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In JoVE (1)
Other Publications (9)
- Cardiovascular Research
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Molecular and Cellular Cardiology
- American Journal of Physiology. Heart and Circulatory Physiology
- European Journal of Pharmacology
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Cardiovascular Pharmacology
- Journal of Biomolecular Screening
- Frontiers in Pharmacology
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Articles by Kenneth B. Walsh in JoVE
JoVE General
En fluorescerande screeningsanalys för att identifiera modulatorer av GIRK kanaler
En i realtid screening för identifiering läkemedel som interagerar med G-protein-gated inåt likriktare K
Other articles by Kenneth B. Walsh on PubMed
Changes in Cardiac Myocyte Morphology Alter the Properties of Voltage-gated Ion Channels
The goal of this study was to determine if the properties of the transient outward potassium (I(to)), TTX-resistant sodium (I(Na)) and L-type calcium (I(Ca)) currents are altered during changes in cardiac cell shape.
Intracellular Ca(2+) Regulates Responsiveness of Cardiac L-type Ca(2+) Current to Protein Kinase A: Role of Calmodulin
The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.
Overexpression of the Integrin Beta(1A) Subunit and the Beta(1A) Cytoplasmic Domain Modifies the Beta-adrenergic Regulation of the Cardiac L-type Ca(2+)current
Integrins are a family of cell-surface receptors that link the extracellular matrix (ECM) to the cellular cytoskeleton. The goal of this study was to determine the importance of the integrin beta(1) subunit in regulating cardiac L-type Ca(2+) channel function. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing either the human beta(1A) integrin (Adbeta(1A)) or a chimeric protein consisting of the cytoplasmic tail domain of the beta(1A) integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (AdTAC-beta(1)). Expression of the free beta(1) integrin tail (TAC-beta(1)), but not the full-length beta(1A) integrin, altered cell morphology and disrupted normal cell adhesion. When compared with myocytes infected with control virus, neither Adbeta(1A) nor AdTAC-beta(1) infection produced any significant change in the current vs. voltage relationship of the whole-cell Ca(2+) current (I(Ca)) or the kinetics of I(Ca) decay. Expression of TAC-beta(1), but not beta(1A), induced a negative shift in the Ca(2+) channel steady-state inactivation curve. Application of the beta-adrenergic receptor agonist isoproterenol produced over a 90% increase in I(Ca) in control cells, but caused only an 18% increase in myocytes overexpressing the full-length beta(1A) integrin. In addition, beta-adrenergic stimulation resulted in a 5-10-fold increase in intracellular cAMP levels in control cells, but produced no significant response in Adbeta(1A)-infected cells. In contrast, expression of TAC-beta(1) was associated with an augmentation in the Ca(2+) channel response to isoproterenol (160% increase) and the Ca(2+) channel agonist BayK8644. Thus, integrin/ECM interactions may be critical in the regulation of I(Ca)
Regulation of Cardiac Volume-sensitive Chloride Channel by Focal Adhesion Kinase and Src Kinase
The volume-sensitive chloride current (ICl,swell) is found in the mammalian myocardium and is activated by osmotic swelling. The goal of this study was to examine the importance of the tyrosine kinases focal adhesion kinase (FAK) and Src kinase in cardiac ICl,swell regulation. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing beta-galactosidase (AdLacZ), FAK, or FAK-related nonkinase. FAK-related nonkinase (FRNK) is an endogenous cardiac protein, which functions as an inhibitor of FAK. Whole cell patch-clamp recordings demonstrated that osmotic swelling was associated with the activation of an outward rectifying current in uninfected and AdLacZ-infected cells. Consistent with the properties of ICl,swell, this current displayed a reversal potential close to the equilibrium potential for Cl-; was inhibited by the Cl- channel blockers 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and tamoxifen; and was eliminated in hypertonic solution. In addition to activating ICl,swell, hypotonic swelling enhanced the tyrosine phosphorylation of multiple cardiac proteins including those in the range of 68-70 and 120-130 kDa. Pretreatment of the cells with the drug 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of FAK and Src, diminished swelling-induced phosphorylation of these proteins but paradoxically increased ICl,swell. Furthermore, overexpression of FRNK but not FAK caused a twofold augmentation in I(Cl,swell) and increased the rate of current activation. Thus the tyrosine kinases FAK and Src contribute to the regulation of ICl,swell.
Adenoviral-mediated Expression of Dihydropyridine-insensitive L-type Calcium Channels in Cardiac Ventricular Myocytes and Fibroblasts
Cardiac voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive CaV1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant CaV1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+ current (ICa) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC50 of 66 nM and complete block at 250 nM. In contrast, ICa in cells infected with AdCaV1.2DHPi was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 microM diltiazem. In order to study CaV1.2DHPi in isolation, myocytes infected with AdCaV1.2DHPi were incubated with nisoldipine. Under this condition the cells expressed a large ICa (12 pA/pF) and displayed Ca2+ transients during field stimulation. Furthermore, addition of 2 microM forskolin and 100 microM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased IBa in the AdCaV1.2DHPi-infected cells. A Cd2+-sensitive IBa was also recorded in cardiac fibroblasts infected with AdCaV1.2DHPi. Thus, expression of CaV1.2DHPi will provide an important tool in studies of cardiac myocyte and fibroblast function.
Neonatal Rat Cardiac Fibroblasts Express Three Types of Voltage-gated K+ Channels: Regulation of a Transient Outward Current by Protein Kinase C
Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K(+) (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K(+) currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K(+) current (I(to)) that activated at potentials positive to -40 mV and partially inactivated during depolarizing voltage steps. I(to) was inhibited by the antiarrhythmic agent flecainide (100 microM) and BaCl(2) (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K(+) currents [I(K) fast (I(Kf)) and I(K) slow (I(Ks))] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited I(to) but had no effect on I(Kf) and I(Ks). Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 alpha-subunits but not Kv4.2 or Kv1.6 alpha-subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca(2+) mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.
Development of a High-throughput Assay for Monitoring CAMP Levels in Cardiac Ventricular Myocytes
G-protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors involved in cell signal transduction. Many of these GPCRs convey their pharmacological actions by regulating intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP). Although the heart expresses more than 100 GPCRs, drug agonists for approximately one third of these GPCRs have not been identified. The goal of this project was to initiate the development of a high-throughput screening assay for monitoring cAMP in the heart. Neonatal rat cardiac ventricular myocytes were isolated and cultured on coverslips (whole-cell patch clamp recording) or in 96-well plates (fluorescent imaging plate reader measurements). Cells were infected with adenovirus expressing either beta-galactosidase (AdLacZ) or a mutant cyclic nucleotide-gated (CNG) channel containing the double mutation C460W/E583M (AdCNG). Addition of 2 microM forskolin along with 100 microM 3-isobutyl-1-methylxanthine, to increase intracellular cAMP, activated a cation current in myocytes infected with the AdCNG. In myocytes loaded with the fluorescent Ca indicator Fluo-4, stimulation with forskolin, epinephrine, norepinephrine, or the beta-adrenergic receptor agonist isoproterenol increased the fluorescent signal indicative of Ca influx through the CNG channel. In conclusion, CNG channels are readily expressed in cultured cardiac myocytes and may be utilized in high-throughput screening assays of intracellular cAMP.
A Real-time Screening Assay for GIRK1/4 Channel Blockers
The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF.
Targeting GIRK Channels for the Development of New Therapeutic Agents
G protein-coupled inward rectifier K(+) (GIRK) channels represent novel targets for the development of new therapeutic agents. GIRK channels are activated by a large number of G protein-coupled receptors (GPCRs) and regulate the electrical activity of neurons, cardiac myocytes, and β-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the patho-physiology of neuropathic pain, drug addiction, cardiac arrhythmias, and other disorders. However, the pharmacology of these channels remains largely unexplored. In this paper we describe the development of a screening assay for identifying new modulators of neuronal and cardiac GIRK channels. Pituitary (AtT20) and cardiac (HL-1) cell lines expressing GIRK channels were cultured in 96-well plates, loaded with oxonol membrane potential-sensitive dyes and measured using a fluorescent imaging plate reader. Activation of the endogenous GPCRs in the cells caused a rapid, time-dependent decrease in the fluorescent signal; indicative of K(+) efflux through the GIRK channels (GPCR stimulation versus control, Z'-factor = 0.5-0.7). As expected this signal was inhibited by addition of Ba(2+) and the GIRK channel toxin tertiapin-Q. To test the utility of the assay for screening GIRK channel blockers, cells were incubated for 5 min with a compound library of Na(+) and K(+) channel modulators. Ion transporter inhibitors such as 5-(N,N-hexamethylene)-amiloride and SCH-28080 were identified as blockers of the GIRK channel at sub-micromolar concentrations. Thus, the screening assay will be useful for expanding the limited pharmacology of the GIRK channel and in developing new agents for the treatment of GIRK channelopathies.
