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R Numann

Publications and source records attributed to R Numann.

17 recordsLinked to original sources
PubMed2001

High-throughput screening strategies for cardiac ion channels.

In this article we discuss new technologies for high-throughput screening (HTS) of cardiac ion channels. We review the current state of cell based HTS and discuss new technologies and approaches using voltage-sensing Fluorescence Resonance Energy Transfer (FRET) dyes and "native" cell lines. The advantages and disadvantages of a gene-centric approach in which a target protein is overexpressed in a non-native cell line are addressed and the role of such primary screens in the drug discovery process outlined. Primary and secondary screens using "native" type cell lines either endogenously expressing the ion channel of interest or overexpressing it are discussed with respect to HTS capacity and sensitivity for FRET voltage sensing dyes and other technologies. Finally the advantages and approaches of screening against multiple targets in an endogenous cardiac cell line are discussed.

Anti-Arrhythmia Agents↗
PubMed2000

Histamine increases [Ca(2+)](in) and activates Ca-K and nonselective cation currents in cultured human capillary endothelial cells.

We characterized the effects of histamine on intracellular Ca(2+) and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca(2+). The transient response was mediated largely through intracellular Ca(2+) release and the sustained response was due to extracellular Ca(2+) entry. The increase in intracellular Ca(2+) by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca(2+) and this current has several properties of a nonselective, Ca(2+) permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca(2+) levels. A Ca(2+)-activated K(+) current (BKCA) is activated by the increase in intracellular Ca(2+) and this current is blocked by the selective BKCA blocker iberiotoxin.

Calcium↗
PubMed1999

The effects of ZD6169 on the ATP-dependent K(+) current (I(K)(ATP)) in isolated cat ventricular myocytes.

The effect of the K(ATP) channel opener ZD6169 [(S)-N-(4-benzoyl-phenyl)-3,3, 3-trifluoro-2-hydroxy-2-methyl-propionamide] currently under development for the treatment of urinary incontinence was explored in acutely isolated adult feline ventricular myocytes. ZD6169 activated a current over a wide range of concentrations (0.1-100 microM) that is completely blocked by 10 microM glyburide thereby identifying it as I(K(ATP)). The maximum activation of K(ATP) current was observed at 10 microM; higher concentrations decreased current activation. In contrast, the standard K(ATP) channel opener cromakalim showed a more usual concentration-response relationship, with increasing current for increased concentrations and no signs of saturation or reversal. The bell-shaped dose-response relationship for ZD6169 activation of I(K(ATP)) has also been seen in bladder myocytes, albeit at a lower concentration, and it has been proposed to contribute to the reported lack of in vivo cardiovascular side effects. We compared the effects of ZD6169 to cromakalim and showed that both compounds dramatically shorten cardiac myocyte action potential duration and that ZD6169 does so in spite of the bell-shaped concentration-response relationship for activation of K(ATP) current.

ATP-Binding Cassette Transporters↗
PubMed1999

Fluid flow modulates calcium entry and activates membrane currents in cultured human aortic endothelial cells.

Human aortic endothelial cells (HAEC) respond to flow with Ca2+ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca2+]i) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca2+, fluid flow was unable to evoke the [Ca2+]i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca2+. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release.

Aorta↗
PubMed1999

Induction of Ca2+-activated K+ current and transient outward currents in human capillary endothelial cells.

Human capillary endothelial cells (HCEC) in normal media contain noninactivating outwardly rectifying chloride currents, TEA-sensitive delayed rectifier K+ currents and an inward rectifier K+ current. Two additional ionic currents are induced in HCEC when the media are allowed to become conditioned: A Ca2+-activated K+ current (BKCA) that is sensitive to iberiotoxin is induced in 23.5% of the cells, a transient 4-AP-sensitive K+ current (A current) is induced in 24.7% of the cells, and in 22.3% of the cells both the transient and BKCA currents are coinduced. The EC50 for Ca2+ activation of the BKCA current in HCEC from conditioned media is 213 nM. RNA message for BKCA (hSlo clone) is undetecable after PCR amplification in control cells but is seen in those from conditioned cells. The induction of BKCA current is not blocked by conditioning with inhibitors of nitric oxide synthase, cyclo-oxgenase or lypo-oxygenase pathways. Apparently the characteristics of human endothelial cells are highly malleable and can be easily modified by their local environment.

Calcium↗
PubMed1999

Cloning and expression of the human kv4.3 potassium channel.

We report on the cloning and expression of hKv4.3, a fast inactivating, transient, A-type potassium channel found in both heart and brain that is 91% homologous to the rat Kv4.3 channel. Two isoforms of hKv4.3 were cloned. One is full length (hKv4.3 long), and the other has a 19 amino acid deletion (hKv4.3 short). RT-PCR shows that the brain contains both forms of the channel RNA, whereas the heart predominantly has the longer version. Both versions of the channel were expressed in Xenopus oocytes, and both contain a significant window or noninactivating current seen near potentials of -30 to -40 mV. The inactivation curve for hKv4.3 short is shifted 10 mV positive relative to hKv4.3 long. This causes the peak window current for the short version to occur near -30 mV and the peak for the longer version to be at -40 mV. There was little difference in the recovery from inactivation or in the kinetics of inactivation between the two isoforms of the channel.

Animals↗
PubMed1998

Independent and exclusive modulation of cardiac delayed rectifying K+ current by protein kinase C and protein kinase A.

Expression of minK in Xenopus oocytes results in a current similar to the cardiac slow delayed rectifying K+ (IKs) current. Modulation of the IKs current in cardiac myocytes has been studied extensively because of its role in shaping the cardiac action potential. The human and cat minK cDNA have been cloned, but their regulation by protein kinases has not been characterized. We report here on the complex modulation of human and cat IKs currents by protein kinase C (PKC) and protein kinase A (PKA). Activation of PKC by phorbol ester (100 nmol/L phorbol 12,13-didecanoate [PDD]) produces an increase in IKs current that peaks after 20 minutes and then subsequently decreases to approximately 50% of the control level after 1 hour. PKA activation only produces a sustained increase in IKs current. Interestingly, premodulation by PKC prevents IKs current modulation by PKA, and PKC has no effect on IKs current after potentiation by PKA. This shows that the IKs current is modulated by PKC and PKA in a mutually exclusive manner and suggests that multiple interacting phosphorylation sites are involved. Activation of PKC by diacylglycerol analogues only produces a slow decrease in IKs current. The biphasic effects of PKC on IKs current activated by PDD can also be separated by dose and duration. Low doses of PDD (5 nmol/L) or brief applications (5 minutes) of 100 nmol/L PDD only produces IKs current activation. These data suggest that there are at least 2 independent PKC phosphorylation sites in the minK-KvLQT1 channel. Additionally, long-term activation of PKC strongly attenuates the IKs current expression even when the corresponding changes in capacitance are taken into account.

Animals↗
PubMed1998

Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential.

1. Cultured human capillary endothelial cells (HCEC) contain a large inward rectifier current, IK(IR), that can be abolished by removing external K+ or by adding 50 microM Ba2+. 2. We show that IK(IR) is responsible for maintaining the hyperpolarized potential (-60.6 +/- 0.5 mV, n = 83) of HCEC. Blocking IK(IR) with 50 microM Ba2+ shifts the zero current level and depolarizes HCEC by 36.5 +/- 1.3 mV (n = 4). 3. Increasing external Ca2+ concentration ([Ca2+]o) from 0.5 to 7 mM reduces the magnitude of IK(IR) by 36.5 +/- 2.3 % (n = 5) and depolarizes the cells by 10.33 +/- 2.4 mV (n = 3), whereas decreasing [Ca2+]o from 1.8 to 0.5 mM increases the amplitude of IK(IR) by 6.9 +/- 1.9 % (n = 4). The relationship between [Ca2+]o and the percentage block of IK(IR) gives a Kd value of 5.4 +/- 0.6 mM at -120 mV. 4. IK(IR) is also blocked by other divalent ions, with Ba2+ >> Sr2+ > Mg2+ > Mn2+ = Ca2+, and the block of peak current at -120 mV being 85.3 +/- 3.2 % (n = 5) for 50 microM Ba2+, 62.9 +/- 2.2 % (n = 5) for 5 mM Sr2+, 40.7 +/- 2.5 % (n = 9) for 5 mM Mg2+, 33.4 +/- 2.1 % (n = 5) for 5 mM Mn2+ and 32.9 +/- 2.1 % (n = 5) for 5 mM Ca2+. 5. The voltage dependence of Sr2+ block of peak IK(IR) occurred with a Kd value of 1.0 +/- 0.09 mM for -140 mV, 1.9 +/- 0.16 mM for -130 mV, 3.1 +/- 0.28 mM for -120 mV, 4.6 +/- 0.34 mM for -110 mV and 6.4 +/- 0.5 mM for -100 mV (n = 5), with a calculated electrical distance (delta) of 0.44 from the outside.

Barium↗
PubMed1998

The airway-epithelium: a novel site of action by guanylin.

UNLABELLED: We studied the activation of a chloride channel in normal human bronchial epithelial cells (NHBE) by guanylin. We have observed a background Cl current (ICl,background) and a guanylin-induced outward rectifying chloride currents (ORCC) in NHBE. ICl,background was present in 93% of cells (n = 114), was outwardly-rectifying, and could be completely blocked by 100 microM NPPB (5-Nitro-2(3-phenyl-propylamino)-benzoic acid. Activation of cAMP-activated Cl current with 200 microM CPT-cAMP (8-(4-Chlorophenylthio) adenosine-3',5'-monophosphate) occurred in only 35.3% of cells (n = 34). Gyanylin activated an ORCC in 78.6% (n = 11) of cells. Gyanylin also induced chloride currents in cells that had failed to respond to CPT-cAMP (n = 5). Both CPT-cAMP and the guanylin-induced chloride currents showed strong outward rectification. 500 microM DIDS (4,4'-diisothiocyanostibene-2,2'-disulfonic acid) blocked the guanylin-induced ORCC (n = 10). CONCLUSION: Guanylin activates a DIDS-sensitive ORCC in the NHBE cell which is only modestly activated by cAMP. The guanylin receptor in the NHBE might be of major importance in the regulation of chloride channel activity and transepithelial fluid transport in normal and abnormal airways.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Characterization of the human and rat phospholemman (PLM) cDNAs and localization of the human PLM gene to chromosome 19q13.1.

Previous reports have demonstrated that the phospholemman (PLM), a 72-residue plasma-membrane protein enriched in skeletal muscle and heart, is a major substrate phosphorylated in response to insulin and adrenergic stimulation. Here we describe the isolation and characterization of human and rat PLM cDNA from the heart. Both PLM proteins share significant nucleotide and amino acid sequence and structural similarities with the previously published canine PLM and, to a lesser degree, with Na+/K(+)-ATPase gamma subunit, Mat-8 protein, and CHIF protein. Despite the functional diversity, all these proteins are quite small and possess a single transmembrane domain. Human PLM appears to be a unique gene localized on chromosome 19q13.1. The PLM mRNA is widely distributed in human tissues, with the highest expression in skeletal muscle and heart, suggesting a functional role in muscle contraction. Like canine PLM, both human and rat PLM induce a hyperpolarization-activated chloride current when expressed in Xenopus oocytes. The high degree of sequence and functional conservation among the mammalian PLM proteins indicates that this gene is conserved throughout evolution.

Amino Acid Sequence↗
PubMed1994

Modulation of skeletal muscle sodium channels in a satellite cell line by protein kinase C.

Adult vertebrate skeletal muscle sodium channels are responsible for the spread of excitation from the end-plate through the muscle membrane and transverse tubular system that ultimately leads to contraction. These channels can be distinguished from other sodium channels by their sensitivity to both mu-conotoxin and TTX. The mouse satellite muscle cell line MM14 expresses only TTX- and mu-conotoxin-sensitive sodium channels having the physiological characteristics of adult skeletal muscle channels in both undifferentiated myoblasts and differentiated myotubes. Using undifferentiated and differentiated MM14 cells as well as primary cultures of rat skeletal muscle, we have examined modulation of adult skeletal muscle sodium channels by activators of protein kinase C (PKC). Stimulation of PKC by 1-oleoyl-2-acetyl-sn-glycerol (OAG) slows sodium current macroscopic inactivation rate by up to 70% and reduces the peak sodium current as much as 88%. Single-channel analysis reveals prolonged single channel openings and greatly increased probability of multiple channel openings during sustained depolarizations. These effects are due to PKC activation since they are blocked by a specific peptide inhibitor of PKC. The two effects of OAG are sequential. Low OAG concentrations can cause slowed macroscopic sodium current inactivation in the absence of peak current reduction, and intermediate concentrations of OAG cause slowing of inactivation followed by reduction of peak current. The separation of these two effects indicates that PKC modulation of the skeletal muscle sodium channel may occur by phosphorylation at two independent sites. PKC modulation of muscle sodium channels is expected to have important effects on muscle excitability and resultant contractile activity. Detection of adult skeletal muscle ion channels in replicating MM14 cells suggest that satellite cells may express a distinct subset of muscle-specific genes prior to activation of the terminal differentiation program.

Animals↗
PubMed1993

Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase.

The function of voltage-gated sodium channels that are responsible for action potential generation in mammalian brain neurons is modulated by phosphorylation by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (cA-PK) and by protein kinase C (PKC). Reduction of peak sodium currents by cA-PK in intact cells required concurrent activation of PKC and was prevented by blocking phosphorylation of serine 1506, a site in the inactivation gate of the channel that is phosphorylated by PKC but not by cA-PK. Replacement of serine 1506 with negatively charged amino acids mimicked the effect of phosphorylation. Conversion of the consensus sequence surrounding serine 1506 to one more favorable for cA-PK enhanced modulation of sodium currents by cA-PK. Convergent modulation of sodium channels required phosphorylation of serine 1506 by PKC accompanied by phosphorylation of additional sites by cA-PK. This regulatory mechanism may serve to integrate neuronal signals mediated through these parallel signaling pathways.

Action Potentials↗
PubMed1993

Inhibition of Na+ channels by the novel blocker PD85,639.

This study examined the actions of the novel Na+ channel blocker PD85,639. In whole-cell voltage-clamp recordings from Chinese hamster ovary cells transfected with a cDNA encoding the rat brain type IIA Na+ channel and from dissociated rat brain neurons, PD85,639 attenuated Na+ currents when applied either in the external bath or in the internal pipette solution. Block had a tonic component that occurred in the absence of stimulus pulses and an additional use-dependent component that developed during a train of pulses. The EC50 for tonic block was 30 microM and was not strongly dependent on the holding potential. Use-dependent block was first detectable at 1 microM and was pronounced at higher concentrations, even at stimulus frequencies as low as 1 pulse/2 min. The marked use-dependent block was due to rapid drug binding during depolarizing pulses and very slow recovery of drug-bound channels between the pulses (tau = 11 min at -85 mV). Use-dependent block was greater at more depolarized potentials, suggesting that the drug binding site was partway across the membrane electric field. The block that developed with strong depolarizations was rapidly reversed by opening channels with trains of unblocking pulses to more negative potentials. These characteristics of block by PD85,639 suggest that it is a local anesthetic drug with novel properties.

Animals↗
PubMed1991

A phosphorylation site in the Na+ channel required for modulation by protein kinase C.

Voltage-gated sodium channels are responsible for generation of action potentials in excitable cells. Activation of protein kinase C slows inactivation of sodium channels and reduces peak sodium currents. Phosphorylation of a single residue, serine 1506, that is located in the conserved intracellular loop between domains III and IV and is involved in inactivation of the sodium channel, is required for both modulatory effects. Mutant sodium channels lacking this phosphorylation site have normal functional properties in unstimulated cells but do not respond to activation of protein kinase C. Phosphorylation of this conserved site in sodium channel alpha subunits may regulate electrical activity in a wide range of excitable cells.

Amino Acid Sequence↗
PubMed1991

Functional modulation of brain sodium channels by protein kinase C phosphorylation.

Voltage-gated sodium channels, which are responsible for the generation of action potentials in the brain, are phosphorylated by protein kinase C (PKC) in purified form. Activation of PKC decreases peak sodium current up to 80 percent and slows its inactivation for sodium channels in rat brain neurons and for rat brain type IIA sodium channel alpha subunits heterologously expressed in Chinese hamster ovary cells. These effects are specific for PKC because they can be blocked by specific peptide inhibitors of PKC and can be reproduced by direct application of PKC to the cytoplasmic surface of sodium channels in excised inside-out membrane patches. Modulation of brain sodium channels by PKC is likely to have important effects on signal transduction and synaptic transmission in the central nervous system.

Animals↗