Jan Eggermont

KU Leuven | ku leuven · Department of Cellular and Molecular Medicine

ATP13A2 is a late endolysosomal transporter that exports the polyamines spermine and spermidine from the organellar lumen to the cytosol. Loss-of-function variants in ATP13A2 are causative for Kufor-Rakeb syndrome (KRS, a recessive juvenile-onset parkinsonism with dementia) and have also been identified in early-onset PD (EOPD) and hereditary spast...

The polyamines putrescine, spermidine, and spermine are abundant polycations of vital importance in mammalian cells. Their cellular levels are tightly regulated by degradation and synthesis, as well as by uptake and export. Here, we discuss the delicate balance between the neuroprotective and neurotoxic effects of polyamines in the context of Parki...

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). So far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that p...

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine subst...

Cells acquire the polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary action of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine su...

Polyamines (PAs) are physiologically relevant molecules that are ubiquitous in all organisms. The vitality of PAs to the healthy functioning of a cell is due to their polycationic nature causing them to interact with a vast plethora of cellular players and partake in numerous cellular pathways. Naturally, the homeostasis of such essential molecules...

Polyamines, such as putrescine, spermidine and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system (PTS) using CHO-MG cells, a widely used model to study alternative polyamine...

Significance Mutations in ATP13A2 cause a spectrum of related neurodegenerative disorders. ATP13A2 is a lysosomal exporter of polyamines that contributes to lysosomal health and controls cellular polyamine content. Conversely, loss of ATP13A2 leads to lysosomal dysfunction, a hallmark of neurodegeneration. Here, we show that polyamines transported...

Parkinson’s disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of α-synuclein-positive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD p...

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome—a parkinsonism with dementia1—and early-onset Parkinson’s disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson’s disease, whereas loss of ATP13A2 c...

The Ca2+/Mn2+ transport ATPases 1a and 2 (SPCA1a/2) are closely related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and are implicated in breast cancer and Hailey-Hailey skin disease. Here, we purified the human SPCA1a/2 isoforms from a yeast recombinant expression system and compared their biochemical properties after reconstitution. W...

Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallm...

Purification of yeast Ypk9p (WT and D781N). A-B. The N-terminal 10xHis-tagged versions of Ypk9p (A) and Ypk9 (D781N) (B) were purified to relative homogeneity as described in materials and methods by utilizing Ni2+ affinity chromatography. The resulting proteins were found at the expected size of ~170 kDa at high purity. 15 μl of sample for each fr...

Characterization of the affinity-purified ATP13A1 antibody for immunoblotting. A. Topology model of ATP13A1 indicating the peptide recognition sequences of the ATP13A1 SY2459 (AA 543–557 in hATP13A1) B-C. Microsomal fractions of COS-8 cells transiently transfected with ATP13A1 or ATP13A1 fusion proteins (N-terminal GFP-tag) were applied. As a negat...

The P5B ATPases ATP13A3 and ATP13A4 share topology with ATP13A2. A. Topology model of ATP13A2 comprising 10 TM helices and one membrane-associated helix, not spanning the membrane. B-E. Fluorescence protease protection (FPP) assay. WT ATP13A3 (N- (B) or C-terminal (C) GFP-tag) or WT ATP13A4 (N- (D) or C-terminal (E) GFP-tag) were transiently transf...

Complementation assay with GFP-labeled ATP13A1 constructs. Both the N- and C-terminal GFP labeled ATP13A1 constructs provide a functional complementation of the spf1- deletion phenotype in yeast. This is not seen with a catalytic dead mutant of ATP13A1 (D530N). This proves that the N- and C-terminal fusion constructs remain functionally active and...

Accession numbers of sequences included in the phylogenetic analysis. Sequence information of the P5 isoforms included in the phylogenetic analysis of Figs 1 and 2. n.s. not specified; A1, P5A isoform; B1-5, number of P5B isoforms per species; P5BINV, P5B invertebrates. (PDF)

Overview of P5B gene duplication events in animal evolution. The phylogenetic tree depicts (1) Gene duplication in deuterostomia evolution of the P5BANCESTOR orthologue resulted in an ATP13A2 and ATP13A3 isoform. A single P5BINV isoform (invertebrates) is present in most protostomia species. (2) Gene duplication of ATP13A3 into an ATP13A3 and ATP13...

Tissue distribution of P5-type ATPase mRNA expression. Analysis of publically available mRNA expression data shows a P5-type ATPase expression profile that is tissue and development specific in human and mouse. A. Comparing expression of ATP13A1-5 mRNA in human tissue samples shows a general presence of ATP13A1 and ATP13A3 mRNA across all tissues w...

Catalytic dead mutants display similar targeting as WT proteins. A. The catalytically inactive ATP13A1 mutant D530N is targeted to the ER, overlapping with the ER marker SERCA2b. B. Like WT ATP13A2, the catalytically inactive mutant hATP13A2-D508N reaches the late endosomal compartment (visualized with GFP-labeled RAB7). Scale bar represents 20 μm....

ATP13A1 contains an additional N-terminal hairpin. A. Predicted topology model of ATP13A1 with 12 TM helices (M) (TMHMM v2). On top of the figure, the numbering of membrane helices is indicated: M1 corresponds to the first M helix that is present in all P-type ATPases, whereas more upstream helices are referred to as Ma and Mb, with Mb the most N-t...

The Secretory Pathway Ca2+ATPases SPCA1 and SPCA2 transport Ca2+and Mn2+into the Golgi and Secretory Pathway. SPCA2 mediates store-independent Ca2+entry (SICE) via STIM1-independent activation of Orai1, inducing constitutive Ca2+influx in mammary epithelial cells during lactation. Here, we show that like SPCA2, also the overexpression of the ubiqui...

The Golgi/secretory pathway Ca2+/Mn2+ transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+ dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and l...

As a result of the decrease of 60 credit points of the master's programme of medicine, imposed by the Government of Flanders (Belgium), all Flemish universities have implemented a curriculum reform in the medicine programme since the academic year 2012-2013. This curriculum reform has a major impact on the educational set up and content of both the...

The late endo-/lysosomal P-type ATPase ATP13A2 (PARK9) is implicated in Parkinson’s disease (PD) and Kufor-Rakeb syndrome, early-onset atypical Parkinsonism. ATP13A2 interacts at the N-terminus with the signaling lipids phosphatidic acid (PA) and phosphatidylinositol (3,5) bisphosphate (PI(3,5)P2), which modulate ATP13A2 activity under cellular str...

Significance ATP13A2 is a lysosomal transporter that is genetically linked to an autosomal recessive variant of Parkinson’s disease and confers protection against α-synuclein toxicity in neurons. Here we show that an N-terminal hydrophobic domain of ATP13A2 specifically recognizes signaling lipids. Interactions with these signaling lipids enhance c...

Anoctamin 6 (Ano6) is a homologue of Ano1 that functions as a calcium‐activated chloride channel. Recently, Ano6 has been identified as an essential component of outwardly rectifying chloride channels. It has been also shown that Ano6 and Ano1 are activated by hypotonic cell swelling (HTS) and might be a part of an epithelial volume‐regulated anion...

Doctoral programmes at LERU universities aim to train researchers to the highest skill levels to become creative, critical and autonomous intellectual risk takers in pushing the boundaries of frontier research.The modern doctorate is at its core determined by an interplay between professional research experience and personal development, the most i...

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes...

The family of TMEM16 (or anoctamin) ion channels comprises 10 members that seem to be permeable for anion and activated by intracellular calcium. The best characterized members of this family, TMEM16A and TMEM16B, function as calcium‐activated chloride channels (CaCC) in many physiological processes such as sensory transduction, transepithelial sec...

Plasma membrane chloride (Cl(-)) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl(-) uptake mechanism) in Cl(-) handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated pa...

The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In...

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K(+), Cl(-), and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat prot...

The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallogra...

The epithelial Na+ channel (ENaC) is modulated by various extracellular factors, including Na+, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni2+ cation on ENaCs, we compared the Na+ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus...

The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as the apical and basolateral membranes. To obtain directional transepithelial solute transport, membrane transporters (i.e., ion channels, cotransporters, exchangers, and ion pumps) need to be targeted selectively to either of these membrane doma...

We have used the patch‐clamp technique to study modulation of the inwardly rectifying K ⁺ current ( I K(IR) ) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole‐cell mode, I K(IR) was defined as the Ba ²⁺ ‐sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K ⁺ ‐selective channel...

• The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling-activated Cl− current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole-cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5 % (mild hypotonicity) or 25 % (st...

• We have studied the modulation of volume-regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. • RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. • ICl,swell, the chloride current through VRACs, was activate...

In neuronal cells, GABA evokes an increase in chloride conductance by activating GABA(A) and GABA(C) receptors. In mature neurons, this increase in conductance generally has a hyperpolarizing and inhibitory action. Using gramicidin-based perforated patch recordings, we show that in cultured motor neurons GABA-induced currents are significantly alte...

Calcium-activated chloride channels (CaCCs) participate in many different physiologic processes such as transepithelial transport, excitability of neurons and muscle cells, and oocyte fertilization. Within the airways, they contribute to epithelial fluid secretion. This review focuses on three outstanding questions about CaCCs. First, although thei...

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed i...

the cytoskeleton, so the textbooks say, consists of three fiber types: actin-containing microfilaments, intermediate filaments, and microtubules. One of the functions assigned to the cytoskeleton is to preserve cell shape and to protect against mechanical stress. Because changes in cell volume

AMPA receptor-mediated excitotoxicity has been implicated in the pathogenesis of stroke, neurotrauma, epilepsy, and many neurodegenerative diseases such as motoneuron disease. We studied the role of Cl- in AMPA receptor-mediated Ca2+-dependent excitotoxicity in cultured rat spinal motoneurons. Using the gramicidin perforated patch-clamp technique,...

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rec...

Cell swelling triggers in most cell types an outwardly rectifying anion current, I(Cl,swell), via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I(C...

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rectifi...

We expressed the three subunits of the epithelial amiloride-sensitive Na+ channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na+ accumulation we performed all experiments in low-Na+ solutions (15 mM)....

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I(Cl,swell), the swelling-induced Cl- current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I(Cl,swe...

Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the...

Using the whole-cell patch-clamp technique, we have studied the electrophysiological and pharmacological properties of the Ca(2+)-activated Cl- current present in Ehrlich cells. The currents activated slowly upon depolarization, deactivated upon hyperpolarization, and showed strong outward rectification. An increase in [Ca2+]i activated the current...

Restoration of cell volume after cell swelling in mammalian cells is achieved by the loss of solutes (K+, Cl-, and organic osmolytes) and the subsequent osmotically driven efflux of water. This process is generally known as regulatory volume decrease (RVD). One pathway for the swelling induced loss of Cl- (and also organic osmolytes) during RVD is...

This study investigated the volume-regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outw...

The availability of ion-channel cDNAs has greatly increased our insight in the structure, function, pharmacology, and regulation of ion channels at the molecular level. Much of this knowledge has been obtained by expressing wild-type or mutant ion channels in a heterologous host system, thereby facilitating functional approaches and analyses, which...

A whole range of cell functions are regulated by the free cytosolic Ca(2+)concentration. Activator Ca(2+)from the extracellular space enters the cell through various types of Ca(2+)channels and sometimes the Na(+)/Ca(2+)-exchanger, and is actively extruded from the cell by Ca(2+)pumps and Na(+)/Ca(2+)-exchangers. Activator Ca(2+)can also be release...

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not b...

Many important aspects of our life are regulated by the free cytosolic Ca2+ concentration. The intracellular Ca2+ signal is regulated both in space, frequency and amplitude. Each cell chooses a unique set of Ca2+ signals to control its function. Ca2+ signal transduction is based on rises in free cytosolic Ca2+ concentration. Ca2+ can come from the...

We discuss in this short review properties of a volume-regulated anion channel that we have described in vascular endothelial cells. This channel, VRAC (volume-regulated anion channel) is outwardly rectifying with single channel conductances between 10-20pS for inward currents and 40-50pS for outward currents. VRAC is permeable for a wide range of...

The Rho/Rho-associated kinase (ROK) pathway has been shown to modulate volume-regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (...

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significant...

1. Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume-regulated anion channels (VRACs). 2. ICl,swell, the cell swelling-induced chloride current through VRACs, was stud...

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl– channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant ΔF508 CFTR. Currents through CaCC were significantly r...

1. We combined patch clamp and fura-2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2. ATP, bradykinin and intracellular InsP3 activated a non-selective cation cu...

Most mammalian cell types, including endothelial cells, respond to cell swelling by activating a Cl- current termed ICl,swell, but it is not known how the physical stimulus of cell swelling is transferred to the channels underlying ICl,swell. We have investigated the precise relation between cell volume and ICl,swell in endothelial cells by perform...

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild...

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extrac...

It has been proposed that the pI(Cln) protein forms a nucleotide-sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide-binding site. To evaluate this hypothesis, we have performed voltage-clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pI(Cln) in w...

The human CLCN6 gene contains a 167 bp exon that is optionally included or excluded in ClC-6 mRNAs. The corresponding region (3.4 kbp) of the human CLCN7 gene has now been cloned and sequenced. a comparison of the human CLCN1, CLCN5, CLCN6 and CLCN7 genes indicates that there is no homologue of the optional CLCN6 exon in the CLCN1, CLCN5 or CLCN7 g...

We have used the whole-cell patch-clamp technique to study the effects of inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] and inositol 1,3, 4,5,6-pentacisphosphate [Ins(1,3,4,5,6)P5] on volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary artery (CPAE ce...

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in m...

1. We have studied the effects of intracellular ionic strength (gamma 1) on the swelling-activated whole-cell Cl- current (ICl,swell) in cultured calf pulmonary artery endothelial cells (CPAE cells). 2. Reducing gamma 1 from 155 to 95 mM at constant osmolarity and Cl- concentration activates an outwardly rectifying current that is mainly carried by...

The human CLCN6 gene contains a 167 bp exon that is optionally included or excluded in ClC-6 mRNAs. The corresponding region (3.4 kbp) of the human CLCN7 gene has now been cloned and sequenced. A comparison of the human CLCN1, CLCN5, CLCN6 and CLCN7 genes indicates that there is no homologue of the optional CLCN6 exon in the CLCN1, CLCN5 or CLCN7 g...

Ca2+-signals in endothelial cells are determined by release from intracellular stores and entry through the plasma membrane. In this review, the nature of Ca2+ entry and mechanisms of its control are reviewed. The following ion channels play a pivotal role in regulation of the driving force for Ca2+ entry: an inwardly rectifying K+ channel, identif...

This chapter discusses differential expression and the physiological meaning of the Ca2+-transport ATPase isoforms involved in intracellular Ca2+ accumulation. The chapter describes the diversity of the eukaryotic Ca2+-transport ATPases in intracellular Ca2+–storage sites. Data on vertebrates, as well as on invertebrates and non-animal species is a...

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel w...

The aim of the present study is to elucidate the effects of the expression of large conductance Ca2+ activated K+ channels (BK[Ca]) in an endothelial cell type normally lacking this channel. The human homologue hslo of BK(Ca) was expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which have no endogenous BK(Ca). Membrane potent...

Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green...

We have studied the kinetic and pharmacological properties of the Ca(2+)-activated Cl- current (ICl,Ca) in cultured cell pulmonary artery endothelial (CPAE) cells by means of combined patch clamp and Fura-2 micro fluorescence measurements. The current was activated by loading the cells via the patch pipettes with Ca(2+)-buffered solutions. Currents...

ClC-6 is a protein that structurally belongs to the family of ClC-type chloride channels. We now report the identification of three additional ClC-6 isoforms that are truncated because of alternative splicing. We have isolated, from human K562 cells, four types of ClC-6 cDNAs that encode four distinct ClC-6 protein isoforms. ClC-6a (869 amino acids...

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3' end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3' end of the SERCA2 gene....

We have used the whole cell configuration of the patch-clamp technique to investigate volume-activated Cl- currents in BC3H1 and C2C12 cells, two mouse muscle cell lines that can be switched from a proliferating to a differentiated musclelike state. Reducing the extracellular osmolality by 40% evoked large Cl- currents in proliferating BC3H1 and C2...

pICln is a protein that induces an outwardly rectifying, nucleotide-sensitive chloride current (ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains controversial. We now report that a chloride current identical to ICln is induced when Xenopus oocytes are i...

1. An increase in cell volume activates, in most mammalian cells, a Cl- current, ICl,vol. This current is involved in a variety of cellular functions, such as the maintenance of a constant cell volume, pH regulation, and control of membrane potential. It might also play a role in the regulation of cell proliferation and in the processes that contro...