Melanopsin signalling in mammalian iris and retina.

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCß4, a vertebrate homologue of the Drosophila NorpA phospholipase C which mediates rhabdomeric phototransduction. The Plcb4(-/-) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCß4-mediated pathway that nonetheless diverges in the two locations.

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Expression and function of the gene encoding the voltage-dependent calcium channel beta3-subunit in the mouse placenta.

Voltage-dependent Ca(2+) channels (VDCC) exist in most excitable cells and their properly regulated activity is essential for critical biological processes as many of these are sensitive to cellular Ca(2+) ion concentration. The ancillary cytoplasmic Ca(2+) channel beta subunits (CACNB) modulate Ca(2+) channel function and are required to enhance the number of functional channels in the plasma membrane. There are four genes encoding CACNB subunits and the gene encoding CACNB3 is over expressed in hyperplastic placentas of mouse interspecies hybrids. To determine the role of CACNB3 in the mouse placenta, we performed an expression and function analysis. Our results show that Cacnb3 exhibits specific spatial and temporal expression in the mouse placenta. Deletion of Cacnb3 does not produce a strong placental phenotype, which may be due to expression of other CACNB subunit encoding genes; however, sporadic occurrence of a labyrinthine architecture phenotype, characterized by reduced density of fetal blood vessels and decrease in pericyte number, could be observed. Down-regulation of Cacnb3 expression did not rescue placental hyperplasia in a model of interspecies hybrid placentas, which indicates that up-regulation in the hyperplastic placentas is a downstream event.

Analysis of TRPV channel activation by stimulation of FCεRI and MRGPR receptors in mouse peritoneal mast cells.

The activation of mast cells (MC) is part of the innate and adaptive immune responses and depends on Ca2+ entry across the plasma membrane, leading to the release of preformed inflammatory mediators by degranulation or by de novo synthesis. The calcium conducting channels of the TRPV family, known by their thermo and osmotic sensitivity, have been proposed to be involved in the MC activation in murine, rat, and human mast cell models. So far, immortalized mast cell lines and nonspecific TRPV blockers have been employed to characterize the role of TRPV channels in MC. The aim of this work was to elucidate the physiological role of TRPV channels by using primary peritoneal mast cells (PMCs), a model of connective tissue type mast cells. Our RT-PCR and NanoString analysis identified the expression of TRPV1, TRPV2, and TRPV4 channels in PMCs. For determination of the functional role of the expressed TRPV channels we performed measurements of intracellular free Ca2+ concentrations and beta-hexosaminidase release in PMCs obtained from wild type and mice deficient for corresponding TRPV1, TRPV2 and TRPV4 in response to various receptor-mediated and physical stimuli. Furthermore, substances known as activators of corresponding TRPV-channels were also tested using these assays. Our results demonstrate that TRPV1, TRPV2, and TRPV4 do not participate in activation pathways triggered by activation of the high-affinity receptors for IgE (FcεRI), Mrgprb2 receptor, or Endothelin-1 receptor nor by heat or osmotic stimulation in mouse PMCs.

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.

Trp12, a novel Trp related protein from kidney.

A novel member of the transient receptor potential (Trp) family of ion channels, Trp12, was identified. The Trp12 mRNA is abundantly expressed in mouse kidney and encodes a protein of 871 amino acid residues. Trp12 transfected cells reveal an elevated cytosolic Ca(2+) and respond with a further increase of cytosolic Ca(2+) to perfusion with hypoosmotic solutions. The human orthologue of murine Trp12 was localized on a genomic clone derived from human chromosome 12. It is composed of 15 translated exons. The intron placement within that primary structure does not correlate with the previously postulated splice sites in transcripts encoding the stretch-inhibitable channel which shares a high degree of amino acid sequence identity with Trp12 and the vanilloid receptor type 1.

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.

Primary structure and functional expression of a cyclic nucleotide-gated channel from rabbit aorta.

Sequences specific for cyclic nucleotide-gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sino-atrial node, cerebellum, C-cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes.

Signaling of the human calcium-sensing receptor expressed in HEK293-cells is modulated by protein kinases A and C.

In this study, the human calcium-sensing receptor (CaR) stably expressed in HEK293 cells was investigated with regard to the phosphorylation-induced desensitization of its signaling pathway. The receptor is known to activate the phospholipase C/inositol-1,4,5-trisphosphate (IP 3 ) signaling cascade, thus stimulating protein kinase C (PKC). In contrast, the adenylylcyclase/cAMP signaling pathway that activates protein kinase A (PKA) is believed to be coupled to the receptor via an inhibitory G-protein. We elucidated the roles of PKC and PKA by measuring Ca 2+o -stimulated accumulation of total inositol phosphates and by individually and simultaneously inhibiting the two kinases pharmacologically in HEK293 cells, which stably expressed the human CaR. Pharmacological inhibition of PKC resulted in a 5-fold enhancement of IP 3 signaling, whereas blocking PKA had almost no effect. IP 3 signaling activity increased even more (10-fold) however, when the two kinases were inhibited simultaneously. Apart from validating the role of PKC as a potent down-regulator of signaling of the human CaR in this cell system, this study suggests that both kinases synergize in inhibiting Ca 2+o -stimulated IP 3 signaling in CaR-transfected HEK293 cells.

Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.

BACKGROUND: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC). RESULTS: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. CONCLUSIONS: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

Biological functions of TRPs unravelled by spontaneous mutations and transgenic animals.

The identification of the biological functions of TRP (transient receptor potential) proteins requires genetic approaches because a selective TRP channel pharmacology to unravel the roles of TRPs is not available so far for most TRPs. A survey is therefore presented of transgenic animal models carrying mutations in TRP genes, as well as of those TRP genes that when mutated result in human disease; the chromosomal locations of TRP channel genes in the human and mouse are also presented.

Paradoxical block of parathormone secretion is mediated by increased activity of G alpha subunits.

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G alpha subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G alpha subunits of heterotrimeric G-proteins.

Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+.

Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.

Mutations of calcium channel beta subunit genes in mice.

Ca2+ influx through high voltage activated Ca2+ channels initiates a number of physiological processes including e.g. excitation-contraction coupling in cardiac myocytes and excitation-transcription coupling in neurones. The Ca2+ channels involved are complexes of a pore-forming alpha1 subunit, a transmembrane delta subunit disulfide-linked to an extracellular alpha2 subunit, a intracellular beta subunit and, at least in some tissues, a gamma subunit. Experimental analysis of beta subunit function comprises functional coexpression of its cDNA together with the cDNAs of the other subunits. This experimental approach can be supplemented by investigating functional alterations that result from the genetic elimination of Ca2+ channel beta genes in mice. Here we summarize the phenotype of mice deficient in the beta1 subunit, the beta3 subunit or the beta4 subunit, respectively.

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.

Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models.

In order to elucidate the functional role of TRPC genes, in vivo, the targeted inactivation of these genes in mice is an invaluable technique. In this review, we summarize the currently available results on the phenotype of TRPC-deficient mouse lines. The analysis of mice with targeted deletion in three TRPC genes demonstrates that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent Ca2+ entry channels in primary cells. Furthermore, from the deficits observed in these TRPC-deficient mouse lines a striking number of biological functions could already be ascribed to TRPC2, TRPC4, and TRPC6, not only on the cellular level but also for complex organ functions and integrative physiology. Accordingly, TRPC2 proteins are critically involved in pheromone sensing by neurones of the vomeronasal organ and, thereby, in the regulation of sexual and social behavior of mice, TRPC4 proteins are essential determinants of endothelial-dependent regulation of vascular tone, endothelial permeability, and neurotransmitter release from thalamic interneurones, and TRPC6 proteins are supposed to have a fundamental role in the regulation of smooth muscle tone in blood vessels and lung.

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.

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.

Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain.

Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L-type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties.

In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.