From the * Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106; Department of
Physiology, University of Maryland, Baltimore, Maryland 21201; and § Department of Pharmacology, University of Tokyo, Tokyo 113, Japan
The ryanodine receptor (RyR)/Ca2+ release channel is an essential component of excitation-contraction coupling in striated muscle cells. To study the function and regulation of the Ca2+ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca2+]. Unlike the native RyR channels in muscle cells, which display localized Ca2+ release events (i.e., "Ca2+ sparks" in cardiac muscle and "local release events" in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca2+ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca2+ release events observed in muscle cells may involve gating of a group of Ca2+ release channels and/or interaction of RyR with muscle-specific proteins.
Key words: excitation-contraction coupling; calcium sparks; Chinese hamster ovary cells; Fura-2; green fluorescent proteinRyanodine receptor (RyR)1 is an essential component of
excitation-contraction (E-C) coupling in striated muscles, where it functions as a Ca2+ release channel in the
sarcoplasmic reticulum (SR) membrane (Fleischer and
Inui, 1989; McPherson and Campbell, 1993
; Sutko and
Airey, 1996
). RyR is a single polypeptide of ~560 kD,
and exists in a homotetrameric structure with at least
two functional domains: a carboxyl-terminal hydrophobic domain containing the conduction pore of the
Ca2+ release channel (Takeshima et al., 1989
; Zorzato
et al., 1990
; Bhat et al., 1997b
), and a large amino-terminal cytoplasmic domain referred to as the "foot
structure" (Block et al., 1988
; Lai et al., 1989
; Wagenknecht et al., 1989
; Franzini-Armstrong and Jorgensen, 1994
). The activity of the skeletal muscle Ca2+ release
channel is modulated by a wide spectrum of endogenous and exogenous ligands (Coronado et al., 1994
;
Meissner, 1994
; Zucchi and Ronca-Testoni, 1997
). Cytoplasmic Ca2+ activates the channel in nanomolar to
micromolar concentrations, while in the micromolar to
millimolar concentrations it inactivates the channel
(Lai et al., 1988
; Ma et al., 1988
; Smith et al., 1988
; Ma
and Zhao, 1994
). This dual effect of Ca2+ is thought to
involve two independent high and low affinity Ca2+
binding sites on the RyR protein (Chu et al., 1993
; Laver et al., 1995
). Caffeine is one of the most widely used
exogenous activators of the Ca2+ release channel (Zucchi and Ronca-Testoni, 1997
). Rousseau et al. (1988)
demonstrated a direct activation by caffeine of the isolated Ca2+ release channel reconstituted in planar lipid
bilayer. Caffeine has been shown to activate Ca2+ release from SR by increasing the apparent affinity of the
Ca2+ activation site for Ca2+ (Rousseau et al., 1988
;
Meissner et al., 1997
). However, the site(s) on the Ca2+
release channel protein responsible for caffeine effect
have not yet been identified.
Understanding the function and regulation of the
Ca2+ release process is important in elucidating the
mechanism(s) of E-C coupling. Recently, spontaneous
local increases in the concentration of intracellular
Ca2+, called "Ca2+ sparks," have been observed in cardiac muscle (Cheng et al., 1993). The Ca2+ release process during normal cardiac E-C coupling has been suggested to result from the spatial and temporal summation of these elementary events of release (Cheng et al.,
1993
, 1996
; Cannell et al., 1994
, 1995
; Lopez-Lopez et
al., 1994
, 1995
). Studies in skeletal muscle have revealed
these spontaneous Ca2+ release events to be smaller
than cardiac sparks, suggesting that these events are
controlled by different mechanisms in skeletal and cardiac muscles (Tsugorka et al., 1995
; Klein et al., 1996
; Blatter et al., 1997
). It is not yet clear as to whether the
"functional Ca2+ release units" underlying these events
consist of a single or a cluster of Ca2+ release channels.
The RyRs of skeletal (Takeshima et al., 1989; Zorzato
et al., 1990
) and cardiac (Nakai et al., 1990
; Otsu et al.,
1990
) muscle are coded by different genes, and share a
high degree of sequence identity, especially in the carboxyl-terminal regions where the two proteins are well
conserved in their amino acid sequence (Sorrentino and Volpe, 1993
; Takeshima, 1993
). This region of the
protein contains several putative transmembrane segments (Takeshima et al., 1989
; Zorzato et al., 1990
),
and the binding site(s) for Ca2+ and ryanodine (Callaway et al., 1994
; Witcher et al., 1994
). In recent studies,
we have successfully used heterologous expression system to study the structure-function relationship of the
Ca2+ release channel (Bhat et al., 1997a
, 1997b
). Full
length skeletal RyR expressed in Chinese hamster ovary
(CHO) cells exhibits single channel properties similar
to those of RyR from skeletal muscle SR. The carboxyl-terminal ~20% of the RyR (RyR-C) was found to contain structures sufficient to form a functional Ca2+ release
channel. The opening of the truncated RyR channel requires micromolar concentrations of Ca2+. However,
unlike the full length RyR channel, the RyR-C channel fails to close at high cytoplasmic Ca2+ concentrations
(Bhat et al., 1997b
). Moreover, the amino-terminal foot
structure also appears to participate in both ion conduction and Ca2+-dependent regulation of the Ca2+ release channel (Bhat et al., 1997a
, 1997b
).
In the present study, we have investigated the effect of caffeine on the Ca2+ release channel function of the full length and carboxyl-terminal portion of skeletal muscle RyR expressed in CHO cells using three different approaches: (a) single channel measurements using planar lipid bilayer reconstitution, (b) intracellular Ca2+ release measurements using Ca2+-sensitive dye Fura-2, and (c) Ca2+ imaging in single cells using laser scanning confocal microscopy. Caffeine was found to induce release of intracellular Ca2+ in cells expressing full length RyR in a concentration-dependent manner. This cooperative gating of the RyR channel is consistent with the Ca2+-induced Ca2+ release mechanism proposed for E-C coupling in striated muscles. However, caffeine did not show any effect on the function of the carboxyl-terminal portion of RyR, suggesting that the region(s) responsible for caffeine effect reside in the amino-terminal domain of the protein. Cells expressing the truncated RyR were found to have a higher resting intracellular Ca2+ compared with untransfected cells or those expressing full length RyR, which may be the result of spontaneous changes of intracellular Ca2+ observed in these cells. Furthermore, Ca2+ spark signals, which underlie localized Ca2+ release in muscle cells, were not present in CHO cells expressing either the full length or the carboxyl-terminal portion of RyR.
Cells and Expression System
The entire sequence of the rabbit skeletal muscle RyR cDNA
(15.3 kb, RyR-wt) was cloned into the pRRS11 expression vector and the transcription occurs under the control of the SV40 promoter (Penner et al., 1989; Takeshima et al., 1989
). A deletion
mutant of pRRS11, pRyR-C, was generated through digestion
with SalI (nt 546) and XhoI (nt 12018) restriction enzymes and
religation. pRyR-C lacks the nucleotide sequence from 546 to
12018, which corresponds to a deletion of amino acids from 183 through 4006 (Bhat et al., 1997b
). CHO cells were grown at 37°C
and 5% CO2 in Ham's F-12 medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The expression plasmids were introduced into the cells (50-
60% confluent) using lipofectamine reagent (Life Technologies,
Inc., Grand Island, NY). Stable transfectant cells were selected
with G418 (0.5 mg/ml) 48 h after transfection. The level of RyR
protein expression was tested using Western blot analysis.
Western Blot Analyses
Normal and transfected CHO cells were harvested and washed
twice with ice cold PBS and lysed with ice cold modified RIPA
buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1 mM EGTA, 1%
Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% sodium
deoxycholate) in the presence of protease inhibitors (0.5 mM Pefabloc, 1 µM pepstatin, 1 µM leupeptin, 1 µg/ml aprotinin, and
1 mM benzamidine). The proteins in the whole cell lysate were
mixed with the sample buffer (200 mM Tris-Cl, pH 6.7, 9% SDS,
6% -mercaptoethanol, 15% glycerol, 0.01% bromophenol blue)
and separated on a 3-12% linear SDS-PAGE gel after heating the
samples at 85°C for 5 min. The proteins were then transferred to
a polyvinylidene difluoride membrane and blotted with RR2
monoclonal antibody and horseradish peroxidase-linked secondary antibody using the electrochemiluminescence detection system (Amersham Corp., Arlington Heights, IL). The epitope
for RR2 monoclonal antibody is known to reside in the carboxyl-terminal 656 amino acid region of the skeletal muscle RyR
(Takeshima et al., 1993
).
Expression and Detection of Fluorescence of Green Fluorescent Protein Fusion Proteins
The 15.2-kb HindIII-EcoRV fragment cDNA encoding the full
length rabbit skeletal muscle RyR was cloned into pcDNA3 expression vector (Invitrogen Corp., San Diego, CA) to get pcDNA3-RyR. Similarly, a 4.2-kb BamHI-EcoRV fragment (nt 10982-
15230) encoding the carboxyl-terminal (amino acids 3661-5037)
portion of RyR was cloned into pcDNA3 expression vector (Invi-trogen Corp.) to generate pcDNA3-RyR-C. The cDNA encoding
the green fluorescent protein (GFP, 720 bp, 240 residues) was
generated by PCR using pEGFP-N1 (Clontech, Palo Alto, CA) as
template, with HindIII-KOZAK-6xHis sequence added to the 5
terminal end and KpnI to the 3
terminal end for ease of cloning.
This sequence (HindIII-KOZAK-6xHis-GFP-KpnI) was then cloned
in-frame to pcDNA3-RyR, by replacing the sequence (nt 1-866) that encodes the aminoterminal first 289 amino acids of RyR, to give pcDNA3 (GFP-RyR). A similar sequence (HindIII-KOZAK-6xHis-GFP-BamHI) was cloned into pcDNA3-RyR-C in tandem
on the 3
terminal end of RyR sequence to give pcDNA3 (GFP-RyR-C). Transfection of these plasmids into CHO cells was done
as described above, using pEGFP-N1 to express GFP alone as positive control. Cells expressing GFP alone, GFP-RyR, and GFP-RyR-C
fusion proteins were grown on coverslips and fixed with 4%
paraformaldehyde and placed on a glass slide in inverted position for viewing. The cells are visualized with a Zeiss laser scanning confocal microscope using a 100× oil immersion objective
(Carl Zeiss, Inc., Thornwood, NY). An FITC dichroic filter set
with excitation at 450-490 nm and emission at 520 nm was used.
Measurement of Cytoplasmic [Ca2+]
All experiments were performed in saline solution containing
140 mM NaCl, 2.8 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 12 mM
glucose, 0.1% (wt/vol) bovine serum albumin, and 10 mM
HEPES-NaOH, pH 7.2. Normal and transfected CHO cells were
loaded with 2 µM Fura-2 AM (Molecular Probes Inc., Eugene,
OR) for 45 min at room temperature. To remove nonhydrolyzed
dye, cells were washed twice and resuspended at ~106 cells/ml.
Fluorescence (339 nm excitation/500 nm emission) was measured using cells maintained under continuous stirring in a cuvette at room temperature using previously described instrumentation (Dubyak and De Young, 1985). Ca2+-dependent fluorescence was calibrated using standard techniques as described
(Grynkiewiz et al., 1985; Lam et al., 1993
).
Confocal Imaging of Intracellular [Ca2+]
Single rat cardiac ventricular cells were obtained from 2-mo-old
Sprague-Dawley rats by an enzymatic technique described in detail previously (Lopez-Lopez et al., 1995). Both cardiac and CHO
cells were loaded with the Ca2+ indicator Fluo-3 by incubation for
30 min or longer in Tyrode's solution to which 10 µM Fluo-3 AM
was added (Molecular Probes Inc.). Recordings of [Ca2+] were
made in normal Tyrode's solution (composition [mM]: 140 NaCl, 10 dextrose, 10 Hepes, 4.0 KCl, 1 MgCl2, 1 CaCl2, pH adjusted to 7.3-7.4 with NaOH) at room temperature.
For `x-y' or `full-frame' imaging of calcium in the CHO cells,
an MRC 600 confocal microscope (Bio-Rad Laboratories, Richmond, CA) was used. Fluo-3 fluorescence line-scan images were
acquired with the `homebrew' confocal microscope (Parker et
al., 1997) and were expressed as normalized increases in fluorescence compared with `resting' level (F/F0) (see Figs. 5-7), or, in
some cases (see Fig. 8), simply in arbitrary units. Images of F/F0
were produced by dividing the fluorescence intensity (F) of each
pixel in the original fluorescence image by its intensity at the beginning of the image (defined as F0), during a time when the cell
was assumed to be in the `resting' state. All line-scan recordings of Fluo-3 fluorescence on single cells were obtained with the homebrew confocal microscope attached to the camera port of a Nikon Diaphot inverted microscope equipped with a 60× plan-apo oil-immersion objective (numerical aperture 1.4; Nikon Inc.,
Melville, NY), and the resolution was always 3 ms per scan line. As
reported earlier, the spatial resolution of this instrument was
near diffraction limited, at 300 nm laterally and 400 nm vertically
(Parker et al., 1997
). In some cases (see Fig. 5 B), the image was
compressed (using neighborhood averaging) to display the fluorescence over a long period of time. Full frame fluorescence images of Fluo-3 fluorescence (see Fig. 8) were obtained with an
MRC-600 confocal microscope (Bio-Rad Laboratories).
The excitation of Fluo-3 was achieved using the 488-nm line from a 100 megawatt argon-ion laser (Omnichrome, Chino, CA), attenuated to 1-10%, beam expanded to overfill the back aperture of the objective, and deflected by a galvanometer-driven scan mirror (Cambridge Technology, Inc., Cambridge, MA) positioned at a conjugate telocentric plane formed by an eyepiece lens placed in the camera port. Fluorescence emission was de-scanned by the same mirror and wavelengths >510 nm were directed onto a confocal aperture placed in front of an avalanche photodiode. Computations and image analysis were performed on a Risc System/6000 workstation (IBM, Armonk, NY) with the software IDL (Research Systems, Inc., Boulder, CO).
Isolation of Microsomal Membrane Vesicles from CHO Cells
Microsomal membrane vesicles were isolated from transfected
CHO cells as described (Bhat et al., 1997b). The cells were homogenized on ice in hypotonic lysis buffer (10 mM HEPES, pH
7.4, 1 mM EDTA) containing protease inhibitors (0.5 mM pefabloc-SC, 1 µM pepstatin, 1 µM leupeptin, 1 µg/ml aprotinin,
and 1 mM benzamidine) using nitrogen cavitation (300 psi for
15 min) with 10 strokes in tight-fitting Dounce homogenizer
(Wheaton, Milville, NJ), followed by 15 strokes after addition of
an equal volume of sucrose buffer (500 mM sucrose, 10 mM
HEPES, pH 7.4, 1 mM EDTA). Microsome vesicles were collected by centrifugation of postnuclear supernatant (10,000 g, 15 min) at 100,000 g for 45 min at 4°C. The pellet was resuspended in a
buffer containing 250 mM sucrose, 10 mM HEPES, pH 7.2. The
membrane vesicles were stored at a protein concentration of 2-6
mg/ml at
75°C until use. Usually, 1-3 µl of microsomal membrane vesicles was used for reconstitution of Ca2+ release channels
in the lipid bilayer system.
Reconstitution of Ca2+ Release Channels in Lipid Bilayer Membrane
Lipid bilayer membranes were formed across an aperture of
~200 µm diameter using the Muller-Rudin method with a mixture of phosphatidylethanolamine:phosphatidylserine:cholesterol
(6:6:1); the lipids were dissolved in decane at a concentration of
40 mg/ml. Incorporation of the Ca2+ release channel in bilayer
was achieved by addition of membrane vesicles containing RyR-wt or RyR-C proteins to the cis solution, under a concentration
gradient of 200 mM (cis)/50 mM (trans) cesium-gluconate. A
symmetrical 200-mM Cs-gluconate was used as current carrier in
the presence of 1 mM ATP. The pH in both cis and trans solution was maintained throughout the experiment at 7.4 with 10 mM
HEPES-Tris. The free Ca2+ concentrations in both solutions were
buffered with 1 mM EGTA, and measured using a Ca2+-sensitive
electrode (Orion Research, Boston, MA). Orientation of the
Ca2+ release channel in the lipid bilayer, usually in the cis-cytoplasmic trans-lumenal SR manner, was determined by the sensitivity of the channel to cytoplasmic Ca2+ (Bhat et al., 1997b). Channels oriented in the opposite direction, which account for <10%
of the experiments, were not used in the present study. To maintain stability of the bilayer membrane and the Ca2+ release channel activities, designed pulse protocols were used to measure currents through the single Ca2+ release channels. The bilayer membrane was kept at a holding potential of 0 mV, and pulsed to
different test potentials of 0.5-1-s durations. Single channel currents were recorded with an Axopatch 200A patch clamp unit
(Axon Instruments, Inc., Foster City, CA). Data acquisition and
pulse generation were performed with a 486 computer and 1200 Digidata A/D-D/A convertor (Axon Instruments, Inc.). The currents were sampled at 0.05 ms/point and filtered at 1 kHz
through an 8-pole Bessel filter. Single channel data analyses were
performed with pClamp, TIPS, and custom programs.
Full Length and Carboxyl-Terminal RyRs are Expressed in Intracellular Membranes of CHO Cells
c1148 is a stable Chinese hamster ovary cell line that
was transfected with pRRS11, which encodes the entire
sequence of the rabbit skeletal muscle ryanodine receptor (Penner et al., 1989; Takekura et al., 1995
). To
study the function and regulation of the carboxyl-terminal RyR channel, we have generated another clone of CHO cells (ryr-c) that was stably transfected with a
deletion mutant of RyR, pRyR-C. This mutant lacks
3,824 amino acids (amino acids 183-4006) from the cytoplasmic domain, but retains the carboxyl-terminal putative transmembrane domain of RyR, and has a predicted molecular weight of ~130 kD (Bhat et al., 1997b
).
The ryr-c cells expressed detectable amounts of the 130-kD RyR-C proteins (Fig. 1, lanes 3 and 6), recognized
by a monoclonal antibody (RR2) whose epitope resides
in the COOH-terminal region of skeletal muscle RyR
(Takeshima et al., 1989
). The c1148 cells expressed a
high level of the full length RyR proteins (RyR-wt) (Fig.
1, lane 7), which was identical to the native RyRs from
the rabbit skeletal muscle SR (Fig. 1, lane 1), in terms
of molecular weight and immunoreactivity. No proteins
were recognized by RR2 in untransfected CHO cells
(Fig. 1, lane 2), or in those transfected with pcDNA3
alone, suggesting that CHO cells do not contain any detectable amount of endogenous skeletal isoform of RyR
proteins.
For subcellular localization of RyR proteins expressed in CHO cells, a fusion protein (GFP-RyR) was
generated by replacing the amino-terminal first 289 amino acids of RyR-wt with those of the green fluorescent protein. In addition, a GFP-RyR-C construct was
obtained by linking the GFP sequence in tandem with
the COOH-terminal 1,377 amino acids of RyR (amino
acids 3661-5037). For positive controls, CHO cells were
transfected with pEGFP-N1 that encodes only GFP. As
shown in Fig. 2, cells transfected with pEGFP-N1 exhibited a diffuse pattern of fluorescence, as expected for a soluble protein (Fig. 2 A), whereas cells expressing
GFP-RyR and GFP-RyR-C fusion proteins exhibited a
fluorescence signal only in certain subcellular areas,
particularly in the perinuclear region (Fig. 2, B and C),
indicating that the protein is probably localized to the
endoplasmic reticulum (ER) membrane of CHO cells. Both GFP-RyR and GFP-RyR-C proteins were detected
in these cells by the RR2 antibody in a Western blot
(Fig. 1, lanes 5 and 4, respectively). These results suggest that the carboxyl-terminal portion of RyR contains
the signal for retention of the RyR protein in the intracellular membranes.
Caffeine Enhances Activity of the Wild-Type, Not the COOH-Terminal RyR Ca2+ Release Channels
The Ca2+ release channel functions of the expressed
RyR-wt and RyR-C were measured by reconstituting microsomal membrane vesicles from the c1148 and ryr-c
cells into lipid bilayer membranes, using Cs-gluconate
as current carrying ion in the recording solution. In
previous studies, we showed that RyR-C forms a functional Ca2+ release channel that shared some of the single channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine
(Bhat et al., 1997b). But unlike the RyR-wt channel, which
exhibited a linear current-voltage relationship and inactivated at millimolar Ca2+, the channels formed by RyR-C
displayed significant inward rectification and failed to
close at high cytoplasmic Ca2+ (Bhat et al., 1997b
).
Fig. 3 shows that openings of both RyR-wt and RyR-C
channels were sensitive to activation by cytoplasmic
Ca2+, as chelating the free [Ca2+]i from 220 to 0.08 µM
resulted in decrease in open probability (Po) of the
RyR-wt channel from Po = 0.121 ± 0.044 to 0.013 ± 0.002, and that of the RyR-C channel from Po = 0.201 ±
0.036 to 0.008 ± 0.003. It is interesting to note that the
Po of RyR-C is higher than RyR-wt at free [Ca2+] of 220 µM, which could be due to the lack of Ca2+-dependent
inactivation of the RyR-C channel (Bhat et al., 1997b). The RyR-wt channel was sensitive to activation by caffeine, as addition of 10 mM caffeine to the cytoplasmic
solution resulted in a significant increase of Po to 0.198 ± 0.063 at [Ca2+]i = 0.08 µM (Fig. 3 A). In contrast,
the activity of the RyR-C channel did not change upon
the addition of 10 mM caffeine (Fig. 3 B). Furthermore, the lack of effect of caffeine on the RyR-C channel was independent of [Ca2+]i in a concentration
range from 0.08 to 220 µM (data not shown).
To further test the effect of caffeine on the Ca2+ release channels, we measured the change in cytoplasmic
[Ca2+] in c1148 and ryr-c cells after stimulation with caffeine. The measurements were performed with a large
number of cells (1-2 × 106) loaded with a fluorescent
Ca2+ indicator, Fura-2, which provides a macroscopic
assessment of the caffeine sensitivity of the RyR-wt and
RyR-C channels. Untransfected CHO cells had negligible amounts of caffeine-induced Ca2+ release, but they
maintained an intact intracellular Ca2+ pool, as addition of 500 nM thapsigargin resulted in a rise in [Ca2+]i
due to inhibition of the Ca2+-ATPase in the ER (Fig. 4
A). With the c1148 cells, we observed a robust increase
of cytoplasmic [Ca2+] from a resting level of 0.192 ± 0.038 µM to 1.222 ± 0.257 µM upon stimulation with
10 mM caffeine (Fig. 4 B). This rapid rise of [Ca2+]i in
c1148 cells was followed by a slow decline phase, probably reflecting the exit of Ca2+ to the extracellular solution through the plasma membrane Ca2+ pump or Na/
Ca2+ exchanger. Different from the c1148 cells, the ryr-c
cells did not respond to caffeine (Fig. 4 C), and the
thapsigargin-sensitive ER Ca2+ pool appears to be
smaller in the ryr-c cells than in the untransfected CHO
cells (comparing Fig. 4, A and C), although the response to thapsigargin was variable (Table I). The ryr-c
cells, however, had a resting [Ca2+]i = 0.422 ± 0.071 µM, which was significantly higher than that of the
c1148 cells and the untransfected CHO cells ([Ca2+]i = 0.209 ± 0.012 µM). These results are summarized in
Table I.
Table I. Caffeine-induced Ca2+ Release in CHO Cells Expressing RyR-wt and RyR-C* |
Caffeine Exhibits Concentration-dependent Effect on RyR-wt Channels Expressed in CHO Cells
Confocal images of individual c1148 cells expressing
the full length skeletal muscle RyR showed a rapid caffeine-induced Ca2+ release from the intracellular membranes (Fig. 5 A). The effect of caffeine is reversible,
since [Ca2+]i returned completely to the resting level
upon washout of caffeine (Fig. 5 A, c). Having established a positive caffeine response, we next obtained a
series of line-scanning images of the same cell after reapplication of 10 mM caffeine (Fig. 5 B). These line scanning pictures show that the distribution of [Ca2+]i
is inhomogeneous in response to caffeine. There appeared to be `hot spots' where the level of [Ca2+]i is
higher than other places, as indicated by the arrows in Fig. 5 B. These hot spots may correspond to regions
where the Ca2+ release channels are clustered together
(Takekura et al., 1995). Also, the overall increase in
[Ca2+]i is biphasic in that caffeine-induced transient
rise in [Ca2+]i was followed by a slow decaying phase,
consistent with the observation shown in Fig. 4 B. The
rapid increase in [Ca2+]i may underline cooperative
features of the Ca2+ release process.
To further examine the caffeine sensitivity of RyR-wt,
we tested different concentrations of caffeine (ranging
from 0.1 to 10 mM) in c1148 cells. Fig. 6 shows the concentration of intracellular Ca2+ measured at a given position from three c1148 cells exposed to 0.5, 2, and 10 mM caffeine. From the dose-response relationship, it is
clear that caffeine exhibits a concentration-dependent
Ca2+ release in these cells. The threshold for caffeine
activation of the RyR-wt channels occurred at a concentration between 0.1 and 0.5 mM. The time course of increase in [Ca2+]i had a strong correlation with the concentration of caffeinethe lower the caffeine concentration, the slower the time course. It is evident that
there is a plateau phase preceding the rise in [Ca2+]i,
in particular at lower caffeine concentrations (0.5 and
2 mM). The cooperative nature of the response is clear
in the sigmoidal onset of Ca2+ release, which is most evident at 2.0 and 10 mM caffeine (Fig. 6, B and C).
RyR Channels Expressed in CHO Cells Do Not Exhibit "Localized Ca2+ Release" Typical of Muscle Cells
In cardiac muscle cells, local increases in intracellular
Ca2+ have been observed that occur spontaneously
(Cheng et al., 1993), as well as in response to activation
of voltage-gated Ca2+ channels (Lopez-Lopez et al., 1994
,
1995
; Cannell et al., 1994
, 1995
). Elementary Ca2+ release events, similar to those in cardiac muscle, although smaller in size, have also been recorded in skeletal muscle cells (Tsugorka et al., 1995
). It is not known
if a single or a group of Ca2+ release channels acting in
concert constitute the "Ca2+ release units" underlying
the local Ca2+ transients in skeletal and cardiac muscles. We tested for the presence of spontaneous changes
in intracellular Ca2+ in CHO cells expressing skeletal
RyR (Fig. 7). Under resting conditions, no spontaneous
local Ca2+ transients were evident in the line-scan images of c1148 cells (Fig. 7 A), although caffeine was capable of inducing Ca2+ release in these cells (Fig. 7 B).
Furthermore, no local Ca2+ signals were observed in
c1148 cells stimulated with various concentrations of
caffeine (ranging from 0.1 to 10 mM, data not shown).
Under identical spatial and temporal resolution, typical Ca2+ sparks could be recorded in rat cardiac ventricular myocytes (Fig. 7 C). Therefore, the lack of local
Ca2+ transients in the CHO cells was not likely to be
due to inability to resolve such events had they been
present and similar to the calcium sparks typical of cardiac muscle.
CHO Cells Expressing RyR-C Exhibit Spontaneous Changes in [Ca2+]i
While CHO cells expressing the carboxyl-terminal portion of RyR did not respond to stimulation with caffeine (Figs. 3 B and 4 C), the intracellular [Ca2+] showed spontaneous changes in these cells, as shown in Fig. 8. This change in [Ca2+]i appears to occur due to release of Ca2+ from intracellular store since this response is restricted to an area of the cell surrounding the nucleus. During the period of observation (usually 10 min), these transient changes in [Ca2+]i were observed in ~10% of the CHO cells expressing RyR-C. The higher resting [Ca2+]i observed in these cells (Fig. 4 C) may be attributed to these changes in [Ca2+]i. No evidence for such changes in intracellular Ca2+ was found in c1148 cells. These spontaneous changes in intracellular Ca2+ in cells expressing truncated, but not full length, RyR suggest the importance of the amino-terminal foot region of the protein in the regulation of Ca2+ release channel.
The present study examined the function and regulation of skeletal muscle Ca2+ release channel through
heterologous expression of RyR in CHO cells. Using
this approach, we have recently demonstrated that the
carboxyl-terminal portion of RyR contains structures
sufficient to form a functional Ca2+ release channel,
but the amino-terminal portion of the protein also contributes to the ion conduction and regulation of the
channel (Bhat et al., 1997a, 1997b
). In the present
study, we show that the RyR protein is localized to the
intracellular organelle, possibly ER, of CHO cells based
upon the fluorescence pattern of GFP-RyR fusion proteins (Fig. 2). The sequence required for targeting of
the RyR protein to the intracellular membrane is likely
to reside in the carboxyl-terminal end of the protein
since the COOH-terminal RyR protein is also localized
to the perinuclear area of CHO cells. In addition, an
earlier study by Takeshima et al. (1993)
showed that a
75-kD protein containing the carboxyl-terminal 656 amino acids of the skeletal muscle RyR was expressed
in the ER membranes of CHO cells.
Caffeine, an effective activator of Ca2+ release in both
skeletal and cardiac muscles, was found to induce release of Ca2+ in cells expressing full length RyR in a
dose-dependent manner. However, it failed to show
any effect on cells expressing the carboxyl-terminal
portion of RyR. The sigmoidal onset of Ca2+ release induced by caffeine in c1148 cells reveals the cooperative nature of the response (Fig. 6). There appears to be a
threshold level of intracellular [Ca2+] required before
a robust increase in [Ca2+]i can occur, especially at
lower concentrations of caffeine. It is possible that Ca2+
released from one single or a cluster of RyRs triggers
activation of adjacent Ca2+ release channels in these
cells. The affinity of Ca2+ activation site for Ca2+ in RyR
protein is increased in the presence of caffeine, causing a cooperative interaction between these sites. This is
consistent with the Ca2+-induced Ca2+ release mechanism proposed for activation of Ca2+ release in both
skeletal and cardiac muscles (Fabiato, 1983). The lack
of effect of caffeine in activating Ca2+ release from cells
expressing RyR-C suggests that the site(s) responsible
for caffeine effect are lost in the truncated RyR protein.
While the caffeine-binding site on the Ca2+-release
channel protein has not been identified, our data suggest the following possibilities: first, the caffeine binding site resides within the amino-terminal foot region
of the RyR protein; second, the binding site is formed
by the ensemble of both the amino- and the carboxyl-terminal portion of RyR; and third, the spatial configuration of the binding site is disrupted in the absence of
the amino-terminal portion of the protein. The lack of
effect of caffeine in cells expressing RyR-C is not likely due to the higher resting [Ca2+]i in these cells, since
caffeine was not able to influence the activity of single
RyR-C channels in cytoplasmic [Ca2+] ranging from
0.08 to 220 µM.
In cells expressing the RyR-C protein, the resting intracellular [Ca2+] was found to be higher than that in
control cells and in those expressing RyR-wt. Furthermore, spontaneous transient changes in intracellular
[Ca2+] were recorded in cells expressing the truncated
RyR, but not in those expressing the full length protein. The high level of resting [Ca2+]i and spontaneous
changes in Ca2+ in these cells could be due to several
possible factors. First, inability of the RyR-C channel to
close at higher cytosolic [Ca2+]. Unlike the native RyR
from skeletal muscle SR and the full length RyR expressed in CHO cells, RyR-C channels lack the apparent Ca2+-dependent inactivation property (Bhat et al.,
1997b). Recent studies by other investigators have demonstrated that dyspedic myotubes (skeletal muscle cells
lacking native RyR), when transfected with cardiac subtype of RyR, exhibit spontaneous oscillation of intracellular Ca2+ (Nakai et al., 1997
; Yamazawa et al., 1996
,
1997
). Thus, a sustained level of activation of the Ca2+
release channel may cause a higher intracellular Ca2+
concentration. It is interesting to note that the cardiac
Ca2+ release channels, unlike the skeletal subtype, are
less susceptible to inactivation by cytoplasmic Ca2+
(Chu et al., 1993
; Laver et al., 1997). It remains to be
seen if CHO cells expressing the cardiac RyR also exhibit any spontaneous changes in intracellular Ca2+.
Second, abnormally large conductance states associated with the RyR-C protein. In a recent study, we have
shown that RyR-C forms a large conductance channel
in ~20% of the single channel experiments, which
seems to be less sensitive to Ca2+ regulation (see Bhat
et al., 1997b
; Fig. 5). This large conductance channel
may constitute a leak in the ER membranes, which may
result in an elevated level of cytoplasmic [Ca2+]. Although the mechanism of this phenomenon is not understood, this may result from a defective or unstable
oligomerization of the truncated RyR protein. Thus,
the instability of the oligomeric structure together with
a greater tendency of the truncated RyR channel to
opening (see Fig. 3) may contribute to spontaneous increase in intracellular [Ca2+]i in cells expressing RyR-C
protein. Third, the truncated RyR protein may have altered interaction with accessory proteins, such as FK506
binding protein (FKBP12), which is known to regulate the function of the Ca2+ release channel (Brillantes et
al., 1994
; Ma et al., 1995
; Ahern et al., 1997
). While the
interaction of FKBP appears to be essential for setting
normal gating properties of RyR, channels depleted of
FKBP show a higher probability of channel opening, as
well as an increased sensitivity to activation by cytoplasmic Ca2+ (Brillantes et al., 1994
; Ahern et al., 1997
). It
is likely that the site of FKBP interaction with the Ca2+
release channel is missing in the truncated RyR protein
(Wagenknecht et al., 1996
).
In cells expressing full length or truncated RyR, no
evidence was found for the presence of spontaneous local increase in intracellular Ca2+ such as described in
cardiac muscle (i.e., sparks) (Cheng et al., 1993) and
skeletal muscles (Tsugorka et al., 1995
; Klein et al.,
1996
). In cardiac cells, Ca2+ sparks occur at about the
rate of 1/100 µm/s per cell, or ~100/s per cell (Cheng
et al., 1993
). In this study, at least 50 CHO cells expressing wild-type RyR were scanned over a distance of 25 µm (usually somewhat more than their diameter) for at
least 25 s. This amount of scanning of cardiac cells
would have produced 312.5 sparks. As shown in Fig. 7,
had such sparks occurred in the CHO cells (Fig. 7, A
and B), they would have been readily detectable since
Ca2+ sparks were readily detected in cardiac cells when
scanned at the same spatial and temporal resolution
(Fig. 7 C). The release of Ca2+ from each RyR in the
CHO cells seems to be too small to produce a resolvable
local Ca2+ transient, perhaps similar to the situation in
cardiac muscle, where small, unresolvable Ca2+ transients, termed "quarks" are thought to arise from Ca2+
released from a single RyR (Lipp and Niggli, 1996
).
The absence of local Ca2+ transients associated with
the expressed RyR, similar to those in muscle, may be
attributed to the following possible factors. First, the
absence of local cooperative opening of multiple channels in CHO cells, which is thought to underlie the
origin of sparks. Although the channels exhibit Ca2+-
induced Ca2+ release, as seen in response to stimulation with caffeine, the spatial distribution of protein
may not mimic that seen in muscle cells. Second, the
absence (in CHO cells) of muscle-specific proteins involved in E-C coupling. In skeletal muscle, the dihydropyridine receptor (DHPR) functions as a voltage sensor
and is thought to be in close interaction with the RyR
(Rios et al., 1991). Recent studies have suggested that
RyR not only receives a signal from DHPR, but also
transmits a retrograde signal back to regulate the activity of DHPR in skeletal muscle and neurons (Chavis et al., 1996
; Nakai et al., 1996
). The interaction between
DHPR and RyR appears to be tissue specific since coexpression of these two proteins in CHO cells failed to
form junctions between plasma and ER membranes
such as typically exists between transverse tubule and
SR membranes in muscle cells (Takekura et al., 1995
).
Thus, an intimate juxtaposition of RyR, DHPR, and/or
other accessory proteins may be essential to form a "local response element" responsible for generation of detectable local Ca2+ release events (Yue, 1997
; Suda et
al., 1997
). Alternatively, Ca2+ sparks require a close apposition among channels and other related proteins in
a restricted diffusional space provided by the dyads and
triads of cardiac and skeletal muscle. The absence of
this structural arrangement in CHO cells may result in
the lack of spontaneous or caffeine-induced Ca2+
sparks.
Address correspondence to Dr. Jianjie Ma, Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106. Fax: 216-368-5586; E-mail: jxm63{at}po.cwru.edu
Received for publication 3 June 1997 and accepted in revised form 9 September 1997.
1 Abbreviations used in this paper: CHO, Chinese hamster ovary; DHPR, dihydropyridine receptor; E-C, excitation-contraction; ER, endoplasmic reticulum; GFP, green fluorescent protein; Po, open probability; RyR, ryanodine receptor; RyR-C, carboxyl-terminal ~20% of the RyR; SDS, sodium dodecyl sulfate; SR, sarcoplasmic reticulum.We thank Dr. George Dubyak for help with the Fura-2 measurement of cytoplasmic Ca2+ shown in Fig. 4, and Dr. Maryanne Pendergast for help with the confocal imaging of cells expressing GFP fusion proteins.
This work was supported by grants from National Institutes of Health (AG-15556 to J. Ma and HL-55280 to W.G. Wier), a Pilot Project from Howard Hughes Medical Institute, and an Established Investigatorship from the American Heart Association to J. Ma.
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