Agonist-Induced Ca Signaling in HEK-293-Derived Cells Expressing a Single IP Receptor Isoform.

In mammals, three genes encode IP receptors (IPRs), which are involved in agonist-induced Ca signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IPR genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IPR1, IPR2, and IPR3, respectively. All engineered cells responded to ACh with Ca transients in an "all-or-nothing" manner, suggesting that each IPR isotype was capable of mediating CICR.

Proliferation, migration, and resistance to oxidative and thermal stresses of HT1080 cells with knocked out genes encoding Hsp90α and Hsp90β.

Heat shock protein 90 (Hsp90) fulfils essential housekeeping functions in the cell associated with the folding, stabilization, and turnover of various proteins. In mammals, there exist two Hsp90 isoforms, stress-inducible Hsp90α and constitutively expressed Hsp90β. In an attempt to identify cellular processes dependent on Hsp90α and Hsp90β, we generated a panel of clones of human fibrosarcoma HT1080 cells with the knocked out HSP90AA1 or HSP90AB1 genes encoding, respectively, Hsp90α and Hsp90β.

Contribution of TRPC3-mediated Ca entry to taste transduction.

The current concept of taste transduction implicates the TASR/PLCβ2/IPR3/TRPM5 axis in mediating chemo-electrical coupling in taste cells of the type II. While generation of IP has been verified as an obligatory step, DAG appears to be a byproduct of PIP cleavage by PLCβ2. Here, we provide evidence that DAG-signaling could play a significant and not yet recognized role in taste transduction.

Taste Cells of the Type III Employ CASR to Maintain Steady Serotonin Exocytosis at Variable Ca in the Extracellular Medium.

Type III taste cells are the only taste bud cells which express voltage-gated (VG) Ca channels and employ Ca-dependent exocytosis to release neurotransmitters, particularly serotonin. The taste bud is a tightly packed cell population, wherein extracellular Ca is expected to fluctuate markedly due to the electrical activity of taste cells. It is currently unclear whether the Ca entry-driven synapse in type III cells could be reliable enough at unsteady extracellular Ca.

Modeling of Ca transients initiated by GPCR agonists in mesenchymal stromal cells.

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP/Ca-regulated Ca release via IP receptors (IPRs) as a key mechanism mediating agonist-dependent Ca transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca mobilization for mathematical modeling.

Calcium signaling mediated by aminergic GPCRs is impaired by the PI3K inhibitor LY294002 and its analog LY303511 in a PI3K-independent manner.

The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY294) and its much less active analog LY303511 (LY303) constitute the paired probe that is commonly used to demonstrate the involvement of PI3K in intracellular signaling. We studied effects of LY294 and LY303 on Ca signaling initiated by certain GPCR agonists in cells of several lines, including CHO cells expressing the recombinant serotonin receptor 5-HT2C and mesenchymal stromal cells derived from the human adipose tissue (AD-MSCs) and umbilical cord (UD-MSCs). The LY294/LY303 pair exerted apparently specific effects on responsiveness of AD-MSCs to ATP, suggesting the involvement of PI3K in ATP transduction.

Arachidonic acid hyperpolarizes mesenchymal stromal cells from the human adipose tissue by stimulating TREK1 K channels.

The current knowledge of electrogenesis in mesenchymal stromal cells (MSCs) remains scarce. Earlier, we demonstrated that in MSCs from the human adipose tissue, transduction of certain agonists involved the phosphoinositide cascade. Its pivotal effector PLC generates DAG that can regulate ion channels directly or via its derivatives, including arachidonic acid (AA).

Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex.

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers.

Coupling of P2Y receptors to Ca mobilization in mesenchymal stromal cells from the human adipose tissue.

The purinergic transduction was examined in mesenchymal stromal cells (MSCs) from the human adipose tissue, and several nucleotides, including ATP, UTP, and ADP, were found to mobilize cytosolic Ca. Transcripts for multiple purinoreceptors were detected in MSC preparations, including A, A, A, P2Y, P2Y, P2Y, P2Y, P2Y, P2Y, P2Y, P2X, P2X, and P2X. Cellular responses to nucleotides were insignificantly sensitive to bath Ca, pointing at a minor contribution of Ca entry, and were suppressed by U73122 and 2-APB, implicating the phosphoinositide cascade in coupling P2Y receptors to Ca release.

Calcium-gated K channels of the K1.1- and K3.1-type couple intracellular Ca signals to membrane hyperpolarization in mesenchymal stromal cells from the human adipose tissue.

Electrogenesis in mesenchymal stromal cells (MSCs) remains poorly understood. Little is known about ion channels active in resting MSCs and activated upon MSC stimulation, particularly, by agonists mobilizing Ca in the MSC cytoplasm. A variety of Ca-gated ion channels may couple Ca signals to polarization of the plasma membrane.

Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells.

Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue.

Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca(2+) imaging.

The ATP permeability of pannexin 1 channels in a heterologous system and in mammalian taste cells is dispensable.

Afferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly.

Modulation of P2X3 receptors by spider toxins.

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors.

Stimulation of the extracellular Ca²⁺-sensing receptor by denatonium.

The extracellular Ca(2+)-sensing receptor (CASR) is a promiscuous G-protein-coupled receptor closely related to the taste receptors T1R1-T1R3. Here we analyzed the possibility that apart from being stimulated by external Ca(2+) and amino acids, the substances effective as tastants, CASR might serve as a receptor for other sapid compounds. CASR was heterologously expressed in HEK-293 cells, and their responsivity to a variety of bitter and sweet substances was examined.

Functional expression of the extracellular-Ca2+-sensing receptor in mouse taste cells.

Three types of morphologically and functionally distinct taste cells operate in the mammalian taste bud. We demonstrate here the expression of two G-protein-coupled receptors from the family C, CASR and GPRC6A, in the taste tissue and identify transcripts for both receptors in type I cells, no transcripts in type II cells and only CASR transcripts in type III cells, by using the SMART-PCR RNA amplification method at the level of individual taste cells. Type I taste cells responded to calcimimetic NPS R-568, a stereoselective CASR probe, with Ca(2+) transients, whereas type I and type II cells were not specifically responsive.

Afferent neurotransmission mediated by hemichannels in mammalian taste cells.

In mammalian taste buds, ionotropic P2X receptors operate in gustatory nerve endings to mediate afferent inputs. Thus, ATP secretion represents a key aspect of taste transduction. Here, we characterized individual vallate taste cells electrophysiologically and assayed their secretion of ATP with a biosensor.

P2Y isoforms operative in mouse taste cells.

Recent functional evidence indicates that mouse taste cells express P2Y receptors coupled to IP(3) production and Ca(2+) mobilization. Our studies of the expression profile of particular P2Y isoforms in the taste tissue of the mouse have revealed that ATP and UTP equipotently mobilize intracellular Ca(2+) at saturating concentrations, suggesting that common receptors for both nucleotides, i.e.