4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

Background  

Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.     

Altering bioelectricity on inhibition of human breast cancer cells

Background

Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer.

Methods

In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive).

Results

Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10–20 μM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 μM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil.

Conclusions

Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.

     

The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

Background  

Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium.     

Network dynamics in nociceptive pathways assessed by the neuronal avalanche model

Background

Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.

Findings

When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K⁺ channels, K_v1.1 and K_v1.2 (voltage-gated K⁺ channels) and TREK1 and TRAAK (K_2P channels). IQ effectively reduced the currents mediated by both K⁺ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K_v1.1 and K_v1.2.

Conclusions

Our results demonstrate that IQ blocks the voltage-gated K⁺ channels to increase AP duration and K_2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.     

Expression of ATP-sensitive potassium channels in human pregnant myometrium

Background  

Potassium channels play critical roles in the regulation of cell membrane potential, which is central to the excitability of myometrium. The ATP-sensitive potassium (KATP) channel is one of the most abundant potassium channels in myometrium. The objectives of this study were to investigate the protein expression of KATP channel in human myometrium and determine the levels of KATP channel in lower and upper segmental myometrium before and after onset of labour.     

Molecular and functional characterization of voltage-gated sodium channels in human sperm

Background  

We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility.     

P80, the HinT interacting membrane protein,is a secreted antigen of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT.     

Fine mapping of an epilepsy modifier gene on mouse Chromosome 19

Mutations in voltage-gated sodium channels are associated with several types of human epilepsy. Variable expressivity and penetrance are common features of inherited epilepsy caused by sodium channel mutations, suggesting that genetic modifiers may influence clinical severity. The mouse model Scn2a ^Q54 has an epilepsy phenotype due to a mutation in Scn2a that results in elevated persistent sodium current. Phenotype severity in Scn2a ^Q54 mice is dependent on the genetic background. Congenic C57BL/6J.Q54 mice have delayed onset and low seizure frequency compared to (C57BL/6J × SJL/J)F1.Q54 mice. Previously, we identified two modifier loci that influence the Scn2a ^Q54 epilepsy phenotype: Moe1 (modifier of epilepsy 1) on Chromosome 11 and Moe2 on Chromosome 19. We have constructed interval-specific congenic strains to further refine the position of Moe2 on Chromosome 19 to a 5-Mb region. Sequencing and expression analyses of genes in the critical interval suggested two potential modifier candidates: (1) voltage-gated potassium channel subunit subfamily V, member 2 (Kcnv2), and (2) SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 2 (Smarca2). Based on its biological role in regulating membrane excitability and the association between ion channel variants and seizures, Kcnv2 is a strong functional candidate for Moe2. Modifier genes affecting the epilepsy phenotype of Scn2a ^Q54 mice may contribute to variable expressivity and penetrance in human epilepsy patients with sodium channel mutations.     

Rice Hypersensitive Induced Reaction Protein 1 (OsHIR1) associates with plasma membrane and triggers hypersensitive cell death

Background  

In plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1.     

Computational identification of residues that modulate voltage sensitivity of voltage-gated potassium channels

Background  

Studies of the structure-function relationship in proteins for which no 3D structure is available are often based on inspection of multiple sequence alignments. Many functionally important residues of proteins can be identified because they are conserved during evolution. However, residues that vary can also be critically important if their variation is responsible for diversity of protein function and improved phenotypes. If too few sequences are studied, the support for hypotheses on the role of a given residue will be weak, but analysis of large multiple alignments is too complex for simple inspection. When a large body of sequence and functional data are available for a protein family, mature data mining tools, such as machine learning, can be applied to extract information more easily, sensitively and reliably. We have undertaken such an analysis of voltage-gated potassium channels, a transmembrane protein family whose members play indispensable roles in electrically excitable cells.     

Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

Background  

Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish).     

The IBR5 phosphatase promotes Arabidopsis auxin responses through a novel mechanism distinct from TIR1-mediated repressor degradation

Background  

In Arabidopsis, INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5), a putative dual-specificity protein phosphatase, is a positive regulator of auxin response. Mutations in IBR5 result in decreased plant height, defective vascular development, increased leaf serration, fewer lateral roots, and resistance to the phytohormones auxin and abscisic acid. However, the pathways through which IBR5 influences auxin responses are not fully understood.     

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109 ± 6 pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg²⁺ complex and Ca²⁺ ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.     

Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs

Background  

By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.     

Single potassium channel of anomalous (inward) rectification in mollusk neurons

Currents passing through individual potassium channels with anomalous (inward) rectification were recorded at the neuronal membrane ofPlanorbarius corneus using the patch clamp technique. These currents could be detected, whether in "right side out" or "inside out" configurations in the presence of 50 mM potassium ions or one of the potassium channel blockers: tetraethylammonium (TEA), barium, or cesium (2–20 mM) on the external side of the membrane. Inward currents were observed in individual channels at potentials more negative than level of potassium equilibrium potential (E_k); conductance of these measured 81±12 pS (n=11). At more positive potentials than E_k, conductance fell to zero. Potassium channels with anomalous (inward) rectification inPlanorbarius corneus resemble equivalent channels in other cells in their kinetics: time scale of the open state may be described by a single exponential function. This would imply that the ionic channel has a single open state. Time scale of the closed state was biexponential, thus indicating the possible existence of two kinetically different nonconducting states of the potassium channel with anomalous (inward) rectification at the neuronal membrane ofPlanorbarius corneus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 31–38, January–February, 1989.     

The calcium channel β2 (CACNB2) subunit repertoire in teleosts

Background  

Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology [1]. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility [2]. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts.     

MICA: desktop software for comprehensive searching of DNA databases

Background  

Molecular biologists work with DNA databases that often include entire genomes. A common requirement is to search a DNA database to find exact matches for a nondegenerate or partially degenerate query. The software programs available for such purposes are normally designed to run on remote servers, but an appealing alternative is to work with DNA databases stored on local computers. We describe a desktop software program termed MICA (K-Mer Indexing with Compact Arrays) that allows large DNA databases to be searched efficiently using very little memory.     

Rybp,a polycomb complex-associated protein,is required for mouse eye development

Background  

Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp.     

The Arabidopsis Mei2 homologue AML1 binds AtRaptor1B,the plant homologue of a major regulator of eukaryotic cell growth

Background  

TOR, the target of the antibiotic rapamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.