) of receptors that differed in the alpha-subunit subtype, alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S. cDNAs encoding rat brain subunits were co-expressed heterologously in HEK-293 cells and the resultant receptors studied with the whole-cell patch clamp technique and rapidly applied GABA pulses (5-10 s). 2. IGABA of both receptors showed a loosely similar dependence on GABA concentration over a wide range (1-5000 microM). Generally, IGABA manifested activation reaching an early current peak, subsequent slower spontaneous desensitization, and deactivation of open channels at pulse termination. Lowering GABA concentrations reduced peak currents and slowed activation and desensitization kinetics. 3. The presence of alpha 3 altered the peak IGABA concentration-response relationship by shifting the fitted Hill equation to tenfold greater GABA concentrations (GABA concentration at half amplitude: alpha 1, 7 microM; and alpha 3, 75 microM) without affecting Hill coefficients (alpha 1, 1.6; alpha 3, 1.5). These findings indicate a reduction in the apparent activating site affinity and are consistent with previous reports. 4. To investigate differences in gating, we normalized for apparent activating site affinities by analysing the time course of macroscopic gating at equi-activating GABA concentrations. The presence of alpha 3 slowed activation fourfold (time to current peak (means +/- S.E.M.): alpha 1, 1.2 +/- 0.06 s (2 microM); alpha 3, 4.7 +/- 0.5 s (20 microM)), desensitization nearly twofold (reciprocal of time to 80% decay: alpha 1, 2.5 +/- 0.48 s-1 (100 microM); alpha 3, 1.5 +/- 0.15 s-1 (1000 microM)) and deactivation threefold (monoexponential decay time constant: alpha 1, 0.22 +/- 0.026 s (2 microM); alpha 3, 0.68 +/- 0.1 s (20 microM)). 5. To gain an insight into the gating mechanisms underlying macroscopic desensitization, we extended a previous gating model of GABAA receptor single-channel activity to include a desensitization pathway. Such a mechanism reproduced empirical alpha 1 beta 2 gamma 2S activation, desensitization and deactivation kinetics. 6. To identify molecular transitions underlying the gating differences between alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S receptors, we explored parameter alterations of the alpha 1 beta 2 gamma 2S gating model that provided an accounting of alpha 3 beta 2 gamma 2S empirical responses. Remarkably, alteration of rates and rate constants involved in ligand binding alone allowed reproduction of alpha 3 beta 2 gamma 2S activation, desensitization and deactivation. 7. These results indicate that substitution of the alpha 3 subunit variant in an alpha 1 beta 2 gamma 2S receptor alters transition rates involved in ligand binding that underlie changes in apparent activating site affinity and macroscopic current gating. Furthermore, they argue strongly that the structural determinants of these functional features reside on the alpha-subunit.

Animals↗
PubMed1995

Inhibition of cardiac L-type calcium channels by quaternary amlodipine: implications for pharmacokinetics and access to dihydropyridine binding site.

We have used whole cell patch clamp procedures to investigate the inhibition of L-type calcium channel currents in guinea pig ventricular cells by the permanently charged dihydropyridine (DHP)compound UK-118,434-05 (quaternary amlodipine, QA). The location of the charge group of this drug molecule is approximately three times closer to the active DHP moiety than is the case for SDZ-207-180, the only other previously-investigated quaternary DHP molecule. Like SDZ-207-180, QA inhibits channel activity only by external application, consistent with an externally, but not internally, accessible binding site, and once blocked, channels do not recover availability by membrane hyperpolarization independent of extracellular pH. However inhibition by QA occurs at roughly 20 x lower potency than comparable inhibition by SDZ-207-180. Low affinity binding to the DHP binding site was confirmed directly with radioligand binding. The permanently charged amlodipine derivative inhibited radioligand DHP binding in partially purified rabbit skeletal muscle transverse tubule membranes with a pseudo-Hill slope close to unity and an IC50 value of 4.2 +/- 0.6 microM. These results indicate that the characteristically slow pharmacokinetics of tertiary amlodipine are due to the unusually stable inhibition of L-channels caused by the ionized fraction of drug molecules. Furthermore, because the distance between the ionized head group and the DHP moiety is so short, the low affinity binding and channel inhibition by QA suggests that the DHP binding site is not on the extracellular domain of the L-channel alpha 1 subunit, but instead must reside within the bilayer or channel pore at a location closer to the extracellular rather than the intracellular face of the membrane.

Amlodipine↗
PubMed1995

Cholinergic inhibition of slow delayed-rectifier K+ current in guinea pig sino-atrial node is not mediated by muscarinic receptors.

We studied the effects of cholinergic agonists on slow delayed-rectifier K+ current (IKs) in isolated cells from the sino-atrial node (SAN) region of guinea pig heart, using patch-clamp procedures. Carbachol (5 nM to 10 microM) inhibited IKs in guinea pig SAN cells in the absence of previous beta-adrenergic stimulation and in cells pretreated with 8-(4-chlorophenylthio)-cAMP. Neither the muscarinic antagonist atropine nor the nicotinic antagonist hexamethonium antagonized carbachol inhibition of the current. Similar results were obtained with other cholinergic agonists. Cholinergic stimulation of the muscarinic K+ current was successfully antagonized by atropine in SAN cells where inhibition of IKs persisted. Therefore, the lack of antagonist effects on inhibition of IKs cannot be attributed to either an absence of muscarinic cholinoceptors on SAN cells or a loss of antagonist activity under our experimental conditions. These data demonstrate that cholinergic agonists, including the endogenous neurotransmitter acetylcholine, decrease the amplitude of IKs in guinea pig SAN cells via a non-muscarinic, non-nicotinic, cAMP-independent mechanism. Although the precise nature of this signal transduction pathway has not been elucidated, it is clearly different from those described for regulation of other nodal currents. Differential regulation of IKs in guinea pig SAN and ventricle cannot be attributed to higher basal adenylate cyclase activity in SAN cells. The inhibitory effect of carbachol on IKs was not additive with that of verapamil, a drug that is both an allosteric muscarinic antagonist and a potassium channel-blocking agent. Cholinergic agonists may inhibit IKs in SAN cells via a direct interaction with the SAN IKs channel.

Allosteric Regulation↗
PubMed1994

Polymorphism of the gene encoding a human minimal potassium ion channel (minK).

A gene (minK) that encodes a minimal potassium channel has been cloned recently. We describe in this paper a human minK sequence which differs from the original sequence with a single A-->G at position 112. This resulted in a change from a Ser codon (AGT) to a Gly codon (GGT) and created a new MspAI restriction site. Of the 32 alleles from 16 subjects studied, 25 had this newly discovered sequence and 7 had the previously described sequence.

Alleles↗
PubMed1994

Inhibition of L-type calcium-channel activity by thapsigargin and 2,5-t-butylhydroquinone, but not by cyclopiazonic acid.

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+]i). Higher concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗