Biomedical subjects

V Flockerzi

Publications and source records attributed to V Flockerzi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells.

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 current (IhTRP3) in htrp3-transfected CPAE cells but not in non-transfected wild-type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3-transfected cells than in control CPAE cells. 3. The permeability for monovalent cations was PNa > PCs approximately PK >> PNMDG and the ratio PCa/PNa was 1.62 +/- 0.27 (n = 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist-activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24.4 +/- 0.7 microM). 4. The single-channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5. Thapsigargin and 2,5-di-tert-butyl-1, 4-benzohydroquinone (BHQ), inhibitors of the organellar Ca2+-ATPase, failed to activate IhTRP3. U-73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor-sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6. It is concluded that hTRP3 forms non-selective plasmalemmal cation channels that function as a pathway for agonist-induced Ca2+ influx.

Adenosine Triphosphate↗

PubMed1999

Ca2+-dependent interaction of the trpl cation channel and calmodulin.

The transient receptor potential-like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential-like fusion proteins to characterize calmodulin binding regions in the transient receptor potential-like channel through the use of [125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C-terminus of the transient receptor potential-like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S710-I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) and binds both [125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent Ki values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 microM (calmodulin binding site 1) and 3.3 microM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+-calmodulin-dependent mechanism contributes to transient receptor potential-like cation channel modulation when expressed in CHO cells.

Amino Acid Sequence↗

PubMed1999

Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling.

We describe here the isolation and primary culture of endothelial cells from mouse aorta ("primary explant technique"). These cells provide an excellent model for functional studies in transgenic mice. The primary explant method delivers cells that grow out from small pieces of mouse aorta placed on Matrigel enriched with endothelial growth factors. Cells can be studied on the Matrigel after removing the pieces of aorta or after passages by using dispase and reseeding the cells on gelatine-coated cover-slips. Cells on Matrigel or from the first and second passages were characterised using the combined patch-clamp and fura-2 fluorescence methods. Cells had a mean membrane resting potential of -19+/-3 mV (n=21), a membrane capacitance of 49+/-5 pF (n=37) and a resting cytosolic free [Ca2+] ([Ca2+]i) of 103+/-8 nM (n=30). Adenosine 5'-triphosphate (ATP), acetylcholine and bradykinin, but not histamine, induced fast release of intracellular Ca2+ followed by a sustained rise in [Ca2+]i. Oscillations in [Ca2+]i were observed at lower agonist concentrations. In nearly all cells (93%, n=30), these agonists activated charybdotoxin-sensitive, Ca2+-activated K+ channels and induced hyperpolarisation. In 84% of the cells (n=32), an increase in [Ca2+]i also activated strongly outwards-rectifying Cl- channels. These activated slowly at positive potentials and inactivated rapidly at negative potentials. Increasing [Ca2+]i to 1 microM activated a non-selective cation channel in 86% of the cells (n=28). Each tested cell responded to a challenge with hypotonic solution by activating a Cl- current that was modestly outwards rectifying and inactivated at positive potentials. This current is similar to the well-described swelling-activated current through volume-regulated anion channels (VRAC) in endothelial cells. However, its activation is slower, its inactivation faster and the current density lower than in cultured endothelial cells. It is concluded that the primary explant technique provides a reliable cell model for studying mouse vascular endothelial cell function.

Acetylcholine↗

PubMed1999

Store-operated cation channels in the heart and cells of the cardiovascular system.

In many nonexcitable cells, activation of phospholipase C (PLC)-linked receptors results in a release of Ca(2+) from intracellular stores followed by a transmembrane Ca(2+) entry. This Ca(2+) entry underlies the sustained phase of [Ca(2+)](i) increase, is important for various cellular functions including gene expression, secretion and cell proliferation, and is supported by agonist-activated Ca(2+)-permeable ion channels. Ca(2+)-permeable channels which are activated by store depletion and which are therefore referred to as store- operated channels or SOCs form a major pathway for agonist-induced Ca(2+) influx. So far, the molecular structures of these channels have not been identified. Potential candidates are encoded by members of the TRP family, a class of ion channels initially discovered in Drosophila and involved in the PLC-dependent transduction of visual stimuli. Here, we review recent evidence that agonist-induced Ca(2+) influx and especially SOCs are present in different cell types of the heart and of the cardiovascular system and compare these findings with the possible functions and tissue-specific expression of mammalian TRP proteins.

Animals↗

PubMed1998

A novel capacitative calcium entry channel expressed in excitable cells.

In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.

Amino Acid Sequence↗

PubMed1998

Structure and mRNA expression of a bovine trp homologue related to mammalian trp2 transcripts.

Mammalian homologues of the transient receptor potential (trp) gene product from Drosophila melanogaster function as Ca2+-selective or non-selective cation channels. Complementary DNA was isolated from a bovine testis cDNA library which encodes bovine trp2 (btrp2), a protein of 432 amino acid residues comprising four predicted transmembrane segments. Btrp2 mRNA is expressed in bovine testis, spleen and liver but not in brain, heart, adrenal gland or retina. In bovine testis expression of btrp2 mRNA is restricted to spermatocytes but is not present in spermatogonia, Leydig or Sertoli cells suggesting that btrp2 may contribute to the formation of ion channels in sperm cells.

Amino Acid Sequence↗

PubMed1998

Alternative splicing and tissue specific expression of the 5' truncated bCCE 1 variant bCCE 1delta514.

In many non-excitable as well as electrically excitable cells, depletion of intracellular Ca2+ stores after stimulation of G protein coupled receptors or receptor tyrosine kinases is followed by Ca2+ entry across the plasma membrane, a mechanism referred to as capacitative calcium entry (CCE) [Putney, J.W., Cell Calcium 11 (1990) 611-624; Fasolato, C. et al., Trends Pharmacol Sci. 15 (1994) 77-83]. Recently, we reported that bCCE 1, a homologue of the Drosophila protein trp, exhibits the characteristics of CCE channels [Philipp, S. et al., EMBO J. 15 (1996) 6166-6171]. In this study, we report the cloning of a 5' truncated splice variant (bCCE 1delta514) of the full-length bCCE 1. The bCCE 1delta514 cDNA encodes a protein of 486 amino acids with the ATG triplet encoding M514 of bCCE 1 as translation initiation codon and, therefore, comprises two putative transmembrane segments corresponding to the predicted transmembrane segments 5 and 6 of bCCE 1. bCCE 1delta514 transcripts appear to be specifically expressed in the adrenal gland and genome analysis reveals an alternative splice site within an exon of the CCE 1 gene leading to the formation of bCCE 1delta514.

Adrenal Glands↗

PubMed1998

The structure of the murine calcium channel gamma-subunit gene and protein.

The gamma-subunit of voltage-gated calcium channels is a membrane protein that is associated with the skeletal muscle type of voltage gated calcium channels. Using a subunit-specific polyclonal antibody, the gamma-protein was detected in mouse skeletal muscle but not in brain, where at least five additional types of voltage-gated calcium channels are expressed. Murine genomic clones containing the full coding sequence of the gamma-subunit were isolated, the exons were mapped and sequenced. The murine gamma-subunit is encoded by a single copy gene containing 4 translated exons which are distributed over approximately 14 kilobases of DNA. The intron placement within the mouse gene correlates with the previously revealed organization of the human gamma-subunit gene, although the primary structures of the gamma-subunits are only moderately conserved between the murine, human, rat and rabbit proteins (75% identity).

Alternative Splicing↗

PubMed1997

Molecular characterization of a novel human PDZ domain protein with homology to INAD from Drosophila melanogaster.

PDZ domains are thought to act as protein-binding modules mediating the clustering of membrane and membrane-associated proteins. The INAD protein has been shown to interact via a PDZ domain with the calcium channel TRP which contributes to capacitative calcium entry into Drosophila photoreceptor cells. We have cloned the cDNA of a human INAD-Like protein (hINADL) of 1524 amino acids in length containing at least five PDZ domains. Additionally, two truncated versions hINADL(delta304) and hINADL(delta853) were identified. hInadl transcripts of differing size are expressed in various tissues including brain, where transcripts are abundant in the cerebellum.

Amino Acid Sequence↗

PubMed1997

alpha1-beta interaction in voltage-gated cardiac L-type calcium channels.

The beta subunits of voltage-gated calcium channels normalize current amplitude, kinetics and voltage dependence of these channels by interacting with the channel's pore forming subunit alpha1. By screening an epitope expression library of alpha1Ca fusion proteins, a beta2a binding site of 22 amino acids was identified within the I-II cytoplasmic linker but not on other cytoplasmic sequences of alpha1Ca. This binding site overlaps by 14 amino acids with the conserved 18 amino acid peptide assumed to be essential for alpha1-beta interaction. The common 14 amino acid motif of alpha1Ca is sufficient to bind beta2a, and in addition beta1a, beta3 and beta4.

Amino Acid Sequence↗

PubMed1997

Regional expression and cellular localization of the alpha1 and beta subunit of high voltage-activated calcium channels in rat brain.

The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different alpha1 and four different beta subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The beta subunit-specific antibodies identified the presence of beta1a in skeletal muscle; beta2 in heart; and beta2, beta3, and beta4 in brain. The beta3 protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The beta4 protein was also widely expressed, most prominently in the cerebellum. beta2 protein was expressed at only low levels. In situ hybridization with beta subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the alpha1A, alpha1B, alpha1C, and alpha1D subunits showed a broad distribution of alpha1A and alpha1B transcripts, whereas the expression level of alpha1C and alpha1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that beta4 may associate predominantly, but probably not exclusively, with the alpha1A subunit, and beta3 with the alpha1B subunit. In certain brain areas such as the habenula, the beta3 subunit may associate with other alpha1 subunits too. Furthermore, the beta2 subunit may form complexes with different alpha1 subunits in brain and cardiac muscle. These results demonstrate that a given beta subunit may associate with different alpha1 subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.

Animals↗

PubMed1997

Alternatively spliced IS6 segments of the alpha 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels.

Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca2+ channel at lower concentrations than the cardiac Ca2+ channel, although their alpha 1 subunit, which binds the DHPs, is derived from the same gene. This alpha 1C gene gives rise to several splice variants, among which the alpha 1C-b variant is affected by lower concentrations of nisoldipine than the alpha 1C-a variant. Functional expression of chimeras of alpha 1C-a and alpha 1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the alpha 1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the alpha 1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 alpha 1C-a and IS6 alpha 1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca2+ channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the alpha 1C gene.

Alternative Splicing↗

PubMed1996

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.

Amino Acid Sequence↗

PubMed1996

Gene structure of the murine calcium channel beta3 subunit, cDNA and characterization of alternative splicing and transcription products.

The beta3 subunit of high-voltage-gated calcium channels is a peripheral membrane protein that copurifies with neural N-type calcium channels. Murine genomic clones containing the full coding sequence of beta3 were isolated and the exons were mapped and sequenced. The murine calcium channel beta3 subunit is encoded by a unique gene composed of 13 translated exons that spread over approximately 8 kb of genomic sequence. Alternatively spliced transcripts of the beta3 gene were identified and characterized. The primary structure of beta3 is highly conserved between the murine, human, rabbit and rat proteins (98% identity). The intron placement within that primary structure correlates with the previously postulated exon positions in transcripts encoding the members of the calcium channel beta subunit family and confirm a close evolutionary relationship of the beta3, beta1, beta2 and beta4 subunit genes.

Alternative Splicing↗

PubMed1996

Expression of a calcium-sensing receptor in a human medullary thyroid carcinoma cell line and its contribution to calcitonin secretion.

An extracellular Ca(2+)-sensing mechanism consisting of a G protein-coupled receptor linked to phosphoinositide turnover and inhibition of PTH secretion, has recently been identified in bovine parathyroid cells. In C cells, voltage-dependent L-type calcium channels are thought to be involved in calcium-sensing mechanisms, but evidence exists for additional calcium-sensing mechanisms, such as via a calcium-sensing receptor (CaSR). Using the human medullary C cell carcinoma cell line TT, which lacks L-type calcium channels, we found that Ca2+ or cations specific for the CaSR lead to the release of calcium ions from intracellular stores and to an increase in calcitonin secretion. By molecular cloning we isolated the complete protein-coding complementary DNA of a CaSR from human TT cells, which are derived from a human medullary thyroid carcinoma. The CaSR is derived from the same CaSR gene expressed in the parathyroid gland. In addition, TT cells contain an alternative receptor form of CaSR, CaSRb. These findings provide strong evidence for the presence of a functional CaSR in the human C cell line TT. This receptor contributes not only to the inhibition of PTH secretion in the parathyroid, but also to the stimulation of calcitonin secretion in C cells.

Amino Acid Sequence↗

PubMed1996

Reconstitution of the skeletal muscle dihydropyridine receptor. Functional interaction among alpha 1, beta, gamma and alpha 2 delta subunits.

The L-type voltage-dependent Ca2+ channel purified from skeletal muscle by virtue of its dihydropyridine (DHP) binding activity, is composed of alpha 1, alpha 2 delta, beta and gamma subunits. The alpha 1 subunit has the ability to function alone as a Ca2+ channel and a receptor for DHP and other Ca2+ channel antagonists. In this study, the non-alpha 1 components coexpressed with alpha 1 in COS cells were investigated for their effects on DHP binding and suppression of an anomalous allosteric regulation of the phenylalkylamine (-)D600 in complexes lacking one or more subunits. (-)D600 increased DHP binding to membranes of COS cells expressing alpha 1 beta while it did not affect DHP binding to skeletal muscle membranes. Coexpression of gamma or alpha 2 delta with alpha 1 beta partially suppressed this effect. Coexpression of all the subunits completely eliminated the stimulatory effect of (-)D600, while at the same time increasing the affinity of the complex for DHP to that stabilized in partial complexes by the phenylalkylamine. These results demonstrate that all of the components that co-purify are required for the formation of a functional DHP receptor having the properties of the native skeletal muscle DHP receptor.

Animals↗

PubMed1995

The block of the expressed L-type calcium channel is modulated by the beta 3 subunit.

The alpha 1C subunit of the L-type calcium channel was stable, expressed alone or in combination with the beta 3 subunit in Chinese hamster ovary cells. The beta 3 subunit enhanced significantly the inactivation of barium currents indicating that both subunits interacted with each other. The beta 3 subunit decreased significantly the half-maximal inhibitory concentration of the calcium channel blockers (-)-gallopamil and verapamil, but did not affect significantly the block caused by isradipine and mibefradil at the holding potentials of -80 mV and -40 mV. These results suggest that the beta 3 subunit affects distinctly the interaction of the expressed alpha 1C subunit with different classes of organic calcium channel blockers.

Animals↗

PubMed1995

Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells.

A member of a new subclass of the voltage-activated sodium channel genes has been cloned from the human medullary thyroid carcinoma (hMTC) cell line. The cDNA of hNE-Na (human neuroendocrine sodium channel) encodes a 1977 amino acid protein which phylogenetically represents a link between sodium channels isolated from skeletal muscle and brain. The hNE-Na alpha subunit was transiently expressed in human embryonic kidney cells either alone or in combination with the human sodium channel beta 1 subunit. The channel exhibited rapid activation and inactivation kinetics, and was blocked by tetrodotoxin and cadmium with IC50 values of 24.5 nM and 1.1 mM, respectively. Action potentials were generated in cells expressing high levels of hNE-Na. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated its expression in hMTC cells, in a C-cell carcinoma, and in thyroid and adrenal gland. Transcripts were not identified in pituitary gland, brain, heart, liver or kidney, indicating that the hNE-Na is a sodium channel solely expressed in neuroendocrine cells.

Amino Acid Sequence↗