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EMBO J. Apr 1, 1997; 16(7): 1575–1581.
PMCID: PMC1169761

Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells.

Abstract

Theoretical models and indirect experimental observations predict that Ca2+ concentrations at the inner surface of the plasma membrane may reach, upon stimulation, values much higher than those of the bulk cytosol. In the past few years, we have shown that the Ca2+-sensitive photoprotein aequorin can be intracellularly targeted and utilized for specifically monitoring the [Ca2+] of various organelles. In this work, we extend this approach to the study of the cytoplasmic rim beneath the plasma membrane. We have constructed a new aequorin chimera by fusing the photoprotein with SNAP-25, a neuronal protein which is recruited to the plasma membrane after the post-translational addition of a lipid anchor. The SNAP-25-aequorin chimera, expressed in the rat aortic smooth muscle cell line A7r5, appears correctly sorted as revealed by immunocytochemistry. Using this probe, we demonstrate that the mean [Ca2+] of this cytoplasmic region ([Ca2+]pm) can reach values >10-fold higher than those of the bulk cytosol ([Ca2+]c) upon activation of Ca2+ influx through plasma membrane channels. In unstimulated cells, the mean [Ca2+]pm appears also to be higher than the bulk cytosol, presumably reflecting the existence of microdomains of high [Ca2+].

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Selected References

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  • Silver RB, Sugimori M, Lang EJ, Llinás R. Time-resolved imaging of Ca(2+)-dependent aequorin luminescence of microdomains and QEDs in synaptic preterminals. Biol Bull. 1994 Dec;187(3):293–299. [PubMed]
  • Skutella M, Rüegg UT. Increase of empty pool-activated Ca2+ influx using an intracellular Ca2+ chelating agent. Biochem Biophys Res Commun. 1996 Jan 26;218(3):837–841. [PubMed]
  • Brini M, Murgia M, Pasti L, Picard D, Pozzan T, Rizzuto R. Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin. EMBO J. 1993 Dec;12(12):4813–4819. [PMC free article] [PubMed]
  • Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R. Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. J Biol Chem. 1995 Apr 28;270(17):9896–9903. [PubMed]
  • Zweifach A, Lewis RS. Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback. J Gen Physiol. 1995 Feb;105(2):209–226. [PMC free article] [PubMed]
  • Byron K, Taylor CW. Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells. J Physiol. 1995 Jun 1;485(Pt 2):455–468. [PMC free article] [PubMed]
  • Clapham DE. Calcium signaling. Cell. 1995 Jan 27;80(2):259–268. [PubMed]
  • Etter EF, Kuhn MA, Fay FS. Detection of changes in near-membrane Ca2+ concentration using a novel membrane-associated Ca2+ indicator. J Biol Chem. 1994 Apr 1;269(13):10141–10149. [PubMed]
  • Etter EF, Minta A, Poenie M, Fay FS. Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5368–5373. [PMC free article] [PubMed]
  • Fasolato C, Innocenti B, Pozzan T. Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends Pharmacol Sci. 1994 Mar;15(3):77–83. [PubMed]
  • Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA, Lerner RA, Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. [PMC free article] [PubMed]
  • Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed]
  • Heidelberger R, Heinemann C, Neher E, Matthews G. Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature. 1994 Oct 6;371(6497):513–515. [PubMed]
  • Hess DT, Slater TM, Wilson MC, Skene JH. The 25 kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS. J Neurosci. 1992 Dec;12(12):4634–4641. [PubMed]
  • Hille B, Woodhull AM, Shapiro BI. Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH. Philos Trans R Soc Lond B Biol Sci. 1975 Jun 10;270(908):301–318. [PubMed]
  • Hoth M, Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. [PubMed]
  • Hoth M, Penner R. Calcium release-activated calcium current in rat mast cells. J Physiol. 1993 Jun;465:359–386. [PMC free article] [PubMed]
  • Inouye S, Noguchi M, Sakaki Y, Takagi Y, Miyata T, Iwanaga S, Miyata T, Tsuji FI. Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc Natl Acad Sci U S A. 1985 May;82(10):3154–3158. [PMC free article] [PubMed]
  • Kimes BW, Brandt BL. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp Cell Res. 1976 Mar 15;98(2):349–366. [PubMed]
  • Llinás R, Sugimori M, Silver RB. Microdomains of high calcium concentration in a presynaptic terminal. Science. 1992 May 1;256(5057):677–679. [PubMed]
  • Llinás R, Sugimori M, Silver RB. The concept of calcium concentration microdomains in synaptic transmission. Neuropharmacology. 1995 Nov;34(11):1443–1451. [PubMed]
  • Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T, Rizzuto R. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J. 1995 Nov 15;14(22):5467–5475. [PMC free article] [PubMed]
  • Rizzuto R, Simpson AW, Brini M, Pozzan T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature. 1992 Jul 23;358(6384):325–327. [PubMed]
  • Rizzuto R, Brini M, Murgia M, Pozzan T. Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science. 1993 Oct 29;262(5134):744–747. [PubMed]
  • Rizzuto R, Bastianutto C, Brini M, Murgia M, Pozzan T. Mitochondrial Ca2+ homeostasis in intact cells. J Cell Biol. 1994 Sep;126(5):1183–1194. [PMC free article] [PubMed]
  • Rizzuto R, Brini M, Bastianutto C, Marsault R, Pozzan T. Photoprotein-mediated measurement of calcium ion concentration in mitochondria of living cells. Methods Enzymol. 1995;260:417–428. [PubMed]

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