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.

An introduction on TRP channels.

The transient receptor potential (TRP) ion channels are named after the role of the channels in Drosophila phototransduction. Mammalian TRP channel subunit proteins are encoded by at least 28 genes. TRP cation channels display an extraordinary assortment of selectivities and activation mechanisms, some of which represent previously unrecognized modes of regulating ion channels. In addition, the biological roles of TRP channels appear to be equally diverse and range from roles in thermosensation and pain perception to Ca2+ and Mg2+ absorption, endothelial permeability, smooth muscle proliferation and gender-specific behaviour.

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.

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.

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.

Excitation-contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit gamma1.

1. In skeletal muscle, dihydropyridine (DHP) receptors control both Ca(2+) entry (L-type current) and internal Ca(2+) release in a voltage-dependent manner. Here we investigated the question of whether elimination of the skeletal muscle-specific DHP receptor subunit gamma1 affects excitation-contraction (E-C) coupling. We studied intracellular Ca(2+) release and force production in muscle preparations of a mouse deficient in the gamma1 subunit (gamma-/-). 2. The rate of internal Ca(2+) release at large depolarization (+20 mV) was determined in voltage-clamped primary-cultured myotubes derived from satellite cells of adult mice by analysing fura-2 fluorescence signals and estimating the concentration of free and bound Ca(2+). On average, gamma-/- cells showed an increase in release of about one-third of the control value and no alterations in the time course. 3. Voltage of half-maximal activation (V(1/2)) and voltage sensitivity (k) were not significantly different in gamma-/- myotubes, either for internal Ca(2+) release activation or for the simultaneously measured L-type Ca(2+) conductance. The same was true for maximal Ca(2+) inward current and conductance. 4. Contractions evoked by electrical stimuli were recorded in isolated extensor digitorum longus (EDL; fast, glycolytic) and soleus (slow, oxidative) muscles under normal conditions and during fatigue induced by repetitive tetanic stimulation. Neither time course nor amplitudes of twitches and tetani nor force-frequency relations showed significant alterations in the gamma1-deficient muscles. 5. In conclusion, the overall results show that the gamma1 subunit is not essential for voltage-controlled Ca(2+) release and force production.

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.

The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM-Sepharose in the presence of Ca(2+), and (2) TRP4-glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688-759 and 786-848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca(2+) concentrations above 10 microM, with half maximal binding occurring at 16.6 microM (domain 1) and 27.9 microM Ca(2+) (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694-728 and 829-853, interacted directly with dansyl-CaM with apparent K(d) values of 94-189 nM. These results indicate that TRP4/Ca(2+)-CaM are parts of a signalling complex involved in agonist-induced Ca(2+) entry.

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer.

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin.

A finely tuned Ca(2+) signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca(2+) channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca(2+) permeation properties suggest an important function in Ca(2+) uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca(2+)-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

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.

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.

Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF.

Mammalian homologues of Drosophila Trp have been implicated to form channels that are activated following the depletion of Ca(2+) from internal stores. Recent studies indicate that actin redistribution is required for the activation of these channels. Here we show that murine Trp4 and Trp5, as well as phospholipase C beta1 and beta2 interact with the first PDZ domain of NHERF, regulatory factor of the Na(+)/H(+) exchanger. We demonstrated the association of Trp4 and phospholipase C-beta1 with NHERF in vivo in an HEK293 cell line expressing Trp4 and in adult mouse brain by immuno-coprecipitation. NHERF is a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family. Thus, store-operated channels involving Trp4 and Trp5 can form signaling complexes with phospholipase C isozymes via interactions with NHERF and thereby linking the lipase and the channels to the actin cytoskeleton. The interaction with the PDZ protein may constitute an important mechanism for distribution and regulation of store-operated channels.

Single-channel currents through transient-receptor-potential-like (TRPL) channels.

Single-channel current recordings were used to examine the properties and modulation of Drosophila transient-receptor-potential-like (TRPL) channels transiently expressed in HEK and COS cells. Recombinant TRPL channels were constitutively active and characterized by a conductance of 104 pS in on-cell membrane patches with 115 mM Na+ and 2 mM Mg2+ in the pipette solution. In inside-out membrane patches exposed to 115 mM Na+ plus 2 mM Mg2+, 115 mM Na+ plus 10 mM Mg2+, 90 mM Ca2+ and 90 mM Ba2+ on both sides, the single-channel conductances were 72 pS, 36 pS, 48 pS and 46 pS, respectively. The single TRPL channel currents reversed close to 0 mV and displayed a linear voltage dependence between -120 mV and +120 mV. Removal of cations from the pipette and bath solutions abolished inward and outward currents, respectively. Similar currents were not observed in mock-transfected and native cells. The opening probability of TRPL channels increased by depolarizing the membrane and accounted for the outward rectification of whole-cell TRPL currents. In on-cell membrane patches, the TRPL channel activity was enhanced by cell dialysis of 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and by a rise of intracellular Ca2+ (>2 microM). Constitutively active TRPL channels depolarized the host cells to -10 mV and the membrane potential was restored by cell dialysis with 10 mM BAPTA. The present results suggest that TRPL forms non-selective cationic channels modulated by intracellular Ca2+ in mammalian cells.

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+.

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.

TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

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.

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.