Ryanodine receptor 1 mediates Ca2+ transport and influences the biomechanical properties in RBCs

ARTICLE IN PRESS Journal of Biomechanics 42 (2009) 2774–2779

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Ryanodine receptor 1 mediates Ca2 + transport and influences the biomechanical properties in RBCs Xianwei Wang a,1, Xi Chen a,1, Zhiyu Tang a, Weijuan Yao a, Xiao Liu a, Risheng Wei a, Xifu Wang b, Weibo Ka c, Dagong Sun c, Dongqi He a, Zongyao Wen a,, Shu Chien d, a

Department of Biophysics, Health Science Center, Peking University, Beijing 100191, PR China Department of Cardiology, Peking University Shougang Hospital, Beijing 100144, PR China c Department of Medical Physics, Health Science Center, Peking University, Beijing 100191, PR China d Department of Bioengineering and Medicine, University of California, San Diego, La Jolla, CA 92093-0412, USA b

a r t i c l e in f o

a b s t r a c t

Article history: Accepted 29 July 2009

Ryanodine receptors (RyRs) are a family of Ca2 + channel proteins that mediate the massive release of Ca2 + from the endoplasmic reticulum into the cytoplasma. In the present study, we manipulated the incorporation of RyR1 into RBC membrane and investigated its influences on the intracellular Ca2 + ([Ca2 + ]in) level and the biomechanical properties in RBCs. The incorporation of RyR1 into RBC membranes was demonstrated by both immunofluorescent staining and the change of [Ca2 + ]in of RBCs. In the presence of RyR1, [Ca2 + ]in showed biphasic changes, i.e., it increased with the extracellular Ca2 + ([Ca2 + ]ex) up to 5 mM and then decreased with the further increase of [Ca2 + ]ex. However, [Ca2 + ]in remained constant in the absence of the RyR1. The results of biomechanical measurements on RBCs, including deformability, osmotic fragility, and membrane microviscosity, reflected similar biphasic changes of [Ca2 + ]in mediated by RyR1 with the increases of [Ca2 + ]ex. Therefore, it is believed that RyR1 can incorporate into RBC membrane in vitro, and mediate Ca2 + influx, and then regulate RBC biomechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca2 + release channel. & 2009 Elsevier Ltd. All rights reserved.

Keywords: Ryanodine receptor Red blood cell Calcium ions RBC deformability Membrane microviscosity

1. Introduction Ca2 + is one of the most important elements that control many cellular events in living body. The elevation of the intracellular free Ca2 + concentration ([Ca2 + ]in) causes a wide variety of changes in cell functions, including the membrane biomechanics and cell deformability (Smith et al., 1981; Mortensen and Novak, 1992; Wang et al., 2001; Watts and Handy, 2007). In eukaryotic cells, the intra- and extra-cellular Ca2 + concentrations ([Ca2 + ]ex) are significantly different, with [Ca2 + ]in 10  8–10 7 mol/L and [Ca2 + ]ex 10  3–10  2 mol/L. The [Ca2 + ]ex level under conditions is one of the factors that regulates [Ca2 + ]in. Moreover, the intracellular Ca2 + , which mainly exists in the intracellular Ca2 + pools (mitochondria and endoplasmic reticulum), also plays an essential role in regulating the [Ca2 + ]in (Sitsapesan and Williams, 1995; Yin et al., 2008). Hence, there are two available pathways for the elevation of [Ca2 + ]in, one via entry from the outside of the cells and the other via release from the intracellular Ca2 + pools.

 Corresponding authors.

E-mail address: [email protected] (Z. Wen). These authors contributed equally to this work.

1

0021-9290/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2009.07.036

Many different types of Ca2 + transporters (channels, exchangers, pumps) participate in modulating the [Ca2 + ]in levels. The Ca2 + pools release the stored Ca2 + through two families of channels, the ryanodine receptors (RyRs) and the inositol trisphosphate receptors (IP3Rs). Calcium plays an important role in regulating the RyR- and IP3R-mediated Ca2 + release (Sonnleitner et al., 1998). RyR-mediated Ca2 + release is substantially triggered by the depolarization of the plasma membrane. In the single channel studies, Ca2 + activates the RyR channel and enhances its open probability (Sonnleitner et al., 1998). A small increase in Ca2 + concentrations can lead to the depolarization of the surface membrane and induce Ca2 + releases. This phenomenon is known as ‘‘Ca2 + -induced Ca2 + release (CICR)’’. Apart from Ca2 + , other compounds, including Ca2 + at micromolar concentrations, ATP, adenosine nucleotides, ryanodine at nano-molar concentrations, caffeine, etc., can also activate RyR channels (Meissner 1994; Sonnleitner et al., 1998). To date, three RyR subtypes, RyR1-3, have been identified in mammal cells. Of the three RyRs, RyR1 is the most well characterized one (Wayne Chen et al., 1997; Yin et al., 2008). The mammalian RBCs do not possess the intracellular Ca2 + pools. Hence, for the mammalian RBCs, [Ca2 + ]in depends on the [Ca2 + ]ex levels and the balance of the Ca2 + influx and efflux across

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the membrane. Previous in vitro studies indicated that the calcium ionophore, such as A23187, could facilitate Ca2 + transport across the membrane in RBCs under in vitro condition (Tanabe and Doi, 1989; Mark et al., 2000; Wang et al., 2001; Friederichs et al., 2006). In these experiments, the researchers incubated RBCs with A23187/Ca2 + containing buffer, and found that [Ca2 + ]in levels increased transiently. The experiments on RyR-mediated Ca2 + release are usually performed on an artificial bilayer apparatus by incorporating RyR proteins into the lipid bilayers or the lipid–protein bilayers in vitro (Sonnleitner et al., 1998; Fill and Copello, 2002). These studies suggested that, with an increase of [Ca2 + ]ex, the incorporated RyR proteins could mediate the Ca2 + release and regulate the Ca2 + levels in the artificial bilayer apparatus. Because of the lack of the intracellular organelles, RBCs can be regarded as a lipid–protein bilayer apparatus. To our knowledge, no experimental works have been done on the interaction between RyRs and RBC membrane, and whether RyR proteins can incorporate into the RBC membrane and mediate the Ca2 + release in RBCs is still unknown. In the present work, we manipulated the incorporation of the purified RyR1 into RBC membrane in vitro through incubating RBCs in RyR1 buffer and investigated its influences on the levels of [Ca2 + ]in, and the biomechanical properties of RBCs.

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2.4. Measurement of the RBC diameter The RBCs were washed three times and re-suspended in 0.1 M PBS (2  106 cells/ml). The diameter (2a0) of RBCs was measured with a high-resolution microruler superimposed on the RBC images. Ten cells were measured for each sample.

2.5. Measurement of small deformation index The washed RBCs were suspended in 1.5% polyvinylpyrrolidone (PVP, MW 30 kDa) buffer and adjusted to 2  107cells/ml, and then 600 ml of the cell suspension was analyzed in an ektacytometer (Model LABY-N6A, Precil Company, China). The small deformation index (DI)d was plotted against the shear rate (g_ ) to obtain the (DI)d-g_ curve. (DI)d was obtained at the maximum shear rate used at 140 s  1.

2.6. Measurement of cellular osmotic fragility RBCs were incubated in solutions with various concentrations of NaCl (range from 0.25% to 0.70%) for 30 min. After centrifugation, the supernatants were analyzed at 540 nm in a spectrophotometer. The hemolytic ratio (Hr) was calculated based on the following formula: Hr ¼

Tr300  Tr  100% Tr300  Tr40

where Tr40 is the transmittance under an osmotic concentration of 40 mOsmol/L (0.1% NaCl). Tr300 is the transmittance under a concentration of 300 mOsmol/L (0.85% NaCl). The hemolytic ratio–osmotic pressure curve was plotted from the data.

2. Materials and methods 2.1. Sample preparation

2.7. Measurement of RBC membrane microviscosity

Three milliliter of venous blood was taken from rabbit ear veins and anticoagulated with heparin. Plasma and the buffy coat were discarded after centrifugation (2000 rpm, 15 min). RBCs were washed three times and then resuspended in 0.1 M isotonic phosphate buffered saline (PBS). Fourteen aliquots of cell suspension (200 ml each) were prepared. Seven aliquots (control group) were added to 100 ml of a buffer (100 mM KCl, 46 mM NaCl, 10 mM citric acid natrium, 10 mM Tris, pH =7.6, 295 mOsmol/kg) supplemented with CaCl2 at different concentrations: 0, 0.5, 1, 10, 100, and 1000 mM. Another 7 aliquots (RyR1 group) were added to the same gradient CaCl2 buffers plus 55 ml of 1.5 mg/ml RyR1 protein (provided by Prof. Changcheng Yin from Peking University Health Science Center). The cells were incubated at 37 1C for 45 min and then washed in PBS prior to various analyses.

The RBCs were washed three times with a wash buffer (150 mM NaCl/5 mM sodium phosphate/20 mg PMSF, pH 7.5), and then were lysed in a hypotonic lysis buffer (7.5 mM sodium phosphate/1 mM EDTA/20 mg PMSF/2 mg pepstatin, pH 7.5) in wet ice. The ghost membrane was washed twice with hypotonic lysis buffer and re-suspended in 0.1 M PBS. The membrane microviscosity was measured according to a previous protocol (Wang et al., 2008) by determining the fluorescent polarization parameter p. Membrane microviscosity Z was calculated according to the formula: Z = 2p/(0.46  p).

2.2. Immunofluorescence staining and microscopy After 45 min incubation, two aliquots of RBCs (one incubated in PBS, the other one incubated in PBS plus RyR1 protein) were washed three times and fixed in a 4% paraformaldehyde in 0.1 M PBS overnight at 4 1C. After washing three times, RBCs were blocked in 0.1 M PBS/10% normal goat serum/1% BSA overnight at 4 1C, and then washed three times. The washed RBCs subsequently were incubated in rabbit anti-rynaodine receptor1 antibody (Millipore, USA) solution diluted in 0.1 M PBS/ 1% normal goat serum/1% BSA (1:1000) at 37 1C for 30 min. The RBCs were washed three times and incubated in FITC-conjugated goat anti-rabbit IgG (KPL, USA) solution overnight at 4 1C in the dark. Then the RBCs were re-suspended in 50 ml 0.1 M PBS, and were dropped in a chamber designed for confocal laser scanning microscopy. Fluorescence measurements were made by using an optical imaging system. The excitation wavelength was 495 nm, and a 525 nm long-pass filter was used in the fluorescence detection path. The fluorescent image was collected using a 60x oil immersion objective, and the corresponded phase contrast image at the same field was also collected.

2.3. Measurement of intracellular free Ca2 + concentration The RBCs were washed three times and re-suspended at 0.5% hematocrit in a 2 ml Eppendorf tubes. The RBC suspensions were loaded with 10 mM fluorescent calcium-binding dye Fluo-3/AM (Molecular probes, Eugene, OR) and then incubated for 40 min at 37 1C in the dark with an intermittent mixing. The cells were washed three times in PBS to remove any extracellular Fluo-3 and resuspended at 0.05% hematocrit. Finally, the cell suspensions were analyzed with a flow cytometer (Becton Dickinson, Mountain View, USA). Approximately 10,000 cells were collected by the flow cytometer. The calcium level in each group was analyzed by determining the fluorescence intensity.

2.8. Data analysis All results were presented as means 7standard deviations (SD). The statistical analysis was performed with SPSS 11.5 software. Statistical significance was assessed by two-tailed Student’s t-test. Po 0.05 was considered to be statistically significant.

3. Results 3.1. Immunofluorescence analysis Immunofluorescence analysis is a method commonly used to detect the localizations of proteins in the cells. Our results show that, in RyR1 group, there are many visible fluorescence spots detected by RyR1 antibody on RBC membranes; however, in the control group, there is no fluorescence spot on the membrane (Fig. 1). The results suggest that RyR1 have incorporated into RBC membrane after incubation. 3.2. Intracellular calcium ion concentration The Fluo-3 fluorescence intensities of the RBCs, which reflect [Ca2 + ]in levels in RBCs, are shown in Fig. 2. The control group showed no change in the fluorescence intensity in RBCs with the changes of [Ca2 + ]ex. However, in RyR1 group, the fluorescence intensity increased with [Ca2 + ]ex increase; it reached a maximum at 5 mM and then began to decrease with a further increase of [Ca2 + ]ex.

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Fig. 1. Immunofluorescence staining of RBCs in the control- and RyR1-group (600  ). A and B show phase contrast and fluorescence staining of RBCs in the control group, respectively; C and D show phase contrast and fluorescence staining of RBCs in the RyR1 group, respectively.

increase with a further increase of [Ca2 + ]ex. The change of (DI)d is negatively related the [Ca2 + ]in. 3.4. RBC diameters and the shear modulus The shear modulus (m) of RBCs was calculated according to a mathematic model newly established by our group (Liu et al., 2007).

m¼ 2

2Zg_ a0

l2  ð1=l2 Þ

lðl2 1Þð4l4 þ 5l2 þ 29Þ 4 6ðl 1Þ2

6 4

Fig. 2. Fluo-3 fluorescence intensities in RBCs under various extracellular CaCl2 concentrations in the control- and RyR1-group.

3.3. Small deformation index The RBC small deformation indexes (DI)d at the maximum shear rate of 140 s  1 are presented in Table 1. There was no change in (DI)d over the range 0–1000 mM [Ca2 + ]ex in the control group. However, in RyR1 group, (DI)d decreased as [Ca2 + ]ex increased and reached a minimum at 5 mM, then began to



5l 4 2ðl 1Þ3=2

2

 lnðl þ

l2

pffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffi3 l ffi arcsin l4  1Þþ p3ffiffiffiffiffiffiffiffi l4  1 4 7 2 l 1 5

where l2 = 1 + (DI)d/1  (DI)d. To calculate the shear modulus of RBCs, we first measured their diameters under stationary condition. There is no significant difference in RBC diameters between the RyR1- and control-group for all levels of [Ca2 + ]ex tested (Table 2). Based on RBC diameters and the (DI)d, the shear modulus (m) was calculated according to the above formula. In the control group, m did not change with an increase in [Ca2 + ]ex, however, in the RyR1 group, m increased first and then decreased as [Ca2 + ]ex increased; the maximum value for the shear modulus was found at a [Ca2 + ]ex of 5 mM (Fig. 3). 3.5. Osmotic fragility The results of RBC osmotic fragility, which is represented by the hemolytic ratio at 167 mOsmol/L, are shown in Fig. 4. The

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Table 1 Small deformation index (DI)d (%) of RBCs in two groups at different extracellular CaCl2 concentrations. CaCl2 con. RyR1 group Control group

0 mM 7.97 0.2 7.97 0.3

0.5 mM 7.07 0.2** 7.9 7 0.3

1 mM 5.97 0.2** 7.87 0.3

5 mM 4.7 7 0.2** 7.8 7 0.3

10 mM 6.87 0.1** 8.07 0.4

100 mM 7.3 70.4 7.8 70.2

1000 mM 8.0 70.2 7.9 70.3

**p o 0.01, compared with the control group.

Table 2 RBC diameter (mm) of RBCs in two groups at different extracellular CaCl2 concentrations. CaCl2 con. RyR1 group Control group

0 mM

0.5 mM

1 mM

5 mM

10 mM

100 mM

1000 mM

6.58 7 .356 6.79 7 .376

6.38 7 .298 6.477 .364

6.51 7.518 6.84 7.500

6.807 .458 6.43 7 .453

6.61 7 .369 6.61 7 .427

6.44 7.503 6.62 7.603

6.34 7 .313 6.64 7 .578

3.6. Membrane microviscosity In the RyR1 group, as shown in Table 3, the RBC microviscosity

Z increased first and then decreased as [Ca2 + ]ex was raised to above 5 mM. The RBC microviscosity was significantly higher in the RyR1 group at [Ca2 + ]ex of 0.5, 1, 5, and 10 mM; but there was no significant difference between the two groups as the microviscosity of the RyR1 group decreased at [Ca2 + ]ex of 100 and 1000 mM.

4. Discussion

Fig. 3. The shear modulus (m) of RBCs treated with various Ca2 + concentrations in the control- and RyR1-group.

Fig. 4. RBC hemolytic ratio curve at different Ca2 + concentration in the controland RyR1-group when the osmotic pressure is 167 mmol/L.

osmotic fragility increase with an increase in [Ca2 + ]ex up to 5 mM, beyond which the osmotic fragility began to decrease. Thus, the changes of osmotic fragility with [Ca2 + ]ex, as the [Ca2 + ]in and the biomechanics parameters, showed a biphasic pattern in response to changes in [Ca2 + ]ex, with an initial increase followed by a decrease when [Ca2 + ]ex was raised beyond 5 mM.

In the present study, we incubated RBCs with RyR1 in a buffer system including KCl, NaCl, acitic acid natrium and Tris. The immunofluorescence analysis shows that RyR1 protein has been incorporated into RBC membrane after the incubation (Fig. 1). The difference of [Ca2 + ]in of RBCs between the control- and RyR1group further confirms the incorporation of RyR1 in membrane. The difference also shows that the incorporated RyR1 plays a role in regulating Ca2 + release in RBCs. Interestingly, in our study, [Ca2 + ]in in the RyR1 group did not increase continuously in response to the increase of [Ca2 + ]ex. It increased from the 0.5 up to 5 mM and then began to decrease with the further increase of [Ca2 + ]ex. It shows a bell-shaped Ca2 + response (Fig. 2). The similar results were also observed by other groups in previous studies performed on the lipid bilayer (Bull and Marengo, 1993; Wayne Chen et al., 1997; Sonnleitner et al., 1998). It is accepted now that RyR channels are activated by Ca2 + at nanomolar level and inhibited by Ca2 + at micromolar level (Lai et al., 1988; Bull and Marengo, 1993; Wayne Chen et al., 1997; Sonnleitner et al., 1998; Friederichs et al., 2006). Several groups reported that the activity of RyR1 could be almost entirely inhibited as [Ca2 + ]ex concentration rises to 1 mM (Bull and Marengo, 1993; Yin et al., 2005). In the single channel study, in the absence of other modulating agents, the apparent KD of single RyR channel Ca2 + activation is typically between 0.5 and 5 mM (Fabiato, 1985). This is consistent with our results of [Ca2 + ]in change in RBCs. The mechanism of the bell-shaped function of RyR channels is very complex and still not clear. In general, the transition threshold of RyR channel’s activity ranges from 100 nM to 1 mM. The variable responses of RyR to Ca2 + concentrations are determined by the channel’s properties and the extracellular physicochemical conditions, such as the media in the buffer system and the temperature. Different channel proteins (such as RyR1, RyR2 and RyR3) have the different transition thresholds (Bull and Marengo, 1993; Tripathy and Gerhard, 1996). It is reported that RyR1 is almost entirely inhibited by 1 mM Ca2 + (in our study, it is between 100 mM and 1 mM); however, RyR2 and RyR3 require higher levels of Ca2 + to be entirely inhibited (Coronado et al., 1997; Fill and

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Table 3 RBC membrane microviscosity (Z) in two groups at different extracellular CaCl2 concentrations.

p

Z

Control 0.019 7 0.001 0.087 7 0.01

0 mM 0.0217 0.002 0.0957 0.02

0.5 mM 0.0237 0.002* 0.1057 0.02*

1 mM 0.027 7 0.001* 0.123 7 0.01

5 mM 0.029 7 0.002* 0.1357 0.02*

10 mM 0.025 70.001* 0.117 70.02*

100 mM 0.02 70.001 0.09 70.01

1000 mM 0.017 7 0.002 0.078 7 0.02

*po 0.05, compared with the sample in which CaCl2 concentration is zero. p is the fluorescent polarization parameter.

Copello, 2002). The presence of other media (besides Ca2 + ) also influences Ca2 + -induced Ca2 + release and the transition thresholds of RyR channels. ATP is a main factor that influences the open state of the channel (Sonnleitner et al., 1998; Fill and Copello, 2002). In the absence of ATP, the open probability (Po) of the channel at 10 mM Ca2 + concentration was 0.16; addition of 5 mM ATP in the buffer system could increase Po to 0.6 (Smith et al., 1988). In the presence of ATP, inhibition of the channel requires a higher Ca2 + concentration, meaning a higher transition threshold. In addition, the concentrations of tris, HEPES, Mg2 + , K + and other chemicals in the buffer system also influence the activation of RyR channel through altering the transmembrane voltages and membrane depolarization (Imagawa et al., 1987; Smith et al., 1988). Finally, the processes governing transitions from open to closed conformations of the channel is sensitive to the variations of temperature in the buffer system. At lower temperature, the channels have a higher Po, i.e., a higher threshold (Sitsapesan et al., 1991). Previous studies have suggested low [Ca2 + ]in is essential for normal RBC deformability (Murakmi et al., 1986; Oonishi et al., 1997). Many studies including ours have revealed that the elevation of [Ca2 + ]in can lead to the increase of intracellular viscosity and the loss of deformability (Smith et al., 1981; Friederichs and Meiselman, 1994; Wang et al., 2001). Therefore, it is important to investigate the effects of RyR1-mediated Ca2 + increase on deformability and other mechanic properties in RBCs. It is noteworthy in our study that RBC osmotic fragility, membrane microviscosity and shear modulus in responding for the changes of [Ca2 + ]in, also presented a bell-shaped response, and the small deformation index (DI)d showed an inverse bell-shaped response. (DI)d is positively related to the lipid fluidity of RBC membrane, and the membrane microviscosity is negatively related to the membrane fluidity (Wen et al., 1997; Yao et al., 2001). Both parameters are consistent with each other in the study. The elevation of [Ca2 + ]in causes the increase of cytoplasmic viscosity and irreversible alterations in the membrane including peroxidation of membrane, cross-linking and degradation of membrane proteins and leads to the attachment of hemoglobins onto the membrane (Smith et al., 1981; Friederichs et al., 2006; Friederichs and Meiselman, 1994). All biphasic variations of RBC’s mechanic properties in responding for the changes of [Ca2 + ]in with [Ca2 + ]ex increase, in our study, may be ascribed to these biochemical and biophysical changes in the membrane. The lipid peroxidation, proteins cross-linking and the attachment of hemoglobins increase the membrane viscoelasticity. Evidence shows that [Ca2 + ]in increase also causes the perturbations of membrane skeletal protein network (Friederichs et al., 2006), which would give rise to membrane instability and lead to the loss of cell surface area and then the deformability, and even cell lysis (Zail, 1986; Kalfa et al., 2006). It was also reported that [Ca2 + ]in could affect the elastic modulus and RBC deformability through changing the transmembrane potential (Trayko and Rakesh, 1986). In the process of RyR1-mediated release, it will inevitably induce the changes of the transmembrane potential, which may be one of the factors that cause the biphasic variations of RBC mechanic properties. Ca2 + -induced increase of cytoplasmic viscosity is another determinant that reduces RBC deformability.

Compared with previous studies performed on the lipid bilayer or the lipid–protein bilayer, the advantage of our approach is that it could reflect the biological effects of RyR-mediated Ca2 + release on an intact living cell or a structure that is similar to ER apparatus. RBC is a relatively simple cell, and is usually utilized to study the characteristics of some endogenous or exogenous channels (apuaporins and urea transporter) due to its lack of nucleolus and organelles. It has been suggested that there are no other proteins that could affect the RyR’s structure. Therefore, RBC membrane can be viewed as lipid bilayer and RBC as bilayer apparatus, which enables us to study the biological effects of RyRmediated Ca2 + release on an intact living cell. In summary, the present work provides the first observation of the incorporation of RyR1 into RBC membrane, and the incorporated RyR1-mediated influx in RBCs. The incorporated RyR1 plays a bell-shaped function in regulating the [Ca2 + ]in level with the increase of [Ca2 + ]ex, and the bell-shaped changes of [Ca2 + ]in give rise to a bell-shaped response in RBC deformability and its mechanical properties. This information suggests that RBCs may serve as a model to study the function of RyR1 as a Ca2 + release channel.

Conflict of interest statement No conflict of interest to disclose.

Acknowledgments This work was supported by the National Natural Science Foundations of China (Grant nos. 10572007 and 30770532). We thank Dr. Changcheng Yin for providing RyR1 protein and valuable suggestions in the preparation of this manuscript.

References Bull, R., Marengo, J.J., 1993. Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence. FEBS Letters 331 (3), 223–227. Coronado, R., Barg, S., Onoue, H., et al., 1997. Heterogeneity of Ca-gating of skeletal muscle and Cardiac ryanodine receptors. Biophysical Journal 73, 141–156. Fabiato, A., 1985. Time and calcium dependence of activation and inactivation of calcium induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. The Journal of General Physiology 85 (2), 247–289. Fill, M., Copello, J.A., 2002. Ryanodine receptor calcium release channels. Physiological Reviews 82, 893–922. Friederichs, E., Farley, R.A., Meiselma, H.J., 2006. Influence of calcium permeabilization and membrane-attached hemoglobin on erythrocyte deformability. American Journal of Hematology 41 (3), 170–177. Friederichs, W., Meiselman, H.J., 1994. Effects of calcium permeabilization on RBC rheologic behavior. Biorheology 31 (2), 207–215. Imagawa, T., Smith, J.S., Coronado, R., Campbell, K.P., 1987. Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2 + permeable pore of the calcium release channel. Journal of Biological Chemistry 262, 1740–1747. Kalfa, T.A., Pushkaran, S., Mohandas, N., Hartwig, J.H., Fowler, V.M., Johason, J.F., Joiner, C.H., Williams, D.A., Zheng, Y., 2006. Rac GTPase regulates the morphology and deformability of the erythrocyte cytoskeleton. Blood 108 (12), 3637–3645.

ARTICLE IN PRESS X. Wang et al. / Journal of Biomechanics 42 (2009) 2774–2779

Lai, F.A., Erickson, H., Rousseau, E., Liu, Q.Y., Meissner, G., 1988. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 331 (6154), 315–319. Liu, X., Tang, Z.Y., Zeng, Z., Chen, X., Yao, W.J., Yan, Z.Y., Shi, Y., Shan, H.X., Sun, D.G., He, D.Q., Wen, Z.Y., 2007. The measurements of shear modulus and membrane surface viscosity of RBC membrane with Ektacytometry: a new technique. Mathematical Biosciences 209 (1), 190–204. Mark, M., Walter, R., Harrris, L.G., Reinhart, W.H., 2000. Influence of parathyroid hormone, calcitonin, 1,25(OH)2 cholecalciferol, calcium, and the calcium ionophore A23187 on erythrocyte morphology and blood viscosity. Journal of Laboratory and Clinical Medicine 135 (4), 347–352. Meissner, G., 1994. Ryanodine receptor/Ca2 + release channels and their regulation by endogenous effectors. Annual Review of Physiology 56, 485–508. Mortensen, A.M., Novak, R.F., 1992. Dynamic changes in the distribution of the calcium-activated neutral protease in human red blood cells following cellular insult and altered Ca2 + homeostasis. Toxicology and Applied Pharmacology 117 (2), 180–188. Murakmi, J., Maeda, N., Kon, K., Shiga, T., 1986. A contribution of calmodulin to cellular deformability of calcium-loaded human erythrocytes. Biochimica et Biophysica Acta – Biomembranes 863, 23–32. Oonishi, T., Sakashita, K., Uyesaka, N., 1997. Regulation of red blood cell filterability by Ca2 + influx and cAMP-mediated signaling pathways. American Journal of Physiology 273, 1828–1834. Sitsapesan, R., Montgomery, R.A., Macleod, K.T., Williams, A.J., 1991. Sheep cardiac sarcoplasmic reticulum calcium-release channels: modification of conductance and gating by temperature. Journal of Physiology 434, 469–488. Sitsapesan, R., Williams, A.J., 1995. The gating of the sheep skeletal sarcoplasmic reticulum Ca2 + release channel is regulated by luminal Ca2 + . The Journal of Membrane Biology 146 (2), 133–144. Smith, B.D., La Celle, P.L., Siefring Jr., G.E., Lowe-Krentz, L., Lorand, L., 1981. Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability. Journal of Membrane Biology 61 (2), 75–80. Smith, J.S., Imagawa, T., Ma, J., Fill, M., Campbell, K.P., Coronado, R., 1988. Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum. Journal of General Physiology 92, 1–26.

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Sonnleitner, A., Conti, A., Bertocchini, F., Schindler, H., Sorrentino, V., 1998. Functional properties of the ryanodine receptor type 3 (RyR3) Ca2 + release channel. The EMBO Journal 17 (10), 2790–2798. Tanabe, K., Doi, S., 1989. Rapid clearance of plasmodium yoelii-infected erythrocytes after exposure to the ionophore A23187. Comparative Biochemistry and Physiology. A, Comparative Physiology 92 (1), 85–89. Trayko, T., Rakesh, K.J., 1986. Effect of transmembrane potential on the deformability of RBC’s suspended in Carbohydrate-saline solutions. Journal of Colloid and Interface Science 114 (1), 273–276. Tripathy, A., Gerhard, M., 1996. Sarcoplasmic reticulum lumenal Ca2 + has access to cytosolic activation and inactivation sites of skeletal muscle Ca2 + release channel. Biophysical Journal 70, 2600–2615. Wang, C.H., Zeng, Y.J., Ka, W.B., 2001. The influence of calcium ions and ionophore A23187 on microrheological characteristics of erythrocytes by new model ektacytometry. Clinical Hemorheology and Microcirculation 24 (1), 19–23. Wang, X.W., Hu, L., Zeng, Z., Yao, W.Y., Han, J.Y., Zhang, Y.Y., Xu, X.F., Liu, Y.Y., Ka, W.B., Sun, D.G., Wen, Z.Y., Chien, S., 2008. Effects of myakuryu on hemorheological charateristics and mesenteric microcirculation of rats fed with a high-fat diet. Biorheology 45 (5), 587–598. Watts, T.J., Handy, R.D., 2007. The hemolytic effect of verapamil on erythrocytes exposed to varying osmolarity. Toxicology in Vitro 21 (5), 835–839. Wayne Chen, S.R., Li, X., Ebisawa, K., Zhang, L., 1997. Functional characterization of the recombinant type 3 Ca release channel (ryanodine receptor) expressed in HEK293 cells. The Journal of Biological Chemistry 26 (272), 24234–24246. Wen, Z.Y., Yan, Z.Y., Gao, T., Dou, H., Sun, D., Lu., Z., 1997. A study of effects of WGA and ConA on RBC membrane receptors using a new ektacytomeric method. Clinical Hemorheology and Microcirculation 17 (6), 467–478. Yao, W., Wen, Z., Yan, Z., Sun, D., Ka, W., Xie, L., Chien, S., 2001. Low viscosity Ektacytometry and its validation tested by flow chamber. Journal of Biomechanics 34 (11), 1501–1509. Yin, C.C., D’Cruz, L.G., Lai, F.A., 2008. Ryanodine receptor arrays: not just a pretty pattern?. Trends in cell biology 18 (4), 149–156. Yin, C.C., Han, H., Wei, R., Lai, F.A., 2005. Two-dimensional crystallization of the ryanodine receptor Ca2 + release channel on lipid membranes. Journal of Structural Biology 149 (2), 219–224. Zail, L., 1986. Clinical disorders of the red cell membrane skeleton. Critical Reviews in Oncology/Hematology 5 (4), 397–453.