14
views
0
recommends
+1 Recommend
0 collections
    4
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          TRPC3 (or Htrp3) is a human member of the trp family of Ca 2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca 2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca 2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca 2+ over Na +. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca 2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca 2+. Likewise, infusion of Ca 2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca 2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca 2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca 2+-permeable channel that supports Ca 2+-induced Ca 2+-entry but should not be considered store operated.

          Related collections

          Most cited references32

          • Record: found
          • Abstract: found
          • Article: not found

          Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

          1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction.

            Recent studies suggest that the fly uses the inositol lipid signaling system for visual excitation and that the Drosophila transient receptor potential (trp) mutation disrupts this process subsequent to the production of IP3. In this paper, we show that trp encodes a novel 1275 amino acid protein with eight putative transmembrane segments. Immunolocalization indicates that the trp protein is expressed predominantly in the rhabdomeric membranes of the photoreceptor cells.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Depletion of intracellular calcium stores activates a calcium current in mast cells.

              In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.
                Bookmark

                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                22 September 1997
                : 138
                : 6
                : 1333-1341
                Affiliations
                Institut für Pharmakologie, Thielallee 69-73, Freie Universität Berlin, D-14195 Berlin, Germany
                Article
                10.1083/jcb.138.6.1333
                2132548
                9298988
                b30914e6-bd1f-47de-a972-9251fccd7287
                Copyright @ 1997
                History
                : 9 May 1997
                : 4 July 1997
                Categories
                Article

                Cell biology
                Cell biology

                Comments

                Comment on this article