RyR1 and RyR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells
Daniela Rossi1,
Ilenia Simeoni1,
Marcella Micheli1,
Martin Bootman2,
Peter Lipp2,
Paul D. Allen3 and
Vincenzo Sorrentino1,*
1 Molecular Medicine Section, Department of Neuroscience, University of Siena,
via Aldo Moro 5, 53100 Siena, Italy
2 Laboratory of Molecular Signalling, The Babraham Institute, Babraham,
Cambridge CB2 4AT, UK
3 Department of Anesthesia Brigham and Women's Hospital, Boston, MA 02115,
USA
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Fig. 1. Westen blot analysis and [3H]ryanodine-binding assay on RyR1-
and RyR3-expressing clones. (A) 50 µg of microsomal proteins obtained from
RyR1- or RyR3-expressing clones were separated on 5% SDS-PAGE and analysed by
western blot with anti-RyR1 or anti-RyR3 polyclonal antibodies. (B) 50 µg
of RyR3 microsomes from RyR1- or RyR3-expressing clones were incubated for 1.5
hours at 36°C with 20 nM [3H]ryanodine in a solution containing
0.2 M KCl, 10 mM Hepes pH 7.4, 10 µM Ca2+. Nonspecific binding
was determined by the addition of a 1000-fold excess of unlabeled ryanodine
(20 µM). The amount of [3H]ryanodine bound was measured by
membrane filtration on Whatman GF-B filters.
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Fig. 2. Subcellular distribution of RyR1 and RyR3 in transfected cells. Cells
decorated with monoclonal antibodies against RyR1 and RyR3 were analysed by
epifluorescence microscopy.
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Fig. 3. Colocalization experiments of RyR3 with endoplasmic reticulum markers.
RyR3-expressing cells were decorated with polyclonal antibodies against
calreticulin or Ins(1,4,5)P3R1 and with monoclonal
antibodies angainst SERCA pump. Calreticulin was detected by a FITC conjugated
anti-goat antibody. Ins(1,4,5)P3R1 was detected by an
Alexa-conjugated anti-rabbit antibody. SERCA pump was detected by a
Cy3-conjugated anti-mouse antibody. The Golgi apparatus was stained
by transfecting the RyR3-expressing cells with the cDNA for a Green
Fluorescent Protein containing a Golgi targeting signal. Cells were
counterstained with monoclonal antibodies against RyR3 and TRITC-,
Cy3- or Alexa-conjugated anti-mouse secondary antibodies. Panels
show representative sections from top to bottom of the cells at about 1 µm
intervals.
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Fig. 4. Caffeine response of RyR1- and RyR3-expressing cells. RyR1- or
RyR3-expressing HEK 293 cells were loaded with 5 µM Fura 2-AM for 30
minutes and analyzed for caffeine-induced Ca2+ release at
excitation wavelength of 340 and 380 nM. Release was normalized on the maximum
Ca2+ release at 5 mM caffeine. Bars indicate the mean±s.e.m.
of the percentage of Ca2+-release increments induced by different
caffeine concentrations.
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Fig. 5. RyR3-expressing cells display spontaneous localized Ca2+
signals. (B) Typical spontaneous Ca2+ signals recorded using
confocal microscopy of single Fluo-3-loaded RyR3-expressing cells. The
subcellular regions from which the localized Ca2+ signals were
recorded are depicted by white circles. The dashed lines indicate the borders
of the cells. A and C illustrate the lack of spontaneous Ca2+
signals in RyR1-expressing cells and control cells, respectively.
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Fig. 6. Variable characteristics of spontaneous Ca2+ signals in
RyR3-expressing cells. A and B illustrate the considerable variability of
spontaneous Ca2+ signals in two RyR3-expressing cells. The traces
in Aa and Ba depict the Ca2+ changes in the regions along the line
scan images marked by the arrows. Panel C depicts the effect of 100 µM
ryanodine treatment on spontaneous Ca2+-release activity in a
single RyR3-expressing cell.
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© The Company of Biologists Ltd 2002
