Marc Freichel

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.

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.

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 Ca2+ and respond with a further increase of cytosolic Ca2+ 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.

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.

TRPC3 Channels Are Required for Synaptic Transmission and Motor Coordination

In the mammalian central nervous system, slow synaptic excitation involves the activation of metabotropic glutamate receptors (mGluRs). It has been proposed that C1-type transient receptor potential (TRPC1) channels underlie this synaptic excitation, but our analysis of TRPC1-deficient mice does not support this hypothesis. Here, we show unambiguously that it is TRPC3 that is needed for mGluR-dependent synaptic signaling in mouse cerebellar Purkinje cells. TRPC3 is the most abundantly expressed TRPC subunit in Purkinje cells. In mutant mice lacking TRPC3, both slow synaptic potentials and mGluR-mediated inward currents are completely absent, while the synaptically mediated Ca2+ release signals from intracellular stores are unchanged. Importantly, TRPC3 knockout mice exhibit an impaired walking behavior. Taken together, our results establish TRPC3 as a new type of postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells and is crucial for motor coordination.

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.

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4

Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcεRI in mast cells triggers the influx of calcium (Ca2+) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca2+ influx in mast cells. Trpm4−/− bone marrow–derived mast cells had more Ca2+ entry than did TRPM4+/+ cells after FcεRI stimulation. Consequently, Trpm4−/− bone marrow–derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4−/− mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca2+ entry in mast cells.

De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury

The role of transient receptor potential M4 (Trpm4), an unusual member of the Trp family of ion channels, is poorly understood. Using rodent models of spinal cord injury, we studied involvement of Trpm4 in the progressive expansion of secondary hemorrhage associated with capillary fragmentation, the most destructive mechanism of secondary injury in the central nervous system. Trpm4 mRNA and protein were abundantly upregulated in capillaries preceding their fragmentation and formation of petechial hemorrhages. Trpm4 expression in vitro rendered COS-7 cells highly susceptible to oncotic swelling and oncotic death following ATP depletion. After spinal cord injury, in vivo gene suppression in rats treated with Trpm4 antisense or in Trpm4−/− mice preserved capillary structural integrity, eliminated secondary hemorrhage, yielded a threefold to fivefold reduction in lesion volume and produced a substantial improvement in neurological function. To our knowledge, this is the first example of a Trp channel that must undergo de novo expression for manifestation of central nervous system pathology.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4α and shorter TRPC4β lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca2+ influx. In common with other “classical” TRPCs, TRPC4 is activated by Gq/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4α but not TRPC4β was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast, several other phosphoinositides (PI), including PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3, had no effect or even potentiated TRPC4α indicating that PIP2 inhibits TRPC4α in a highly selective manner. We show that PIP2 binds to the C terminus of TRPC4α but not that of TRPC4β in vitro. Its inhibitory action was dependent on the association of TRPC4α with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP2 breakdown appears to be a required step in TRPC4α channel activation as PIP2 depletion alone was insufficient for channel opening, which additionally required Ca2+ and pertussis toxin-sensitive Gi/o proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP2/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo

BACKGROUND & AIMS Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. METHODS We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.