J Muscle Res Cell Motil (2013) 34:379–393 DOI 10.1007/s10974-013-9360-y

ORIGINAL PAPER

Quantifying SOCE fluorescence measurements in mammalian muscle fibres. The effects of ryanodine and osmotic shocks Pura Bolan˜os • Alis Guillen • Adriana Ga´mez Carlo Caputo

•

Received: 28 June 2013 / Accepted: 18 September 2013 / Published online: 16 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract We have quantified Ca2? entry through store operated calcium channels in mice muscle fibres, measuring the rates of change of myoplasmic [Ca2?], d[Ca2?]myo/dt, and of Ca2? removal, d[Ca2?]Removal/dt, turning store operated calcium entry (SOCE) ON, and OFF, by switching on or off external Ca2?. In depleted fibres, poisoned with 10 lM cyclopiazonic acid SOCE influx was about 3 lM/s. Ryanodine (50 lM) caused a robust, nifedipine (50 lM) independent, increase in SOCE activation to 8.6 lM/s. Decreasing medium osmolarity from 300 to 220 mOsm/L, decreased SOCE to 0.9 lM/s, while increasing osmolarity from 220 to 400 mOsm/L potentiated SOCE to 43.6 lM/s. Ryanodine inhibited the effectsof hypotonicity. Experiments using 2-aminoethoxydiphenyl borate, nifedipine, or Mn2? quenching, strongly suggest that the increased [Ca2?]myo by ryanodine or hypertonic shock is mediated by potentiated SOCE activation. The Ca2? response decay, quantified by d[Ca2?]Removal/dt, indicates a robust residual Ca2? removal mechanism in sarco-endoplasmic reticulum calcium ATPase poisoned fibres. SOCE high sensitivity to osmotic shocks, or to ryanodine receptor (RyR) binding, suggests its high dependency on the structural relationship between its molecular constituents, Orai1 and stromal interaction molecule and the sarcoplasmic reticulum and plasma membranes, in the triadic junctional region, where RyRs, are conspicuously present. This study demonstrates that SOCE machinery is highly sensitive to structural changes

P. Bolan˜os (&)  A. Guillen  A. Ga´mez  C. Caputo Laboratorio de Fisiologı´a Celular, Centro de Biofı´sica y Bioquı´mica, Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Caracas, Venezuela e-mail: [email protected]

caused by binding of an agonist to its receptor or by imposed osmotical volume changes. Keywords Mammalian skeletal muscle  SOCE  Ryanodine receptors  Osmotic shocks

Introduction The store operated calcium entry (SOCE), an important mechanism that insures the maintenance of a relatively constant Ca2? concentration in the endoplasmic reticulum (ER), was originally described in non-excitable cells (Putney 1986; Berridge 1995), where it constitutes a major pathway for Ca2? influx. In these cells, SOCE activation occurs following the release of Ca2? from the ER through release channels, mainly phospholipaseC-dependent IP3 receptors (Parekh and Penner 1997; Parekh and Putney 2005; Smyth et al. 2010). Store operated calcium entry has also been described in skeletal muscle (Kurebayashi and Ogawa 2001; Pan et al. 2002; Gonzalez Narvaez and Castillo 2007), albeit with important structural and functional differences (Launikonis and Rios 2007). In muscle, the sarcoplasmic reticulum (SR) constitutes the major intracellular Ca2? store, with calcium release channels identified as ryanodine receptors, or RyRs (Smith et al. 1988). In non-excitable cells, the electrical manifestation of SOCE operation is a current, the calcium release activated current, ICRAC, whose measurement with patch clamp techniques has led to the biophysical characterization of SOCE (Hoth and Penner 1992; Hoth et al. 1993; Parekh and Penner 1997). In skeletal muscle, this current, has been object of some controversy (Allard et al. 2006), but is now known as ISkCRAC (Yarotsky and Dirksen 2012).

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Two proteins have been identified as the most important pieces of the SOCE machinery. The first one, the stromal interacting molecule STIM1, present in the ER or SR in muscle fibres membrane, is considered the element that senses the degree of filling of Ca2? (Liou et al. 2005; Roos et al. 2005; Zhang et al. 2005; Srikanth et al. 2012). The second, Orai1, has been identified as the conductive pore sub-unit of the ICRAC channel (Feske et al. 2006; Vig et al. 2006). When Ca2? levels are low, STIM1 clusters in regions of the ER near the plasma membrane, where it interacts with Orai1 (Zhang et al. 2005; Luik et al. 2006; Prakriya et al. 2006; Smyth et al. 2006, 2010). Recently, a new binding partner of the SOCE machinery, junctate, has been described in T cells and identified as an EF-hand protein that favors the clustering of STIM1 at the ER–PM junction (Srikanth et al. 2012). In muscle, the close proximity of the dihydropyridine receptors, DHPR, located in the transverse-tubules (Ttubules), and the RyRs, located in the membranes of the SR in the triadic junctional region, provides the structural basis for excitation contraction coupling (ECC) (Franzini-Armstrong and Protasi 1997). The exclusive localization of SOCE in the same region (Launikonis and Rios 2007) facilitates the interaction of the STIM1 molecules, located in the former, with Orai molecules, located in the latter (Dirksen 2009). The integrity of this complex molecular ensemble would seem to be an absolute requirement not only for ECC but also for SOCE (Ma and Pan 2003). Accordingly, it has been shown that in skeletal muscle, selective deletion of Mitsugumin 29, a protein that confers stability to the junctional region between the plasma membrane, and the SR, causes a severe dysfunction in SOCE, similar to that observed in muscle cells lacking both RyR isoforms 1 and 3 (Pan et al. 2002). More recently the importance of the structural integrity of the junctional region for SOCE activation has been confirmed by the demonstration that decreasing the expression of junctophilin, that helps keeping in close contact the T-tubules and the SR membranes, decreases SOCE activation (Hirata et al. 2006). The involvement of RyR1 in SOCE activation in non-excitable cells (Bennett et al. 1998; Kiselyov et al. 2000, 2001), and the conspicuous presence of RyR1 in the junctional region of muscle fibres, suggested the interaction of RyR1 with SOCE, which was later supported by different pieces of evidence. For instance, SOCE is reduced in skeletal myotubes from dispedic (RyR deficient) mice (Pan et al. 2002); the foot structure of the RyR1 is required for functional coupling to Store Operated Channels, SOCs, and activation of SOCE (Sampieri et al. 2005). Entry of Ca2? into HEK293 cells is stimulated following RyR1 expression (Tong et al. 1999). However, it has also been reported that RyR and azumolene, a RyR agonist, inhibits SOCE in

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different preparations, including muscle (Yarotsky and Dirksen 2012; Zhao et al. 2006). It has been proposed that members of the family of mammalian homologues of the Drosophila transient receptor potential channels (TRPC), and in particular, TRPC1, TRPC5 and TRPC6, might have a role either as components of store operated calcium channels (SOCC) (Vandebrouck et al. 2002b; Liu et al. 2003; Ducret et al. 2006) as mediators of SOCE activation, via a conformational coupling between RyR and TRPC (Parekh and Putney 2005; Dirksen 2009). Although the idea of TRP channels interacting with SOCE machinery is controversial (Parekh and Putney 2005; DeHaven et al. 2009), the conformational interaction of TRP channels with IP3 receptors has suggested that also SOCE channels and RyR1 might be conformationally coupled (Kiselyov et al. 2000; Ma and Pan 2003; Dirksen 2009). Some members of the TRPC family are sensitive to stretch, (Ducret et al. 2006; Spassova et al. 2006; Gomis et al. 2008; Gailly 2012). However, while there are studies on the effects of osmotic changes on muscle fibres (Franco-Obregon and Lansman 1994; Vandebrouck et al. 2002a; Ducret et al. 2006; Suchyna and Sachs 2007; Apostol et al. 2009; Pickering et al. 2009), there is no information on the mechano-sensitivity of the SOCE machinery in this preparation. The aim of this work was to test the hypothesis that structural modifications, imposed on single elements of, or on the whole, SOCE molecular ensemble, would affect its activation. We have used ryanodine to modify the open state conformation of RyRs and osmotic shocks to stress the membrane components of the machinery. We have used mice flexor digitorum brevis (FDB) muscle fibres, loaded with Fura-2 AM, and depleted of Ca2?. By switching ON and OFF the external Ca2? concentration we could measure the rates of increase and decrease of myoplasmic Ca2? concentration, [Ca2?]myo, to evaluate the actual SOCE fluxes. We show that ryanodine (50 lM) and hyperosmotic shocks greatly affect the mechanism of SOCE activation, possibly mediated by structural modifications in its machinery.

Materials and methods Fibres preparation The enzymatic dissociation method was similar to the one previously published (Bekoff and Betz 1977; Carroll et al. 1995; Caputo et al. 2004). Briefly, adult (42 days) male mice [Navy Medicine Research Institute-Venezuelan Institute for Scientific Research (NMRI-IVIC)] were killed by rapid cervical dislocation and FDB muscles were dissected. All manipulations and procedures carried out in

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mice during the development of this work were approved by the local Bioethics Committee on Animal Research (COBIANIM) at the Venezuelan Institute for Scientific Research (IVIC). Muscles were incubated in a modified mammalian Ringer solution containing 1 mM Ca2? and 4 mg/ml type II collagenase (Worthington CLS2, 250 u/mg) for 1 h at 36.5 °C, after which they were washed three times with the 1 mM Ca2? Ringer’s solution and gently separated from tendons and remaining tissue with fire-polished Pasteur pipettes. All the experiments were carried out at room temperature (20–22 °C). Experimental solutions and chemicals The composition of the basic experimental solution (mammalian normal Ringer’s solution, NR) was as follows (in mM): 145 NaCl, 2.5 KCl, 1 MgSO4, 2.5 CaCl2, 10 Dglucose, 10 HEPES, pH 7.4. The 5 Ca2? solution was the same as NR but with 5 mM CaCl2. Ca2? free (0Ca) solution had the following composition (in mM): 145 NaCl, 2.8 KCl, 2 MgSO4, 10 D-glucose, 10 HEPES, 0.5 EGTA, pH 7.4, 300 mOsm. The high K? solution had the following composition (in mM): 75 K2SO4, 5 NaCl, 2 MgSO4, 10 HEPES, 10 glucose, pH 7.4. Cyclopiazonic acid (CPA), thapsigargin (TG), N-benzylP-toluene sulphonamide (BTS), ryanodine (Sigma-Aldrich, St Louis), 2-aminoethoxydiphenyl borate (2-APB) (Calbiochem, La Jolla, CA, USA), 2-[2-[4-(4-nitrobenzyloxy) phenyl]] isothiourea (KB-R7943, EMD Biosciences, MA, USA), and 4-chloro-m-cresol (4-CmC) (Fluka Chemical Corp, RonKonKoma, NY, USA) were added from concentrated stock solutions prepared with DMSO to the indicated concentrations. The fluorescent dye Fura-2 AM, (Molecular Probes, Eugene, OR, USA) was prepared as 5 mM stock in dry DMSO 10 % pluronic F-127 (Molecular Probes, Eugene, OR, USA). Depletion protocol This protocol, previously described (Bolan˜os et al. 2009), consisted of a series of exposures to a high K? solution or to 4-CmC (500 lM) in the absence of Ca2?, and in the presence of CPA or TG until depletion of the SR Ca2? stores was achieved. The criterion for depletion was based on the drastic reduction of the Ca2? transient responses to the high K? or the 4-CmC solutions. Fibre viability was checked by electrical stimulation before and during the depletion. Osmolarity changes All the solutions used in the experiments involving osmotic shocks were prepared with a reduced Na? concentration of

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100 mM, to equally affect excitability in all fibres. The isotonic solution was prepared adding 80 mM Mannitol to reach 300 mOsm/L. For the hypotonic solutions, Mannitol added was either 0 or 30 mM to reach osmolarity values of 220 or 250 mOsm/L. Hypertonic solutions were prepared adding 130 or 180 mM Mannitol to reach osmolarities of 350 or 400 mOsm/L. The osmolarity was checked with a Vapor Pressure Osmometer Vapro 5600 (Wescor, Inc., Utah, USA). Fluorescence recording Dissociated fibres were loaded with the Ca2?-sensitive fluorescent indicator Fura-2 AM 5–7 lM (Life Technologies, Carlsbad, CA, USA) for 30 min at room temperature in the dark and then washed with NR. Once placed in the experimental chamber, the loaded cells were allowed to rest an additional 15 min to allow further de-esterification. Fura-2 fluorescence was measured with a fluorescence imaging apparatus (Ionoptix Co., Milton, MA, USA) mounted on an inverted Nikon Diaphot TMD microscope. For Fura-2 measurements the light from a 100-W xenon lamp was filtered alternating 340/20 and 380/18 nm interference filters (Chroma Technology Corp., Rockingham, VT, USA). The resultant fluorescence was passed through a 400 nm dichroic mirror, filtered at 510/40 nm (Chroma Technology Corp., Rockingham, VT, USA) and collected using an intensified CCD camera. Fluorescence images were taken at a rate of 33 ms/frame, digitalized and analyzed using the IonOptix software. The Ca2? concentration was calculated according to the formula (Grynkiewicz et al. 1985): ½Ca2þ i ¼ Kd  ðR  Rmin Þ=ðRmax  RÞ  Sf 2 =Sb2 where R is the measured fluorescence ratio. The values of Rmax and Rmin and the constant Sf2/Sb2 (fluorescence of free and Ca2?-bound Fura-2 at 380 nm) were calculated in vitro using variable CaEGTA/EGTA ratios to give different [Ca2?] (Kit no. 1, Life Technologies, Carlsbad, CA, USA). The dissociation constant Kd, for the FuraCa2?complex, was taken as 89 nM, which is a value obtained from the on and off rate constants for calcium binding to Fura-2 in muscle fibres (Klein et al. 1988). Since with our configuration, the equipment allowed the acquisition of images at video rate, the Ca2? transients elicited by electrical stimulation were heavily filtered and are only shown as proof of fibre excitability. In order to determine whether and how volume changes, due to osmotic shock, might affect measurements of Fura-2 Ca2? fluorescence, control experiments were carried out, measuring fibres basal fluorescence in different media. In five fibres tested at Fura-2 isosbestic point (360 nm) we observed linear changes on the basal fluorescence of ±2.94

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Table 1 Basal values of [Ca2?]myo Depletion procedure CPA

(A)

(1) Basal (nM)

(2) Partially depleted (nM)

(3) Depleted (nM)

78.0 ± 2.5

125.1 ± 4.2

92.3 ± 3.2

N

(36)

Thapsigargin

(B)

95.0 ± 5.0

214.1 ± 17.2

120.9 ± 6.2

(28)

CPA ?ryanodine

(C)

78.9 ± 3.4

125.9 ± 5.6

85.6 ± 2

(18)

Values of myoplasmic Ca2? levels determined with Fura-2. Ca?2 determination was carried out at the beginning of the experiments, before starting the depletion procedure, column 1; during depletion, at a time when the depleting responses to high K? had reached about half initial the amplitude, column 2; and at the end of depletion procedure, before starting the experiments, column 3. Depletion started with fibre poisoning with 10 lMCPA, row A, or with 10 lM thapsigargin, row B. Row C shows the results obtained in fibres treated with ryanodine, 50 lM after the depletion procedure was completed

and ±5.98 % for ±50 or ±100 mOsm/L, respectively. In contrast, changes in the Ca2? level upon SOCE activation after depletion were from -14 to -61 % and ?139 % for a D osmolarity of -50 or ?50 mOsm/L, respectively. Statistics For comparing mean values, Student’s t test for paired or two independent populations or analysis of variance (ANOVA) were used with the Origin 7.5 software, (Microcal Software Inc., Northampton, MA, USA). Results are given as mean ± SEM. Differences were considered statistically significant at p \ 0.05.

Results Basal Ca2? in depleted fibres The robust experimental procedure employed to deplete SR Ca2? stores in isolated fibres can be expected to conspicuously alter Ca2? homeostasis in this preparation. Fura-2 AM has been shown to reliably report the myoplasmic basal Ca2? concentration, [Ca2?]myo, in enzymatically isolated mammalian muscle fibres, with a value of 64 nM obtained for FDB fibres under normal conditions (Gailly et al. 1993). Table 1 reports the values of the basal [Ca2?] obtained before, column 1, during, column 2, and after, column 3, the depletion procedure carried out in three different ways: row A fibres poisoned with CPA alone; row B fibres poisoned with thapsigargin and row C fibres poisoned with CPA and then exposed to 50 lM ryanodine. The columns distinguish the results of measurements taken at different times. Column 1 shows the values of [Ca2?]myo obtained at the beginning of the experiments, before the SERCA poisoning. Column 2 shows the results of measurements taken during the depletion procedure, while column 3 shows the values taken before starting the experiment proper, i.e. before turning ON SOCE. The higher basal value in box B-1, are unexplained. It is

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important to notice, particularly in the case of fibres treated with thapsigargin, that during depletion the Ca2? values, column 2, are higher than at the beginning, column 1, but at the end of the treatment, tend to recover the initial values, column 3. In 18 resting fibres, totally depleted, ryanodine did not have any effect, i.e. no Ca2? increase was observed, Boxes C-1 and C-3. SOCE activation measurements We have measured the change and the rate of change, of myoplasmic Ca2? concentration, [Ca2?]myo, in mice FDB muscle fibres, loaded with Fura-2 AM, incubated in Ca2? free solutions and poisoned with TG, 10 lM, or CPA, 10–20 lM, to study SOCE activation gated by exposure to 5 mM external Ca2?. [Ca2?]myo can be related to SOCE by :       d Ca2þ myo =dt ¼ d Ca2þ SOCE =dt - d Ca2þ Rem =dt ð1Þ This formulation derives from experimentally determined data but, although correct in its form, it does not take into account the fact that only a small fraction of the Ca2? entering the cell is reported by Fura-2. The remaining, only partially accounted for by the term d[Ca2?]Rem/dt binds to endogenous buffers and to other Ca2? binding sites, causing that the term d[Ca2?]SOCE/dt, will be only a scaled down, proportional measurement of the actual SOCE flux. As suggested by an anonymous referee, taking into account these factors, and correcting for the instantaneous buffering power of the cell, as defined by the ratio of the changes in total and free [Ca2?] (Manno et al. 2013), Eq. (1) becomes:

d½CaTot;myo =dt ¼ B d½CaSOCE =dt - B d½CaRem =dt ð2Þ A compromise value of B = 500 was reached using Kd of 1 lM and a value, for the total Ca2? binding sites, compatible with the measured value of about 80 nM resting Ca2?. Following this new formulation, all the values appearing in the text have been corrected by the

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Table 2 SOCE fluxes under different experimental conditions. The effect of ryanodine SERCA poisoning

(1) TG 10 lM

(2) CPA 10 lM

(3) CPA 10 lM

(4) CPA 10 lM

(5) CPA 20 lM

SOCE turn-ON [Ca2?]

5 mM

5 mM

5 mM

5 mM

5 mM

5 mM ?Ry 50 lM 0Ca2?

SOCE turn-OFF

0Ca2?

0Ca2?

0Ca2?

Ca2?

0Ca2?

CPA

0CPA

0CPA

0CPA

(6) CPA 10 lM

Results

Rate of [Ca2?]myo Increase d[Ca2?]myo/dt

(A)

Rate of [Ca2?]myo Decrease d[Ca2?]Removal/dt

(B)

SOCE

(C)

N

(D)

lM/s (nM/s)

lM/s (nM/s)

lM/s (nM/s)

lM/s (nM/s)

lM/s (nM/s)

lM/s (nM/s)

3.4 ± 0.4

1.8 ± 0.4

1.2 ± 0.2

0.9 ± 0.1

4.5 ± 1.1

5.8 ± 1.3

(6.8 ± 0.9)

(3.6 ± 0.8)

(2.5 ± 0.5)

(1.8 ± 0.3)

(8.9 ± 2.3)

(11.7 ± 2.5)

-2.2 ± 0.3

-2.1 ± 0.5

-2.6 ± 0.4

-1.9 ± 0.5

-4.9 ± 1.3

-2.7 ± 0.6

(-4.5 ± 0.7)

(-4.1 ± 1.1)

(-5.2 ± 0.9)

(-3.9 ± 1.0)

(-9.7 ± 2.6)

(-5.5 ± 1.2)

5.6 ± 0.7

3.9 ± 0.8

3.8 ± 0.6

2.9 ± 0.5

9.3 ± 1.4

8.6 ± 1.6

(11.3 ± 1.5)

(7.7 ± 1.6)

(7.6 ± 1.1)

(5.7 ± 1.1)

(18.7 ± 2.7)

(17.2 ± 3.2)

(32)

(11)

(16)

(23)

(5)

(18)

The upper part of the table columns (1)–(6) indicate the SERCA poisoning procedure, and the solutions used for turning, ON or OFF, SOCE. The lower part resumes the Mean ± SEM values of the rates of [Ca2?]myo increase, row A and [Ca2?]myo decrease, row B, and the sum of the two rates equivalent to the SOCE flux, row C. In row D are listed the number of experiments in each case. In each box, defined by a row and a column, appear two numbers: the upper one represents the value of the two rates and of SOCE estimated as described in the text using the correction procedure based on the use the Ca2? binding factor B = 500, (see text for details), and expressed in lM/s. The lower number (between brackets) represents the corresponding value, obtained without correcting for the myoplasmic Ca2? binding power, and expressed in nM/s as appear in the figures. The values of SOCE, listed in row C, represent the mean of the sum of the rates in A plus the rates in B. In row A: A-1 differs significantly (p \ 0.05) from A-2, A-3, A-4 and A-6; A-5 differs similarly from A-2, A-3 and A-4; A-6 differs similarly from A-2 and A-4. In row B: B-6 differs significantly (p \ 0.05) from B-1, B-2, B-3, B-4 and B-5. In row C, C-5 and C-6 differ significantly (p \ 0.05) from C-2, C-3 and C-4

proportional buffering power factor B = 500, while the figures show the original Fura-2 responses. In Tables 2 and 3 the corrected values, expressed in lM/s, appear above the bracketed original values, expressed in nM/s. Although in Fig. 1a the overall raise and decay phases of [Ca2?]myo follows an exponential time course, they are measured in the linear regions of the rising and falling phase of the Ca2?signal, respectively, as it is schematically shown in Fig. 1a. In all the experiments in which SOCE was estimated, the terms d[Ca]myo/dt and d[Ca]Rem/dt were measured in the same fibre. It is worth noticing that in many cases the rising phase d[Ca2?]myo/dt, starts very slowly and then, after a lag phase, it becomes fast and linear. This effect could be due to the speed of the solution changes. However, the OFF responses, and the fast secondary activation when a fibre was challenged with a second solution change (see Figs. 3, 4, 5), argue against the above possibility. It seems more likely that the effect may be due to a sort of cooperativity in the SOCE activation mechanism, certainly worth of future study. In the experiment of Fig. 1a, during the depletion procedure, the fibre was treated for 300 s with 10 lM TG, to

inhibit the SERCA, after which, it was exposed to 5 mM Ca2? for about 125 s to turn ON SOCE, and then exposed again to the Ca2? free solution, to turn it OFF. External Ca2? caused the [Ca2?]myo to increase, until its withdrawal led to a fast decay to about the initial level. For this fibre, the values of d[Ca2?]myo/dt, and d[Ca2?]Removal/dt, were 4.3 and -4.7 lM/s, respectively, yielding a value of 9.0 lM/s for d[Ca2?]SOCE/dt. As shown in Table 2, boxes A-1 and B-1, for 32 fibres poisoned with TG, the mean values of d[Ca2?]myo/dt and d[Ca2?]Rem/dt were 3.4 ± 0.4 and -2.2 ± 0.3 lM/s respectively, and the mean value of d[Ca2?]SOCE/dt was 5.6 ± 0.7 lM/s, box C-1. A large number of experiments, not shown, was carried out in which, due to experimental reasons, only the activation term (d[Ca2?]myo/dt) could be obtained, with a mean value of 1.1 ± 0.1 lM/s in 140 fibres poisoned with CPA 10 lM. It is important to notice that, contrasting with the experiments using CPA, in the experiments with TG, no Ca2? responses to high K? or electrical stimulation could be elicited at the end of the runs, indicating that the SR Ca2? stores had not been recovered, due to the irreversible action of TG.

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Table 3 Effect of hypertonic stress on SOCE Activation lM/s (nM/s) mOsm/L

Rate of [Ca2?]myo Increase

Isotonic 300 (1) (A)

d[Ca2?]myo/dt

Hypotonic–isotonic 220 ? 300 (2)

Hypotonic–hypertonic 220 ? 400 (3)

0.56 ± 0.14

5.00 ± 1.54

34.40 ± 7.78

(1.1 ± 0.3)

(10.0 ± 3.1)

(68.8 ± 15.5)

Deactivation lM/s (nM/s) mOsm/L

Rate of [Ca2?]myo Decrease d[Ca2?]Removal/dt SOCE N

(B) (C)

Isotonic–hypotonic 300 ? 220 (first)

Isotonic–hypotonic 300 ? 220 (second)

Hypertonic–hypotonic 400 ? 220

-0.34 ± 0.10

-2.18 ± 0.45

-11.07 ± 2.48

(-0.7 ± 0.2)

(-4.4 ± 0.9)

(-22.1 ± 4.9)

0.90 ± 0.23

7.18 ± 1.85

43.61 ± 10.49

(1.8 ± 0.5)

(14.3 ± 3.7)

(87.2 ± 20.9)

(18)

(15)

(12)

The upper part of the table shows the effects of the bathing solution tonicity changes on the rate of [Ca2?]myo increase. In its lower part, the table resumes the results obtained with osmotic shocks. As shown in Figs. 4, 5 and 6, the experiments started with depleted fibre held in isotonic solution and exposed to 5 mM Ca2?, for SOCE activation (A-1). Lowering the solution tonicity to 220 mOsm/L caused a small decrease in [Ca2?]myo (B-1). Stepping up the tonicity from 220 to 300 mOsm/L (A-2) or from 220 to 400 mOsm/L (A-3) caused a robust increase in the SOCE response, as can be seen in row (C) that resumes the results obtained on SOCE under the different tonicity changes, C-1, C-2 ad C-3. Values are Mean ± SEM

Figure 1b shows an experiment in which SOCE turn-ON in a fibre treated with 10 lM CPA was followed by turning it OFF. In this case the values of d[Ca2?]myo/dt and d[Ca2?]Rem/dt were 1.4 and -0.9 lM/s respectively, and the value of d[Ca]SOCE/dt was 2.2 lM/s. The mean value of d[Ca2?]SOCE/dt was 3.9 ± 0.8 lM/s, n = 11, as it is shown in box C-2 in Table 2. The record of Fig. 1b also shows that, washing out CPA after SOCE turn OFF, caused a significant increase in the removal rate, most probably, due to partial recovery of the SERCA activity. The fast reversibility of action was the reason for using CPA, since in principle it should have allowed to determine the contribution of the SERCA to the value of d[Ca2?]Rem/dt. However comparison of boxes B-2 and B-3 of Table 2 does not bear with this expectative. (This point will be further considered later). In this and other similar experiments, in which CPA was used, responses to high K? and to electrical stimulation could be elicited at the end of the run, indicating partial replenishment of the SR Ca2? source. Figure 2 shows examples of experiments in which the myoplasmic Ca2? increases were cut short and reversed, either by washing out CPA and maintaining Ca2? in the external solution (Fig. 2a), or taking out both Ca2? and

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CPA, at the same time (Fig. 2b). As summarized in Table 2, boxes A-2, A-3 and A-4 the mean values of d[Ca2?]myo/dt, obtained in runs of the type shown in Figs. 1b and 2a, b are quite similar, since they were obtained under the same initial conditions. As shown in boxes A-2, A-3and A-4 of Table 2, CPA 10 lM appears to be less effective for SOCE activation than TG at the same concentration (box A-1), since with CPA the values of d[Ca2?]myo/dt are smaller than those obtained with TG (p \ 0.05). Comparison of box C-5 with boxes C-1, C-2, C-3 and C-4 shows that the higher SOCE values are obtained with 20 lM CPA; however, with this concentration of CPA fibre survival was diminished. Unfortunately, as already mentioned, in these experiments, fibre variability did not allow dissecting SERCA contribution to Ca2? removal, when CPA was washed out. The decay of the [Ca2?]myo that occurs when SOCE is turned OFF, can be explained assuming that during SOCE activation the different myoplasmic buffers are in equilibrium with Ca2?; when Ca2? entry is switched OFF by SOCE deactivation the high buffering capacity will continue, binding the excess Ca2? in the myoplasm. Thus, a high buffering capacity, not saturated during SOCE activation, could account for, or contribute to, the decay of [Ca2?]myo.

J Muscle Res Cell Motil (2013) 34:379–393 0Ca

5Ca

0Ca

a

TG 10 µM TG 10µM

600 d[Ca2+]myo/dt

d[Ca2+]Removal/dt

2+

400

5Ca

0Ca

2+

[Ca ]

800

[Ca ] (nM)

2+

[Ca ] (nM)

a

385

200

600

K

5 CPA

500 400 300 T

200

K

100 0

0

600

800

1000

1200

1400

1600 1400

Time (s)

1500

1600

1700

1800

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The next set of experiments was carried out to test whether ryanodine affected SOCE. Figure 3a shows an experiment in which a fibre, poisoned by 10 lM CPA, was submitted to a mild depletion procedure, evidenced by the robust responses to high K? elicited before exposure to ryanodine 50 lM for 600 s. Following exposure to Ca2?, SOCE was turned ON, at a rate of 5.1 lM/s. The procedure Ca2?-ON, Ca2?-OFF was repeated inducing a second response similar to the first one. Figure 3b shows a similar experiment in which a fibre, poisoned with 10 lM CPA and extremely depleted, was treated with 50 lM ryanodine for about 550 s, after which exposures to external Ca2?, caused rapid increases in [Ca2?]myo that proceeded at rate of 6.3 lM/s. As above, a second response could be elicited by external Ca2? switching. In both A and B runs, simultaneous withdrawal of Ca2? and CPA led to a faster decay of [Ca2?]myo. Comparison of the results obtained with a fully depleted fibre and that of Fig. 3a suggests that the effect of ryanodine presented here is not dependent on the degree of depletion. In Table 2, box A-6, B-6 and C-6 summarize the

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Fig. 3 The effect of ryanodine on SOCE. a SOCE activation by 5 mM external Ca2? in a fibre partially depleted, poisoned with 10 lM CPA, and exposed to 50 lM ryanodine in the absence of external Ca2?. Removal of Ca2? from the external medium deactivates SOCE which activates again when Ca2? is reintroduced in the external medium. b SOCE activation by 5 mM external Ca2? in a totally depleted fibre, poisoned with 10 lM CPA and treated with 50 lM ryanodine in 0Ca2?. In both cases the Ca2? switching sequences were: Ca2? ? 0Ca2? ? Ca2? ? 0Ca2? 0CPA

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Effects of osmotic shocks Experiments with hypotonic solutions were initially carried out to test the presence of stretch dependent Ca2? entry in our fibres (Spassova et al. 2006; Gomis et al. 2008; Gailly 2012). In the early experiments the fibres were poisoned with 5 or 10 lM CPA, however, later experiments were performed with higher CPA concentrations or with TG. In the experiment of Fig. 4a, after the depletion procedure, carried out in the presence of 5 lM CPA and in the

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results obtained with 18 fibres poisoned with 10 lM CPA and treated with ryanodine, indicating that the mean value of d[Ca]myo/dt and d[Ca]Rem/dt were 5.8 ± 1.3 and -2.7 ± 0.6 lM/s respectively giving a SOCE influx value of 8.6 ± 1.6 lM/s. In these experiments, and as it is clearly shown in Fig. 3a, b, SOCE activation occurs immediately after the external solution change to the one with 5 mM Ca. It seems fair to conclude that no replenishment of the SR could have occurred during the experimental time of SOCE activation, even supposing that SERCA were partially active. Admittedly, the SOCE activation is by necessity accomplished by having Ca2? in the external medium. But in fibres treated with Ryanodine SOCE activation occurs rapidly, in question of few seconds. In the work of Kurebayashi and Ogawa (2001), SR replenishment is much slower. Comparison of SOCE activation responses in the records of Fig. 3a (not completely depleted fibre) and of Fig. 3b (completely depleted fibre) shows no major differences. This point was further explored in three experiments in which fibres, not completely depleted, as indicated by their responses to high K? or CmC, were tested as in the run of Fig. 3a. The three fibres, showed mean values of d[Ca2?]myo/dt, d[Ca2?]Rem/dt and SOCE equal to 4.2 ± 1.3, -2.3 ± 0.7 and 6.5 ± 1.4 lM/s respectively, that fall in the range of the corresponding values in Boxes A-6, B-6 and C-6 of Table 2, again indicating that the ryanodine responses are not dependent on the degree of fibre depletion. In the two experiments of Fig. 3, two responses could be elicited in each fibre, with the first one cut short by a 0Ca medium, in the presence of CPA, and the second one cut short by a 0Ca -0CPA medium, giving the opportunity to test the contribution of SERCA to Ca2? removal. For the case of Fig. 3a the rates of Ca2? removal, d[Ca]Rem/dt were -2.6 and -3.9 lM/s respectively, while for Fig. 3b the values of the removal rates -4.2 and -4.8 lM/s respectively. In the two cases, the fractional participation of SERCA was 0.34 and 0.12, with a mean fractional participation value of 0.23. These are preliminary results indicating the possibility of quantifying SERCA participation in the Ca2? removal phenomena.

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absence of external Ca2?, turning ON SOCE with Ca2? caused the basal [Ca2?]myo to increase slowly at a rate of 0.6 lM/s. After a while, the external medium (300 mOsm/ L) was substituted by an hypotonic one (220 mOsm/L), equivalent to an osmotic gradient of -80 mOsm/L, which stopped and partially reversed the [Ca2?]myo increase at an initial rate d[Ca2?]myo/dt = -0.6 lM/s. From this condition the fibre was restituted to the isotonic medium, which caused a fast increase of [Ca2?]myo at a rate d[Ca2?]myo/ dt = 10.4 lM/s. To avoid further increase of the [Ca2?]myo, that would have caused fibre contraction and the end of the experiment, the fibre was restituted to the hypotonic medium that stopped and almost reversed the [Ca2?]myo at an initial rate of -2.1 lM/s in spite of the continuous presence of Ca2? and CPA in the external medium. Finally the fibre was exposed to the isotonic solution prepared without CPA, but with 5 mM Ca2? and the initial base line value was exponentially recovered. The record also shows that at the end of the experiment the fibre had recovered its responsiveness to high K?. As shown in Table 3, in a group of 18 fibres, whose initial activation rate by Ca2? was somewhat low, 0.56 ± 0.14 lM/s (box A-1), deactivation of SOCE by the hypotonic solution caused the [Ca2?]myo to decrease

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at a rate of -0.34 ± 0.1 lM/s (box B-1). In 15 fibres the rates of increase and decay of [Ca2?]myo when exposed to hypotonic–isotonic (box A-2) and isotonic-hypotonic (box B-2) osmolarity shocks were 5.0 ± 1.54 and -2.18 ± 0.45 lM/s, respectively, yielding a mean SOCE value of 7.18 ± 1.85 lM/s, (box C-2), indicating an eight fold increase in SOCE. To study the effect of a steeper osmotic gradient on SOCE, experiments like that shown in Fig. 4b were carried out. In this experiment, after treatment with 220 mOsm/L hypotonic solution, the fibre was submitted to a 180 mOsm/L osmotic shock, which caused a very fast increase in the [Ca2?]myo at a rate of 44.7 lM/s. A new exposure to the hypotonic medium stopped and reversed this increase at a rate of -5.8 lM/s. As shown in Table 3, the mean values of the rates of Ca2? increase and removal, obtained with 12 fibres exposed to the osmotic gradient of 180 mOsm/L were 34.4 ± 7.8 lM/s (box A-3) and -11.1 ± 2.5 lM/s (box B-3). This result indicates that under these conditions an increase of almost 50 fold in SOCE flux occurred (Box C-3). Control experiments indicated that the hyperosmotic shock, per se, did not cause appreciable change in the basal [Ca2?]myo, whose increase depended strictly on the presence of

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external Ca2?. This point is shown in a different way in Fig. 5a. In this experiment, after inducing the hypertonicity potentiated response, this was cut short and rapidly reversed by removing the Ca2? from the hyperosmotic solution, in the continuous presence of CPA, demonstrating again the master role of external Ca2?. As stated in Methods, dye concentration changes due to fibre shrinking or swelling, could only account for a small fraction of these effects. The above experiments indicate that the increase in myoplasmic Ca2? in response to the hyperosmotic shock is entirely dependent on the presence of external Ca2? however, it remains to be proved that the extra Ca2? influx observed during the hyperosmotic shock, occurs through the SOCE pathway. Therefore, experiments were carried out to test whether the SOCE inhibitor 2-APB was effective in cutting short the [Ca2?]myo increase caused by SOCE activation during an hyperosmotic shock. Figure 5b shows a run in which, after partial depletion, one fibre was first exposed to 2-APB, and later to a hypotonic medium containing 5 mM Ca2?. It can be observed that under these conditions (hypotonic medium and 2-APB), with this particular fibre, SOCE was not activated. Restoring the isotonic medium in the continued presence of Ca2? and 2-APB, caused a small but sizeable response, which started to be reversed by stepping back to the hypotonic medium. A second challenge with the isotonic medium caused an even smaller response. After 2-APB was washed out, repeating the hypotonic–isotonic procedure caused a larger response, indicating the effectiveness of 2-APB as a reversible inhibitor of the putative SOCE mechanism. In this run the fibre was electrically stimulated, during the second and third exposure to the hypotonic medium, eliciting responses that were potentiated with respect to those obtained at the beginning of the run, labelled T. Similar results (not shown) were obtained with the SOCE blockers KBR7943 and with gadolinium (Arakawa et al. 2000; McElroy et al. 2008). The possibility of eliciting responses to electrical stimuli can only be explained in terms of some refilling of the Ca2? stores. The fact that refilling may take place in the presence of 10 lM CPA indicates that a fraction of the SERCA could still operate. The results presented above also suggest that a robust refilling of the SR occurs during the prolonged (about 100 s) periods of exposure to 5 mM Ca2? in spite of the continuous presence of 10 lM CPA in the external solution, without affecting SOCE activation. This apparent paradox, reuptake of Ca2? by the SR in presence of CPA, could be explained considering that, at 5 or 10 lM CPA, SERCA is not completely inhibited as indicated by the different SOCE activation with 10 or 20 lM CPA (see Table 2).

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Fura-2 quenching experiments The experiments presented so far have shown that the increase in [Ca2?]myo described above, is strictly dependent on the presence of external Ca2?. The effects of 2-APB and other putative SOCE blockers as KB-R7943 and gadolinium, and the lack of effect of nifedipine on SOCE, indicate that such increase occurs via the SOCE pathway. Another, and possibly more reliable validation to this conclusion, can be obtained studying Fura-2 quenching by Mn?2 under the same experimental conditions used in the above experiments (Hirata et al. 2006; Lyfenko and Dirksen 2008; Pan et al. 2012). Mn?2 ions have been shown to selectively pass through the SOC channels, and to quench Fura-2 fluorescence. Since the decrease of Fura-2 fluorescence is a measure of Mn?2 entry into the cell, and of SOCE activation, Mn?2 has been be used as a marker of the SOCE activation (Hirata et al. 2006; Lyfenko and Dirksen 2008; Pan et al. 2012). In order to do so, after being loaded with Fura-2, the fibres were exposed to a solution containing 0Ca2? and 2 mM Mn2?. This caused a small decrease in the fibre fluorescence. While still in the presence of Mn2?, the fibre was exposed to CPA after which, Mn?2 was taken out and 5Ca

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the fibre was subjected to the depletion procedure. Reexposure to Mn2?, under different conditions, activated its entry via SOC, causing quenching of Fura-2. Figure 7a compares the rates of Fura-2 quenching of two fibres, under isotonic control and hypertonic shock conditions, respectively. The black trace shows the decay of fluorescence of a fibre that after the depletion procedure was exposed to Mn2? while bathed in NR. The gray trace shows the decay of fluorescence in a fibre subjected to the hypertonic shock, while exposed to Mn2?. Figure 7b summarizes the results obtained measuring the rates of Fura-2 quenching by Mn2? under different conditions. In this figure, the column labeled iso refers to runs carried out under iso-osmotic conditions. The column labeled hypo refers to results obtained exposing the fibre to the hypotonic solution

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Fig. 7 Manganese quenching of Fura-2 fluorescence. a Quenching records obtained at the isosbestic point of Fura-2 (360 nm) with 2 depleted fibres in which SOCE was activated in control isosmotic (black line) and hypertonic (gray line) solutions. b Column graph comparing the mean rates of Mn2? quenching of fluorescence obtained with several fibres (fibre number in parenthesis) in experiments similar to that shown in a. Each column summarizes the results obtained under a different experimental condition, as indicated

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(220 mOsm/L) before applying Mn2?. The column labeled Hyper refers to runs carried out after subjecting the fibres to the hypertonic shock (400 mOsm/L). The column labeled 2-APB refers to experiments carried out blocking SOCE with this compound. Finally the column labeled Ry, refers to experiments carried out activating SOCE after exposing the fibre to 50 lM ryanodine. It appears that there is good qualitative agreement between the results obtained with the quenching experiments with those obtained measuring fluorescence, except for the inversion of the results with hypertonic shocks and ryanodine, since the highest rates of quenching were obtained with the latter and not with the former treatment. A role for Na/Ca exchange In the course of this work we found that when SOCE was turned OFF by different means, a residual Ca2? clearance mechanism could diminish the [Ca2?]myo to resting levels, under conditions in which SERCA activity was severely reduced. Beside the myoplasmic Ca2? buffering capacity, other mechanisms could have a role in Ca2? removal. Figure 8 shows an experiment designed to test whether the Na?/Ca2? exchange contributed in reducing the [Ca2?]myo. The experiment shows that external Na? withdrawal does not affect neither the Ca2? raise caused by the hypotonic– hypertonic shock nor, more importantly, the decay of the Ca2? response caused by returning the fibre to the hypotonic condition. Similar results were obtained in two other fibres.

Discussion The use of fluorescent Ca2? sensitive dyes, especially when complemented by measurements of fluorescence quenching by Mn2?, has provided important information for the

physiological characterization of the SOCE channels (Bird et al. 2008; Pan et al. 2012). It has been stressed that fluorescence measurements of the rate of rise of intracellular Ca2? during SOCE activation give a better indication of Ca2? influx than measurements of the maximum Ca2? level (Bird et al. 2008). In this work we have measured both the rate of rise of Ca2? during SOCE turn ON and the rate of Ca2? decline following SOCE turn OFF, to account for the Ca2? clearance mechanisms. While SOCE turn ON is achieved by exposing the fibres to external Ca2?, turn OFF, is started by taking out external calcium, or by other maneuvers such as washing out CPA, or exposure to hypotonic media. This novel methodological procedure, applied to mammalian skeletal muscle fibres, has allowed us to measure the rates of increase and decrease of free myoplasmic Ca2?, d[Ca2?]myo/dt and, d[Ca2?]Rem/dt under different experimental conditions and estimate the corresponding values for total Ca2?. The measurements with Fura-2 provide values of the net increase in [Ca2?]myo, and not of the total Ca2? increase that is orders of magnitude larger, but heavily filtered by different myoplasmic components, mainly troponin, parvalbumin, ATP and SERCA, that constitute the myoplasmic buffering power, B, defined as: B = D[Ca]Tot.myo/D[Ca2?]myo (Manno et al. 2013), as explained in the Results section. The values of the SOCE fluxes, estimated this way (see Table 2) are in the same order of magnitude of the store operated Ca2? entry into the myoplasm from the T-tubules, triggered by Ca2? release from the SR (Launikonis and Rios 2007). The main results presented in this work pertain the effect of ryanodine and of osmotic shocks on SOCE in mammalian muscle fibres. In fact, here we show direct enhancing effects of ryanodine and osmolarity increase on SOCE. We also show that osmolarity decrease impairs SOCE. A negative, but nevertheless interesting result, regards the impossibility to obtain evidence in favor of a possible role of stretch sensitive TRP channels in SOCE activation. In this work, the identification of SOCE, as the sole mechanism responsible for increased Ca2? entry in response to ryanodine or osmotic shock, has rested on the use of conventional SOCE inhibitors, 2-APB and gadolinium (Putney 2010; McElroy et al. 2008), the use of another inhibitor, KB-R7943 (Arakawa et al. 2000). The use of the Fura-2 quenching by manganese technique (Hirata et al. 2006; Lyfenko and Dirksen 2008; Pan et al. 2012), corroborates the results obtained with the fluorescence experiments. The sizeable effects of ryanodine and of osmotic stress on SOCE, described in this work, suggest the possibility that RyRs might have some conformational interaction with SOCE, favoured by their co-localization in the

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junctional region of the SR. Such interaction could be interfered with by ryanodine and osmotic shocks. Ryanodine The results presented here show for the first time, and unexpectedly, that in adult mammalian skeletal muscle fibres, ryanodine directly potentiates SOCE activation, without significantly affecting the rate of Ca2? removal, in agreement with the idea of a conformational interaction between RyRs and SOCE machinery. SOCE potentiation by ryanodine described in this work contrasts with the results of Zhao et al. (2006) and Yarotsky and Dirksen (2012). In the first case 20 lM azumolene, an equipotent but more soluble derivate of Dantrolene, were effective in inhibiting a component of SOCE in C1148cells derived from CHO cells, stably transfected with RyR1, and also in myotubes derived from a myogenic cell line. Dantrolene is a blocker of the ryanodine sensitive Ca2? release channel of the SR, and its action can be considered analogous to that of ryanodine itself. In the second case, besides describing the inhibitory effect of 100 lM ryanodine on skeletal myotubes ISkcrac, Yarotsky and Dirksen (2012) also report that in myotubes lacking RyRs, the rate of ISkcrac activation was significantly reduced. Apart from the differences in the preparations, and the experimental techniques, an explanation for the conflicting results could reside in the differential behavior of ryanodine that depending on the concentration could potentiate or inhibit the mechanism of Ca2? induced Ca2? release, in vesicles of the heavy SR (Meissner 1986). Also in another milieu, Ca2? release channels, incorporated in planar lipid membranes, at high concentration of ryanodine are blocked shut while at lower concentration remain open in a sub-conductance state (Smith et al. 1988). Be it as it may, although in contrast with ours, these results support the idea of an interaction between RyRs and SOCE machinery. These considerations suggest that RyR may respond to ryanodine in different ways, possibly associated with conformational changes, that by an allosteric mechanism could affect SOCE without involvement of the Ca2?conducting pore of the RyRs. Additional evidences and suggestions for important role of RyR1 in SOCE operation have been contributed by different authors. Kiselyov et al. (2001) showed that cADPribose, an agonist of RyR, activated Icrac, while 8-N-cADPR, a blocker of RyRs, inhibited Icrac. Genetically modified animals, lacking both RyR1 and RyR3, failed to respond to caffeine or ryanodine (Pan et al. 2002). Removal of the foot structure of the ryanodine receptor, inhibited SOCE without affecting SR calcium release induced by caffeine, (Sampieri et al. 2005). Furthermore, electron microscopy has revealed that in presence of ryanodine, RyRs undergo a conformational

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change in the junctional membrane domain, shifting their position by about 2 nm, with respect to DHPR (Paolini et al. 2004). While it is not clear whether this shift could be sufficient to affect the mechanism of SOCE activation, it has been clearly shown that the cytoplasmic amino terminal of the foot portion of the RyR molecule is needed for conformational coupling between RyR and Orai1 channels (Sampieri et al. 2005). Ryanodine could favour this interaction, affecting SOCE. Under normal conditions the RyRs, located in the sarcoplasmic reticulum membrane at the level of the triadic junctions, are activated by physical interaction with the dihydropyridine receptors located in the transverse tubule membrane (Rios and Brum 1987). More recently it has been shown that also under resting conditions, DHPR may control the activity of RyR (Robin and Allard 2012). Several authors have described a retrograde interaction between these receptors, (Gonzalez and Caputo 1996; Nakai et al. 1996; Dirksen 2009), by which an action on the RyR could provide a mechanism for the activation of Cav1.1, adding an extra Ca2? influx to SOCE, in the ryanodine experiments. While the nifedipine experiments argue against Ca2? entry through the Cav1.1 channels, a retrograde mechanism could increase RyR and DHPR interaction with the SOCE machinery. The possibility of an indirect effect of ryanodine, opening release channels and promoting extra Ca2? release, thus increasing SOCE activation, can be discarded by the experiments in which the fibres were acutely depleted. This conclusion is strengthened by the observation that ryanodine caused the same effects in depleted and in partially depleted fibres. Osmotic shocks In muscle, the close proximity of the SR and the T-tubules membranes, in the junctional region, where SOCE occurs favours the interaction of the STIM1 molecules, located in the former, with Orai molecules, located in the latter (Launikonis and Rios 2007; Lyfenko and Dirksen 2008; Dirksen 2009). In agreement with this, it has been shown that decreasing the expression of junctophilin (Hirata et al. 2006) or selected deletion of mitsugumin 29 (MG29) (Pan et al. 2002) that help keeping in close contact the T-tubules and the SR membranes, decreases SOCE activation. We have used osmotic shocks to alter the topological relationship between the two membrane systems in mammalian muscle fibres. We show for the first time that muscle fibre shrinking, induced by hypertonic solution, causes a robust potentiation of SOCE. In contrast, muscle fibres swelling, induced by hypotonic solutions, depresses it. It is interesting to notice that in the osmotic shock experiments, the time allowed for SOCE activation was 20–40 s, a period sufficient for completion of volume

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changes of frog muscle fibres exposed to similar osmotic gradients (Caputo 1968). When tested upon human salivary gland acinar cells, hypotonic solutions showed two effects, an increased Ca2? influx due to the activation of stretch sensitive TRPV4 channels, and an attenuation of SOCE due to disruption of its membrane domain. In fact the loss of SOCE was accompanied by an increase in the gap separating ER and plasma membranes (Liu et al. 2010). In muscle fibres, hypo-osmotic solutions (50 %) cause enlargements of the space between SR and myofibrils, however the presence of tethers that bind mitochondria to the SR membrane provides some structural stability, due to massive presence of mitochondria that clusters in the space adjacent to the SR terminal cysternae, reducing the possibility of a greater separation between the two membrane systems (Boncompagni et al. 2009). Nevertheless hypotonic solutions do reduce SOCE, suggesting a high sensitivity to swellinginduced structural changes. The high sensitivity of the SOCE mechanism to structural changes in the membranes junctional domains is also shown by the results obtained when fibres are submitted to an hyperosmotic shock, either by stepping from a hypotonic solution to an isotonic one, or from a hypotonic or isotonic solution to an hypertonic one, suggesting that the sign of the change of fibre volume, is the important parameter. The robust increase in SOCE activation, described here with the higher osmotic gradients, could mean that under normal conditions the SOCE mechanism might be down regulated by structural constraints that are eliminated when fibre volume is diminished. It has recently been shown that exposure to hypertonic solutions, prepared with 50 mM CaCl2 (about 450 mOsm/L) induces swelling of the T-tubules localized just beneath the surface membrane without causing major ultra-structural changes in the fibre interior (Apostol et al. 2009; Rossi et al. 2011). This solution also caused the activation of local Ca2? release events, calcium sparks, that occurred in the fibre periphery, suggesting an effect on calcium release units, CRU, in the triadic junctions in the region of structural changes (Wang et al. 2005; Rossi et al. 2011). This could suggest that the mechanism of SOCE potentiation by hypertonic shocks might be localized in the same region. It was thought that fibre swelling by hypotonic solutions might provide eventual information about a possible role of stretch sensitive TRP channels in SOCE activation. From the biophysical and pharmacological point of view, TRP channels appear to be similar to the SOC channels (Ducret et al. 2006). Contrary to the effects described here, hypotonic solutions have been shown to activate stretch dependent channels, also activated by store depletion, in muscle fibres from dystrophic (mdx) mice (Franco-

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Obregon and Lansman 1994; Vandebrouck et al. 2002a; Ducret et al. 2006; Suchyna and Sachs 2007), and also in cultured Human Embrionic Kidney cells (Gomis et al. 2008).These findings suggested an association with SOCE, even before this mechanism of Ca2? entry was demonstrated in this preparation (Kurebayashi and Ogawa 2001). The failure of hypotonic stress to increase Ca2? entry, described here, could mean that there are no TRP channels in normal muscle fibres, or that TRPC might be present but are silenced by some mechanism that disappears in mdx mice, or more simply that their activity is not resolved under our experimental conditions. However myoplasmic Ca2? increase caused by hypotonic solutions may not necessarily be due to activation of stretch activated channels. In fact, recently, it has been shown that muscle membrane stretching caused by hypo-osmotic stress, may affect DHPR receptors, leading to Ca2? release from the SR, which could be inhibited by nifedipine, a blocker of L-type, voltage dependent Ca2? channels (Apostol et al. 2009; Pickering et al. 2009). Finally, a remarkable feature of the experiments presented here is the relatively rapid decay phase of [Ca2?]myo, observed when SOCE was turned off by external Ca2? withdrawal, by washing out CPA or by exposing the fibres to an hypotonic medium following an exposure to an isotonic or hypertonic medium in the continuous presence of 5 mM external Ca2?. A large Ca2? buffering capacity in dynamical equilibrium with intracellular ionized Ca2? could have a primordial role in Ca2? removal after SOCE turning OFF. We have shown that the Na?/Ca2? exchange, operating in its forward mode does not contribute to Ca2? removal. On the other hand, mitochondria might play an important role in this phenomenon, since a sizeable Ca2? uptake has been demonstrated when the [Ca2?]myo is increased under different experimental conditions (Rudolf et al. 2004; Bolan˜os et al. 2008, 2009; Rossi et al. 2011). In conclusion, the results presented in this work demonstrate that the multi-molecular SOCE machinery localized in the narrow gap of the triadic junction is highly sensitive to conformational changes and geometrical distortion caused by binding of an agonist to its receptor or by imposed osmotical volume changes. Acknowledgments We thank Dr. J. C. Caldero´n for useful advice reading the manuscript. This work was supported by IVIC.

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