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1.
J Vis Exp ; (196)2023 06 02.
Article in English | MEDLINE | ID: mdl-37335097

ABSTRACT

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.


Subject(s)
Carrier Proteins , Zinc , Animals , Humans , Carrier Proteins/metabolism , Zinc/metabolism , Biological Transport , Membrane Transport Proteins/metabolism , Mammals/metabolism
2.
Metallomics ; 15(6)2023 06 01.
Article in English | MEDLINE | ID: mdl-37193665

ABSTRACT

ZnT1 is a major zinc transporter that regulates cellular zinc homeostasis. We have previously shown that ZnT1 has additional functions that are independent of its activity as a Zn2+ extruder. These include inhibition of the L-type calcium channel (LTCC) through interaction with the auxiliary ß-subunit of the LTCC and activation of the Raf-ERK signaling leading to augmented activity of the T-type calcium channel (TTCC). Our findings indicate that ZnT1 increases TTCC activity by enhancing the trafficking of the channel to the plasma membrane. LTCC and TTCC are co-expressed in many tissues and have different functions in a variety of tissues. In the current work, we investigated the effect of the voltage-gated calcium channel (VGCC) ß-subunit and ZnT1 on the crosstalk between LTCC and TTCC and their functions. Our results indicate that the ß-subunit inhibits the ZnT1-induced augmentation of TTCC function. This inhibition correlates with the VGCC ß-subunit-dependent reduction in ZnT1-induced activation of Ras-ERK signaling. The effect of ZnT1 is specific, as the presence of the ß-subunit did not change the effect of endothelin-1 (ET-1) on TTCC surface expression. These findings document a novel regulatory function of ZnT1 serving as a mediator in the crosstalk between TTCC and LTCC. Overall, we demonstrate that ZnT1 binds and regulates the activity of the ß-subunit of VGCC and Raf-1 kinase and modulates surface expression of the LTCC and TTCC catalytic subunits, consequently modulating the activity of these channels.


Subject(s)
Calcium Channels, L-Type , Calcium Channels, T-Type , Calcium/metabolism , Calcium Channels, L-Type/metabolism , Calcium Channels, T-Type/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Animals , Xenopus
3.
RSC Chem Biol ; 2(2): 486-498, 2021 Apr 01.
Article in English | MEDLINE | ID: mdl-34458794

ABSTRACT

Divalent d-block metal cations (DDMCs) participate in many cellular functions; however, their accumulation in cells can be cytotoxic. The cation diffusion facilitator (CDF) family is a ubiquitous family of transmembrane DDMC exporters that ensures their homeostasis. Severe diseases, such as type II diabetes, Parkinson's and Alzheimer's disease, were linked to dysfunctional human CDF proteins, ZnT-1-10 (SLC30A1-10). Each member of the CDF family reduces the cytosolic concentration of a specific DDMC by transporting it from the cytoplasm to the extracellular environment or into intracellular compartments. This process is usually achieved by utilizing the proton motive force. In addition to their activity as DDMC transporters, CDFs also have other cellular functions such as the regulation of ion channels and enzymatic activity. The combination of structural and biophysical studies of different bacterial and eukaryotic CDF proteins led to significant progress in the understanding of the mutual interaction among CDFs and DDMCs, their involvement in ion binding and selectivity, conformational changes and the consequent transporting mechanisms. Here, we review these studies, provide our mechanistic interpretation of CDF proteins based on the current literature and relate the above to known human CDF-related diseases. Our analysis provides a common structure-function relationship to this important protein family and closes the gap between eukaryote and prokaryote CDFs.

5.
Metallomics ; 9(3): 228-238, 2017 03 22.
Article in English | MEDLINE | ID: mdl-28091657

ABSTRACT

Traditionally, proteins are considered to perform a single role, be it as an enzyme, a channel, a transporter or as a structural scaffold. However, recent studies have described moonlighting proteins that perform distinct and independent functions; for example, TRPM7 is both an ion channel and a kinase. ZnT-1 is a member of the Carrier Diffusion Facilitator family that is expressed throughout the phylogenetic tree from bacteria to humans. Since its cloning in 1995, ZnT-1 is considered a major extruder of Zn2+ based on its capability to protect cells against zinc toxicity. Recently, we reported that ZnT-1 inhibits the L-type calcium channel (LTCC), a major Zn2+ and Ca2+ entry pathway. Here we show that ZnT-1 is a dual-function protein by demonstrating that its abilities to exchange Zn2+/H+ and to inhibit the LTCC are independent of each other and are mediated by different parts of the protein. Specifically, mutations in the membrane-spanning helices that render ZnT-1 unable to transport zinc do not prevent it from inhibiting the LTCC. Moreover, a fragment consisting of the intracellular ZnT-1 C-terminal, which lacks all ion-transfer segments, inhibits the LTCC as efficiently as wild-type ZnT-1. Our data therefore indicates that ZnT-1 performs two structurally independent functions related to zinc homeostasis.


Subject(s)
Calcium Channels, L-Type/chemistry , Cation Transport Proteins/metabolism , Xenopus/physiology , Zinc/pharmacology , Amino Acid Sequence , Animals , CHO Cells , Calcium Channels, L-Type/metabolism , Cation Transport Proteins/genetics , Cricetinae , Cricetulus , Cytoplasm/drug effects , Cytoplasm/metabolism , HEK293 Cells , Homeostasis , Humans , Ion Transport , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Sequence Alignment
6.
Metallomics ; 6(9): 1656-63, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24951051

ABSTRACT

ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protecting cells against Zn(2+) toxicity. Our results demonstrate that ZnT-1 extrudes zinc from mammalian cells by functioning as a Zn(2+)/H(+) exchanger.


Subject(s)
Cation Transport Proteins/metabolism , Hydrogen/metabolism , Mammals/metabolism , Zinc/metabolism , Animals , Biological Transport/drug effects , Calcium/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cytoprotection/drug effects , Electrochemistry , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Mutant Proteins/metabolism , Sodium/metabolism , Zinc/toxicity
7.
J Pharmacol Exp Ther ; 344(1): 59-67, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23055540

ABSTRACT

Selective pharmacological activation of the adenosine 1 receptor (A(1)R) is a promising new approach to achieve a potent block of atrioventricular (A-V)-nodal conduction without significant cardiovascular side effects. The purpose of the present study was to evaluate the cardiovascular profile of INO-8875, a highly selective A(1)R agonist, and to compare its properties with N-[3(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), which has already been shown to induce negative dromotropic effects with minimal cardiovascular side effects in animals and in clinical studies. Dose-response experiments in the isolated hearts of rats were used to evaluate the functional selectivity of INO-8875 for the slowing of A-V-nodal conduction. Ventilated adult rats were used to study the effects of INO-8875, in vivo, on arterial blood pressure as well as on supraventricular electrophysiology. Ex vivo, INO-8875 (100 nM to 3 µM) progressively prolonged A-V-nodal conduction without reducing left ventricular function or coronary resistance. In vivo, INO-8875 up to a dose of 50 µg/kg did not reduce the carotid arterial blood pressure (n = 4). INO-8875 (1-50 µg/kg) and CVT-510 (20 and 50 µg/kg) both induced a dose-dependent decrease in heart rate and atrial refractoriness, as well as slowing of A-V-nodal conduction. However, compared with CVT-510, the activity of INO-8875 was more pronounced in A-V-nodal function. INO-8875 exhibited a greater duration of action, lasting up to 2.5 hours post dosing, whereas the effects of CVT-510 dissipated over 1 hour. INO-8875 demonstrates functional properties of a highly selective A(1)R agonist. INO-8875 exhibits an increased dromotropic effect and greater duration of action compared with CVT-510.


Subject(s)
Adenosine A1 Receptor Agonists/pharmacology , Adenosine/analogs & derivatives , Anti-Arrhythmia Agents , Heart Rate/drug effects , Hemodynamics/drug effects , Nitrates/pharmacology , Adenosine/pharmacology , Anesthesia , Animals , Atrioventricular Node/drug effects , Dose-Response Relationship, Drug , Furans/pharmacology , Heart/drug effects , Heart Conduction System/drug effects , Male , Rats , Rats, Sprague-Dawley , Refractory Period, Electrophysiological/drug effects , Tachycardia, Supraventricular/drug therapy
8.
Am J Physiol Cell Physiol ; 303(2): C192-203, 2012 Jul 15.
Article in English | MEDLINE | ID: mdl-22572848

ABSTRACT

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.


Subject(s)
Calcium Channels, T-Type/biosynthesis , Cation Transport Proteins/biosynthesis , Gene Expression Regulation , MAP Kinase Signaling System/physiology , Proto-Oncogene Proteins p21(ras)/metabolism , Animals , CHO Cells , Cation Transport Proteins/physiology , Cells, Cultured , Cricetinae , Cricetulus , Female , HEK293 Cells , Humans , MAP Kinase Signaling System/drug effects , Mice , Proto-Oncogene Proteins p21(ras)/physiology , Xenopus laevis
9.
J Mol Med (Berl) ; 90(2): 127-38, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22193398

ABSTRACT

Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.


Subject(s)
Cation Transport Proteins/metabolism , MAP Kinase Signaling System , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Reperfusion Injury/chemically induced , Reperfusion Injury/metabolism , Animals , Cation Transport Proteins/genetics , Cell Death , Cell Line , Cell Survival , Enzyme Activation/drug effects , Flavonoids/pharmacology , Gene Expression , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Protein Structure, Tertiary , Rats , Zinc/toxicity
10.
J Pharmacol Exp Ther ; 2010 Jun 04.
Article in English | MEDLINE | ID: mdl-20494956

ABSTRACT

Publication of this article is suspended until the authors can provide full identification and verification of the chemical structure of INO-8875.

11.
Ann N Y Acad Sci ; 1188: 87-95, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20201890

ABSTRACT

Atrial fibrillation (AF), the highest occurring cardiac arrhythmia in the Western world, is associated with substantial morbidity and increased mortality. In spite of extensive research, the cause of atrial electrical remodeling, a major factor in the self-perpetuating nature of AF, is still unknown. Downregulation of L-type Ca2+ channel (LTCC) activity is the hallmark of atrial electrical remodeling. ZnT-1 is a ubiquitous membrane protein that was recently suggested to inhibit the LTCC. We have studied and shown that ZnT-1 expression inhibits LTCC function in an oocyte expression system as well as in isolated cardiomyocytes. Our data also show that rapid electrical pacing can augment ZnT-1 expression in culture as well as in the atria of rats in vivo. Finally, in a pilot study, ZnT-1 expression was found to be augmented in the atria of AF patients. These findings position ZnT-1 as a probable missing link in the mechanism underlying atrial tachycardia remodeling.


Subject(s)
Calcium Channels, L-Type/metabolism , Cation Transport Proteins/metabolism , Coronary Artery Disease/metabolism , Membrane Proteins/metabolism , Tachycardia/metabolism , Xenopus Proteins/metabolism , Animals , Calcium Channels, L-Type/genetics , Cells, Cultured , Coronary Artery Disease/genetics , Coronary Artery Disease/physiopathology , Electrocardiography , Gene Expression Regulation , Heart Rate , Humans , Male , Membrane Proteins/genetics , Oocytes/metabolism , Pilot Projects , Rats , Rats, Sprague-Dawley , Tachycardia/genetics , Tachycardia/physiopathology , Xenopus laevis
12.
J Biol Chem ; 284(47): 32434-43, 2009 Nov 20.
Article in English | MEDLINE | ID: mdl-19767393

ABSTRACT

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory beta-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the beta-subunit. An interaction between ZnT-1 and the beta-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming alpha(1)-subunit of the LTCC. Similarly, a decrease in the surface expression of the alpha(1)-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC beta-subunit and that it involves a decrease in the trafficking of the LTCC alpha(1)-subunit to the surface membrane.


Subject(s)
Calcium Channels, L-Type/chemistry , Carrier Proteins/physiology , Animals , CHO Cells , Carrier Proteins/metabolism , Cricetinae , Cricetulus , Female , Fluorescence Resonance Energy Transfer , Humans , Models, Biological , Myocytes, Cardiac/cytology , Oocytes/metabolism , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Xenopus
13.
Am J Physiol Heart Circ Physiol ; 295(4): H1460-9, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18660446

ABSTRACT

Studies of atrial electrophysiology (EP) in rodents are challenging, and available data are sparse. Herein, we utilized a novel type of bipolar electrode to evaluate the atrial EP of rodents through small lateral thoracotomy. In anesthetized rats and mice, we attached two bipolar electrodes to the right atrium and a third to the right ventricle. This standard setup enabled high-resolution EP studies. Moreover, a permanent implantation procedure enabled EP studies in conscious freely moving rats. Atrial EP was evaluated in anesthetized rats, anesthetized mice (ICR and C57BL6 strains), and conscious rats. Signal resolution enabled atrial effective refractory period (AERP) measurements and first time evaluation of the failed 1:1 atrial capture, which was unexpectedly longer than the AERP recorded at near normal cycle length by 27.2+/-2.3% in rats (P<0.0001; n=35), 31.7+/-8.3% in ICR mice (P=0.0001; n=13), and 57.7+/-13.7% in C57BL6 mice (P=0.015; n=4). While AERP rate adaptation was noted when 10 S1s at near normal basic cycle lengths were followed by S2 at varying basic cycle length and S3 for AERP evaluation, such rate adaptation was absent using conventional S1S2 protocols. Atrial tachypacing in rats shortened the AERP values on a timescale of hours, but a reverse remodeling phase was noted thereafter. Comparison of left vs. right atrial pacing in rats was also feasible with the current technique, resulting in similar AERP values recorded in the low right atrium. In conclusion, our findings indicate that in vivo rate adaptation of the rodent atria is different than expected based on previous ex vivo recordings. In addition, atrial electrical remodeling of rats shows unique remodeling-reverse remodeling characteristics that are described here for the first time. Further understanding of these properties should help to determine the clinical relevance as well as limitations of atrial arrhythmia models in rodents.


Subject(s)
Atrial Function , Cardiac Pacing, Artificial , Electrophysiologic Techniques, Cardiac/instrumentation , Microelectrodes , Monitoring, Ambulatory/instrumentation , Pacemaker, Artificial , Tachycardia, Supraventricular/physiopathology , Action Potentials , Adaptation, Physiological , Animals , Electric Stimulation , Equipment Design , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Rats , Rats, Sprague-Dawley , Refractory Period, Electrophysiological , Time Factors
14.
J Cardiovasc Electrophysiol ; 19(2): 157-64, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17971132

ABSTRACT

BACKGROUND: Until recently, the membrane protein ZnT-1 was studied mainly in the context of zinc homeostasis. However, new findings indicate that it acts as an inhibitor of L-type calcium channels. We recently found that acute rapid pacing of the rat atria in vivo augments the expression of ZnT-1, while knockdown of ZnT-1 in culture can oppose the inhibition of L-type calcium channels following rapid pacing. This pilot study, the first to assess cardiac ZnT-1 in humans, was designed to look for possible correlation between the atrial expression of ZnT-1 and atrial fibrillation. METHODS: Right atrial appendage tissue was collected from 39 patients (27 with sinus rhythm and 12 with atrial fibrillation; 6-permanent, 6- paroxysmal or persistent) undergoing open-heart surgery. The expression of ZnT-1 was analyzed by Western blot utilizing beta-actin as an internal loading control and a standard rat heart sample (STD) for inter-blot comparison. RESULTS: Overall atrial fibrillation patients (n = 12) had median ZnT-1/beta-actin of 1.80 STD (inter-quartile range 1.26 to 2.85) versus 0.73 STD (0.24 to 1.64) in the sinus rhythm group (P = 0.002). No association was found between ZnT-1 level and most other clinical parameters tested. Multivariate analysis determined that atrial fibrillation and increased body mass index were the only independent variables clearly associated with higher ZnT-1 levels (Standardized coefficients Beta = 0.62, 0.31; P = 0.002, P = 0.04, respectively). CONCLUSIONS: This pilot study provides evidence for increased ZnT-1 expression in the atria of patients with atrial fibrillation.


Subject(s)
Atrial Fibrillation/genetics , Atrial Fibrillation/metabolism , Cation Transport Proteins/biosynthesis , Cation Transport Proteins/genetics , Gene Expression Regulation/physiology , Aged , Female , Heart Atria/metabolism , Humans , Male , Middle Aged , Pilot Projects
15.
PLoS One ; 2(9): e843, 2007 Sep 05.
Article in English | MEDLINE | ID: mdl-17786214

ABSTRACT

BACKGROUND: It has been well accepted that glial cells in the central nervous system (CNS) produce nitric oxide (NO) through the induction of a nitric oxide synthase isoform (NOS2) only in response to various insults. Recently we described rapid astroglial, NOS2-dependent, NO production in the neocortex of healthy mice on a time scale relevant to neuronal activity. To explore a possible role for astroglial NOS2 in normal brain function we investigated a NOS2 knockout mouse (B6;129P2-Nos2(tm1Lau)/J, Jackson Laboratory). Previous studies of this mouse strain revealed mainly altered immune responses, but no compensatory pathways and no CNS abnormalities have been reported. METHODOLOGY/PRINCIPAL FINDINGS: To our surprise, using NO imaging in brain slices in combination with biochemical methods we uncovered robust NO production by neocortical astrocytes of the NOS2 mutant. These findings indicate the existence of an alternative pathway that increases basal NOS activity. In addition, the astroglial mutation instigated modifications of neuronal attributes, shown by changes in the membrane properties of pyramidal neurons, and revealed in distinct behavioral abnormalities characterized by an increase in stress-related parameters. CONCLUSIONS/SIGNIFICANCE: The results strongly indicate the involvement of astrocytic-derived NO in modifying the activity of neuronal networks. In addition, the findings corroborate data linking NO signaling with stress-related behavior, and highlight the potential use of this genetic model for studies of stress-susceptibility. Lastly, our results beg re-examination of previous studies that used this mouse strain to examine the pathophysiology of brain insults, assuming lack of astrocytic nitrosative reaction.


Subject(s)
Astrocytes/metabolism , Neocortex/metabolism , Neurons/metabolism , Nitric Oxide Synthase Type II/physiology , Nitric Oxide/biosynthesis , Animals , Behavior, Animal , Blotting, Western , Calcium/metabolism , Mice , Mice, Mutant Strains , Neocortex/cytology , Nitric Oxide Synthase Type II/genetics , Phenotype
16.
Cell Calcium ; 42(1): 71-82, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17196651

ABSTRACT

Crosstalk between two membrane transport systems is an established mechanism underlying regulation. In this study, we investigated the interaction between ZnT-1, a putative plasma membrane zinc transporter, and L-type voltage-dependent calcium channels (LTCC). In the atrium of the myocardium decreased activity of the LTCC is a dominant feature of patients with atrial fibrillation. The trigger for this inhibition has been attributed to the rapid firing rates and consequent calcium overload in the atrial cardiomyocytes. However, the underlying mechanism of LTCC inhibition is still to be elucidated. Here, we showed that the expression of ZnT-1 inhibits the activity of L-type channels during electrical remodeling induced by rapid pacing. (i) Direct manipulations of ZnT-1 expression in cultured cardiomyocytes either by ZnT-1 overexpression or by ZnT-1 silencing with siRNA, decreased or enhanced, respectively, the barium influx through the LTCC. (ii) Co-expression of ZnT-1 with LTCC in Xenopus oocytes decreased whole cell barium current through LTCC. (iii) Rapid pacing of cultured cardiomyocytes (4 h, 100 ms cycle) increased ZnT-1 protein expression and inhibited the voltage-dependent divalent cation influx through the LTCC. Moreover, silencing ZnT-1 with siRNA prevented the rapid pacing induced inhibition of the LTCC (iv) Atrial pacing of anesthetized adult rats (4 h, 50 ms cycle) led to a significant increase in atrial ZnT-1 protein expression in parallel with the typical decrease of the refractory period in the atria. Taken together, these findings demonstrate that crosstalk between ZnT-1 and the L-type calcium channels may underlie atrial response to rapid pacing, suggesting that ZnT-1 is a significant participant in rate-dependent cardiac electrical remodeling.


Subject(s)
Calcium Channels, L-Type/physiology , Membrane Proteins/physiology , Myocytes, Cardiac/physiology , Ventricular Remodeling/physiology , Animals , Barium/metabolism , Cation Transport Proteins , Cells, Cultured , Male , Membrane Proteins/biosynthesis , RNA Interference , Rats , Rats, Sprague-Dawley , Xenopus laevis
17.
J Exp Bot ; 57(14): 3583-94, 2006.
Article in English | MEDLINE | ID: mdl-16968880

ABSTRACT

SPICK2, a homologue of the weakly-inward-rectifying Shaker-like Arabidopsis K channel, AKT2, is a candidate K+-influx channel participating in light- and clock-regulated leaf movements of the legume, Samanea saman. Light and the biological clock regulate the in situ K+-influx channel activity differentially in extensor and flexor halves of the pulvinus (the S. saman leaf motor organ), and also-though differently-the transcript level of SPICK2 in the pulvinus. This disparity between the in situ channel activity versus its candidate transcript, along with the sequence analysis of SPICK2, suggest an in situ regulation of the activity of SPICK2, possibly by phosphorylation and/or by interaction with cAMP. Consistent with this (i) the activity of the voltage-dependent K+-selective fraction of the inward current in extensor and flexor cells was affected differentially in whole-cell patch-clamp assays promoting phosphorylation (using the protein phosphatase inhibitor okadaic acid); (ii) several proteins in isolated plasma membrane-enriched vesicles of the motor cells underwent phosphorylation without an added kinase in conditions similar to patch-clamp; and (iii) the SPICK2 protein was phosphorylated in vitro by the catalytic subunit of the broad-range cAMP-dependent protein kinase. All of these results are consistent with the notion that SPICK2 is the K+-influx channel, and is regulated in vivo directly by phosphorylation.


Subject(s)
Fabaceae/enzymology , Plant Proteins/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Animals , Cesium/pharmacology , Cyclic AMP/metabolism , Electric Conductivity , Enzyme Inhibitors/pharmacology , Fabaceae/cytology , Fabaceae/physiology , Insecta/genetics , Membrane Proteins/metabolism , Okadaic Acid/pharmacology , Patch-Clamp Techniques , Phosphorylation , Plant Proteins/chemistry , Potassium Channels, Inwardly Rectifying/chemistry , Protein Structure, Tertiary , RNA, Messenger/metabolism
18.
Eur J Neurosci ; 21(6): 1626-34, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15845090

ABSTRACT

Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.


Subject(s)
Astrocytes/drug effects , Astrocytes/metabolism , Calcium Signaling/drug effects , Neurotransmitter Agents/metabolism , Zinc/pharmacology , Animals , Calcium Signaling/physiology , Cells, Cultured , Dose-Response Relationship, Drug , In Vitro Techniques , Mice , Mice, Transgenic
19.
Glia ; 48(2): 145-55, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15378655

ABSTRACT

Zinc ions are emerging as an important factor in the etiology of neurodegenerative disorders and in brain damage resulting from ischemia or seizure activity. High intracellular levels of zinc are toxic not only to neurons but also to astrocytes, the major population of glial cells in the brain. In the present study, the role of ZnT-1 in reducing zinc-dependent cell damage in astrocytes was assessed. Zinc-dependent cell damage was apparent within 2 h of exposure to zinc, and occurred within a narrow range of approximately 200 microM. Pretreatment with sublethal concentrations of zinc rendered astrocytes less sensitive to toxic zinc levels, indicating that preconditioning protects astrocytes from zinc toxicity. Fluorescence cell imaging revealed a steep reduction in intracellular zinc accumulation for the zinc-pretreated cells mediated by L-type calcium channels. Heterologous expression of ZnT-1 had similar effects; intracellular zinc accumulation was slowed down and the sensitivity of astrocytes to toxic zinc levels was reduced, indicating that this is specifically mediated by ZnT-1 expression. Immunohistochemical analysis demonstrated endogenous ZnT-1 expression in cultured astroglia, microglia, and oligodendrocytes. Pretreatment with zinc induced a 4-fold increase in the expression of the putative zinc transporter ZnT-1 in astroglia as shown by immunoblot analysis. The elevated ZnT-1 expression following zinc priming or after heterologous expression of ZnT-1 may explain the reduced zinc accumulation and the subsequent reduction in sensitivity toward toxic zinc levels. Induction of ZnT-1 may play a protective role when mild episodes of stroke or seizures are followed by a massive brain insult.


Subject(s)
Astrocytes/metabolism , Brain Diseases/metabolism , Brain/metabolism , Membrane Proteins/metabolism , Nerve Degeneration/metabolism , Zinc/toxicity , Animals , Animals, Newborn , Astrocytes/drug effects , Brain/physiopathology , Brain Diseases/physiopathology , Calcium Channels, L-Type/drug effects , Calcium Channels, L-Type/metabolism , Cation Transport Proteins , Cells, Cultured , Dose-Response Relationship, Drug , Intracellular Fluid/metabolism , Membrane Proteins/drug effects , Metabolic Clearance Rate/drug effects , Metabolic Clearance Rate/physiology , Microglia/drug effects , Microglia/metabolism , Nerve Degeneration/physiopathology , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Rats , Rats, Wistar , Reaction Time/drug effects , Reaction Time/physiology , Up-Regulation/drug effects , Up-Regulation/physiology , Zinc/metabolism
20.
Biochem Biophys Res Commun ; 323(4): 1145-50, 2004 Oct 29.
Article in English | MEDLINE | ID: mdl-15451416

ABSTRACT

The ZnTs are a growing family of proteins involved in lowering or sequestration of cellular zinc. Using fluorescent measurements of zinc transport we have addressed the mechanism of action of the most ubiquitously expressed member of this family, ZnT-1. This protein has been shown to lower levels of intracellular zinc though the mechanism has remained elusive. The rate of zinc efflux in HEK293 cells expressing ZnT-1 was not accelerated in comparison to control cells, suggesting that ZnT-1 may be involved in regulating influx rather than efflux of zinc. Co-expression of the L-type calcium channel, a major route for zinc influx, and ZnT-1 resulted in a 3-fold reduction in the rate of zinc influx in HEK293 and PC-12 cells, indicating that ZnT-1 modulates zinc permeation through this channel. Immunoblot analysis indicates that ZnT-1 expression does not modulate LTCC expression. Our findings therefore indicate that ZnT-1 modulates the permeation of cations through LTCC, thereby, regulating cation homeostasis through this pathway. Furthermore, ZnT-1 may play a role in cellular ion homeostasis and thereby confer protection against pathophysiological events linked to cellular Ca(2+) or Zn(2+) permeation and cell death.


Subject(s)
Calcium Channels, L-Type/metabolism , Ion Channel Gating , Kidney/metabolism , Membrane Proteins/metabolism , Zinc/metabolism , Animals , Cation Transport Proteins , Cations , Cell Line , Homeostasis , Humans , Kidney/embryology , PC12 Cells , Rats , Recombinant Proteins/metabolism
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