a study of store dependent ca2+ influx in frog skeletal muscle

ORIGINAL PAPER

A study of store dependent Ca2+ influx in frog skeletal muscle

J. F. Olivera • Gonzalo Pizarro

Received: 17 November 2011 / Accepted: 6 April 2012 / Published online: 22 April 2012

� Springer Science+Business Media B.V. 2012

Abstract Ca2? influx across the plasma membrane upon

drastic reduction of the sarcoplasmic reticulum Ca2? con-

tent was studied in voltage clamped frog skeletal muscle

fibers. Depletion was produced by the application of

30 lM cyclopiazonic acid (CPA) in Ca2?-free,

[Mg2?] = 8 mM external salines and produced an increase

in resting free myoplasmic [Ca2?]. Once depletion was

attained the external solution was changed to one con-

taining the same concentration of the drug but with Ca2?

instead of Mg2?. Of 27 fibers studied only nine showed a

secondary increase in free myoplasmic [Ca2?] upon read-

mitting Ca2? in the external perfusate. In the presence of

CPA the resting myoplasmic [Ca2?] in Ca2?-free external

saline was 0.08 ± 0.01 lM (Mean ± SEM), and in Ca2?-

containing external saline 0.10 ± 0.02 lM when the

intracellular solution contained [EGTA] = 5 mM

(n = 18). In cells with lower (0.5 mM) intracellular

[EGTA] resting [Ca2?] went from 0.35 ?/- 0.08 lM in

Ca2?-free external solution to 0.42 ?/- 0.12 lM upon

reapplication of Ca2?(n = 9). In both cases the differences

between means were not statistically significant (paired

t test, p = 0.13 in high EGTA and p = 0.25 in low EGTA).

In the nine fibers that showed a secondary increase of

resting [Ca2?] the holding current measured at -90 mV

did not significantly change. These results suggest the Ca2?

entry secondary to store depletion is a labile mechanism in

frog skeletal muscle and when present does not have an

obvious electrical manifestation.

Keywords SOCE � Calcium � Sarcoplasmic reticulum �Skeletal muscle

Introduction

Store operated Ca2? entry (SOCE) is a process present in a

wide variety of cell types consisting in an influx of Ca2?

secondary to the reduction of the Ca2? content of the

endoplasmic reticulum (Parekh and Putney 2005). The

molecular basis of the mechanism has been recently

established, a Ca2? sensor in the membrane of the intra-

cellular store, the protein STIM1 (Roos et al. 2005),

interacts with a protein in the plasma membrane (ORAI1)

that functions as a Ca2? channel (Feske et al. 2006; Vig

et al. 2006; Zhang et al. 2006). Skeletal muscle cells are

known to maintain the twitch for long periods of time in the

absence of external Ca2? (Armstrong et al. 1972). It is not

clear if under physiological conditions skeletal muscle cells

ever lose enough Ca2? to the external medium as to need

the activation of such a process. The existence of SOCE in

adult skeletal muscle has been clearly shown (Kurebayashi

and Ogawa 2001; Launikonis et al. 2003; Gonzalez Nar-

vaez and Castillo 2007; Launikonis and Rıos 2007), as well

as the presence of the molecules that sustain it (Stiber et al.

2008; Lyfenko and Dirksen 2008). It has been reported

(Ducret et al. 2006; Zhao et al. 2005) than under strenuous

activity there is a dependence on extracellular Ca2? and

that SOCE is involved in maintaining the contractile

function directly or by replenishing the sarcoplasmic

reticulum (SR). This role has been questioned on quanti-

tative grounds (Launikonis et al. 2010) at least for fast-

twitch muscles. Roles for SOCE have been described in

skeletal muscle during development (Stiber et al. 2008) and

disease (Edwards et al. 2010a). Conflicting findings for the

J. F. Olivera � G. Pizarro (&)

Departamento de Biofısica, Facultad de Medicina,

Universidad de la Republica, Gral. Flores 2125,

CP11800 Montevideo, Uruguay

e-mail: [email protected]

123

J Muscle Res Cell Motil (2012) 33:131–143

DOI 10.1007/s10974-012-9293-x

role of SOCE in aged muscle has been reported (Zhao et al.

2008, Edwards et al. 2011).

One point of interest is that SOCE in many cell types

generates plasma membrane currents termed ICRAC or ISOC

depending on their properties (Parekh and Putney, 2005).

In adult mammalian skeletal muscle fibers this has been

studied with negative results (Allard et al. 2006; Berbey

and Allard, 2009). Most of the studies cited above were

done in mammalian muscle. We are aware of only one

study in amphibian muscle (Launikonis et al. 2003) carried

out in mechanically skinned fibers with sealed transverse

tubules. Therefore we found of interest to study this phe-

nomenon in cells with a functional cellular membrane

amenable to electrophysiological study. We did so in adult

frog skeletal muscle twitch fibers under voltage clamp with

the double Vaseline gap technique and simultaneously

measuring intracellular [Ca2?] by means of Fluo-3

fluorescence.

Methods

The experiments were carried out on frog (Rana cates-

beiana) cut fibers voltage clamped in a two-Vaseline gap.

After sedation in 5 % ethanol and double pithing, frogs

were decapitated in accordance with the guidelines of the

Honorary Committee for Animal Experimentation of our

institution. 2 cm-long pieces of fiber were dissected from

the semitendinosus muscle and mounted in a three com-

partment Lucite chamber where the double Vaseline gap

was made. The fibers were mounted at slack length and

contraction was prevented either by high intracellular

EGTA (added to the solutions placed in the end compart-

ments) or the extracellular application of BTS (to fibers

with low intracellular EGTA). Several notches were made

to the regions of the fiber in both end compartments in

order to allow exchange of intracellular solution. The

preparation was cooled to temperatures between 12 and

15 8C by means of a Peltier device. Membrane currents and

optical signals were recorded and analyzed as previously

described (De Armas et al. 1998) and briefly summarized

below.

Solutions

The basic extracellular solution consisted of 130 mM

Tetraethylammonium methansulfonate (TEACH3SO3),

8 mM CaSO4 and 10 mM Tris maleate. To further suppress

ionic currents ion channel blockers were added as follows:

0.05 lM TTX, 1 mM Anthracene-9-Carboxilic (A9C) acid

and 1 mM 3,4-Diaminopyridine. All external solutions

were titrated at pH 7.0 with TEAOH. A Ca2?-free version

of this solution was designed replacing Ca2? with the same

concentration of Mg and 0.1 mM EGTA. In some experi-

ments TEACH3SO3 was replaced isosmostically with

NaCH3SO3. Ca2?-containing and Ca2?-free versions of

this Na external saline were designed. Two intracellular

solutions were used a 5 mM (‘‘high’’) EGTA solution and a

0.5 mM (‘‘low’’) EGTA on. The high EGTA intracellular

solution contained 120 mM Cs-glutamate, 10 mM Cs Tris

maleate and 5 mM MgATP, 5 mM ethylene glycol–bis(b-

aminoethyl ether)–N,N,N0,N0-tetraacetic acid (EGTA) and

0.4 mM fluo-3 (pentapotassium salt, Molecular Probes,

Eugene, OR, USA). Total [Ca2?] was 0.256 mM, added as

CaCl2. This would yield a free [Ca2?] = 20 nM assuming

an apparent KD = 0.37 lM for EGTA, calculated from

Martell and Smith (1982) for pH 7.0, temperature of 20 8Cand 0.1 M ionic strength. The pH of the intracellular

solution was titrated to 7.0 with CsOH. The low EGTA

solution consisted in 128 mM Cs-glutamate, 0.5 mM

EGTA, with the rest of the components at the same con-

centration. 0.026 mM of CaCl2 was added to set the free

[Ca2?] to 20 nM, by the same criteria explained previously.

In some experiments 0.4 mM Fura Red (tetrapotassium

salt, Molecular Probes, Eugene, OR, USA) was used dis-

solved in the low EGTA internal solution at a concentration

of 0.4 mM.

Dissection and mounting of single fibers were carried

out in a relaxing solution containing 130 mM K-glutamate,

2 mM MgCl2, 5 mM Tris-maleate buffer and 0.1 mM

EGTA.

Cyclopiazonic acid (CPA) was used from a 30 mM store

in dimethyl sulfoxide (DMSO). It was added o the extra-

cellular saline at 30 lM, this is a 1/1,000 dilution. In

control experiments up to 4/1,000 dilution of DMSO had

no effect on membrane current or Ca2? release. In the

experiments with low EGTA internal solution, 100 lM of

n-benzyl-p-toluenesulfonamide (BTS) (US Biological,

Swampscott, MA, USA, from a 100 mM stock in DMSO)

was added to the external solutions to prevent movement.

All chemicals were purchased from Sigma (Saint Louis,

MO, USA) except where specifically indicated.

Electronics and data acquisition

Data acquisition was performed with a 16-bit resolution,

100 kHz board (HSDAS-16, Analogic Corporation,

Wakefield, MA, USA). Sampling rates were 20 kHz per

channel. Data was compressed by averaging and stored at

0.2 ms per point. Before acquisition data was filtered by a

one-pole passive filter with 0.5 kHz cut-off frequency.

Command pulses were generated with the D/A channels of

the HSDAS board. Membrane currents, membrane voltage,

and emitted light were measured simultaneously during

fiber activation by clamp pulses.

132 J Muscle Res Cell Motil (2012) 33:131–143

123

Optical measurements and processing of the optical

signals

Fluo-3 was excited by epi-illumination at 490 nm, through

an interference filter placed between a halogen light source

and a 510 nm high pass dichroic mirror. The emitted light

was filtered at 530 nm by a second interference filter and

recorded by a high sensitivity photodiode (HUV-200,

EG&G, Quebec, Canada). The free [Ca2?] transient was

derived from the fluorescence signal as described by Shi-

rokova et al. (1996), with koff = 80 s-1 and

kon = 90 s-1 lM-1 as rate constants of Fluo-3, and

extinction coefficients of Fluo-3 measured in our optical

apparatus (e490 = 5.44 9 104 M-1 cm-1, e530 = 0.97 9

104 M-1 cm-1). The concentration of the dye was deter-

mined from measurements of light path and absorbance

made at regular intervals during the experiment at 510 nm

(e510 = 6.95 9 104 M-1 cm-1).

In a series of experiments Fura Red was used to monitor

the putative entry of Mn2? through an activated SOCE

pathway and subsequent quenching of fluorescence as

previously described (Wu and Clusin, 1997). Since neither

the 450 nm nor the 460 nm filters afforded a perfect isos-

bestic measurement for Ca2?, we chose as excitation

wavelength 450 nm where Ca2? binding to the dye induces

a small increase in fluorescence and therefore the effect of

Mn2? induced fluorescence quenching could be separated.

The fiber was illuminated placing this excitation filter

between the light source and a 525 nm long pass dichroic

mirror. The emitted light was filtered before reaching the

photodiode with a 660 nm interference filter.

Fura Red concentration was obtained by measuring the

absorbance at 460 nm using an extinction coefficient of

e560 = 1.96 9 104 M-1cm-1. The absorbance readings

were taken periodically as described previously for Fluo-3.

To be included in the study the holding current and

linear capacitance of the fibers at the end of the experiment

after returning to the initial conditions (external solution

and holding potential) had to fall within ±10 % of the

respective values at the beginning of the experiment. The

corresponding values of these parameters for all fibers

included in the study are given in Table 2. The stability of

membrane capacitance was considered paramount as the

SOCE mechanism is expected to reside in t-tubular mem-

brane. All fibers studied showed some degree of recovery

of the voltage elicited [Ca2?] transient, in average close to

50 % of the initial value in cells with high intracellular

[EGTA] and about 20 % in low [EGTA]. Complete

recovery was not a requisite for inclusion as we realized

that the SOCE-like response was absent in more than half

of our fibers. If cells lost Ca2? and did not recover it,

setting an arbitrary degree of recovery as criteria for cell

acceptability would bias the study towards those cells that

showed a secondary Ca2? entry.

Statistics

All data is given as Mean ± standard error of the mean

(SEM). Normality of the samples was assessed by Shapiro–

Wilk test. Statistical significance was estimated by two

tailed t test (either paired or independent) at p = 0.05.

Results

A secondary rise in Ca2? after SR depletion

was infrequently observed

All the fibers included in this study were kept in Ca2?-

containing external solution in the central compartment of

the voltage clamp chamber and held at -90 mV during the

period of dye loading. Once a working concentration of the

dye was attained this solution was changed to a Ca2?-free

solution containing 8 mM of Mg2?. After a period of

equilibration in this new condition the pharmacological

depletion protocol was applied. We adopted the usual

protocol for detecting store operated Ca2? entry. We used

treatment with CPA, a reversible inhibitor of the SERCA-

Ca pump, to produce the depletion of the SR. In our hands

30 lM of this drug was effective to produce almost com-

plete depletion, judged by the size of the remaining voltage

elicited [Ca2?] transient that was in average 6.7 % of the

pretreatment transient. After exposure to Ca2?-free CPA

external (no less than 10 min, regularly 20 min) the

external solution in the middle compartment was changed

to one with 8 mM of Ca2? and CPA. Thus, the increase in

myoplasmic [Ca2?] during depletion could only be due to

Ca2? coming from the SR as there was no Ca2? in the

external solution. In this way the increase in [Ca2?] due to

the emptying of the SR could be separated from the

increase due to Ca2? influx through the plasma membrane

secondary to depletion of the intracellular store.

The application of CPA to voltage clamped cut fibers

produced a relatively slow increase in resting [Ca2?]. The

central compartment of our experimental chamber with a

cover slip placed above the fiber has a volume of 0.25 ml,

it takes approximately 30 s to fully exchange this volume

with our perfusion system, as shown in the inset of Fig. 1A.

The response to CPA application was much slower. The

time course of resting [Ca2?] during the first 5 min of the

application of CPA in Ca2?-free saline is shown in

Fig. 1A, record 2. In many cells exposed to Ca2?-free

external solution the resting [Ca2?] declined during pro-

longed exposures, with some variability.

J Muscle Res Cell Motil (2012) 33:131–143 133

123

If the depletion of the SR activated a plasma membrane

Ca2? permeability pathway, Ca2? influx should occur as a

consequence of reapplication of external Ca2?. This should

be detected as an increase in resting free [Ca2?] as the

SERCA pump was inhibited. This simple protocol was

applied using solutions where the main extracellular cation

was TEA and the intracellular solution contained 5 mM

EGTA, the usual conditions in our voltage clamp experi-

ments. In Fig. 1A, record labeled 4, the application of CPA

and Ca2?-containing external solution is shown. [Ca2?]

slowly rose during the first 5 min of the application and

continued to do so until it reached a steady level (Fig. 1A,

record 5). Upon wash out of CPA the resting [Ca2?] went

down and voltage elicited release recovered as shown in

Fig. 1 record labeled 6. Under this experimental condition

only 2 out of 9 cells showed a secondary increase in [Ca2?]

of 30 % or more. In the rest of the cells [Ca2?] was barely

modified, and in some cases it even decreased (see

Table 1). In Fig. 2 a cell in which the application of same

protocol gave a negative result is shown.

We carried out a series of experiments closer to physi-

ological conditions, with a normal Na? gradient as it is

possible that the outward Ca2? transport might be depen-

dent on it and in turn would help to maintain SR depletion.

Under this condition the outcome of the application of the

depletion protocol was not substantially different. Out of

nine fibers studied, 4 gave a secondary [Ca2?] rise upon

readmission of Ca2? in the external solution. In Fig. 3 both

a positive and negative outcome are shown.

Given the high Ca2?-buffering power of this intracel-

lular solution the measurement of the effect a low intensity

Ca2? influx could be somehow hampered. Therefore we

Fig. 1 The figure shows the secondary Ca2? influx upon SR

depletion in a fiber in Ca2?-free TEA external solution and 5 mM

EGTA internal solution. In A selected records during the experiment

are plotted. B the time course of resting [Ca2?] measured as the

average of the first 20 points of each acquisition, before the clamp

depolarization was applied). The vertical bars indicate the changes of

solution in the central pool. The values corresponding to the records

in a are indicated with the same numbers. In A record 1 is the last

Ca2? transient elicited by clamp depolarization to 0 mV before

treatment with CPA. Then 30 lM CPA was added to the central pool

dissolved in the same media. In A record 2 shows how the resting

[Ca2?] slowly increases during the application of the drug at the

holding potential of -90 mV. Note the different time scale respect to

1. The effect of CPA is much slower than the change of solutions in

the experimental chamber, the time course of which is shown in the

inset on the upper right. The central pool was filled with a solution

containing carboxy-fluorescein, the light emitted by the dye was used

to monitor the solution change. The application of 1 ml of water fully

exchange the solution bringing the fluorescence to background level,

the application of another ml indicated by the arrow did not further

change the emitted light. In A record 3 is the last voltage triggered

Ca2? transient in response to a clamp depolarization to 0 mV, same

time scale as in 1a applies. The amplitude of the Ca2? transient is

drastically reduced, likely due to SR depletion. In A record 4 the time

course of resting [Ca2?] during the application of Ca2?-containing,

CPA external solution. In a records 5 and 6 are voltage triggered

transients, the last in Ca2? CPA and the last after washing out CPA in

Ca2?-containing solution respectively. Fiber identifier 100211,

[Fluo3] went from 275 to 372 lM, optical path = 120 lm,

diameter = 118 lm


123

repeated the study with lower intracellular [EGTA].

Although in this series of experiments the changes in free

[Ca2?] were wider and better detected the occurrence of a

secondary [Ca2?] rise was observed in only 2 out of 5

fibers studied. The rise in [Ca2?] elicited by CPA went

through a maximum and decayed to a lower level in most

fibers, suggesting that Ca2? was lost to the extracellular

space (see Fig. 4). This was also observed in the high

EGTA internal condition (see Fig. 3) although not as

clearly. It is possible that the Ca2? that abandoned the SR

would redistribute into other intracellular compartments

(i.e. mitochondria) but given the size of the decline

(approximately one half of the maximum) it is unlikely that

intracellular compartments could account for all the

reduction. The loss of cellular Ca2? might have been due to

passive leak although the driving force was very small as

the intracellular [Ca2?] never went above 2 lM. Thus the

outward transport was likely due to the activity of the

plasma membrane Ca2? pump and the Na?/Ca2? exchange

mechanism. We have also studied a group of fibers in low

intracellular EGTA and TEA external saline. Under this

condition also a clear reduction in free [Ca2?] attained

during the treatment with CPA was observed putting some

limits to the contribution of the Na?/Ca2? exchange to the

net outward transport of Ca2?. Of the four fibers in this

group only one showed a secondary rise in [Ca2?] con-

sistent with SOCE. Thus, the rare occurrence of SOCE-like

responses was essentially independent of the experimental

conditions and was not favored by net cellular Ca2? loss.

In Table 1 we present the collected data from the 27

fibers studied, they were subdivided in two groups, one

with high and the other with low intracellular [EGTA].

Although the proportion of SOCE-like responses in

Na?-containing external solution was higher it was not

significantly different (by Fisher exact-test) from the pro-

portion in TEA external solution, justifying pooling them

together. The effect of returning to Ca2?-containing solu-

tions was not statistically significant in any case (see leg-

end of Table 1).

In Table 2 values of different parameters of the same

fibers are given (resting [Ca2?], the amplitude of the

[Ca2?] transient, holding current and linear capacitance),

measured in Ca2? containing external solutions, before the

application of the depletion protocol and after washout of

CPA. Within each group in Table 1 the fibers were sub-

divided in two groups, one consisting in those that gave a

SOCE- like response and the other including those that did

not. For fibers in high intracellular [EGTA] there was no

significant difference between the two groups either in

resting [Ca2?] or in the amplitude of the [Ca2?] transient

under the initial condition. In the case of the fibers in low

intracellular [EGTA] the resting [Ca2?] of the fibers that

showed SOCE was significantly higher than in those that

lacked the response while the amplitude of the [Ca2?]

transient was not. Taking the amplitude of the transient as a

rough indication of healthiness of the excitation contraction

coupling process and the load of the SR we tend to rule out

differences in the initial physiological state of the fibers to

account for the differential response to SR depletion. The

proportion of recovery of the [Ca2?] transient was not

Table 1 Resting myoplasmic [Ca2?] (measured as the average of the

first 20 ms of the acquisition of voltage triggered transients, pulses

started at 50 ms) at the end of the exposure of CPA in Ca2?-free

solution and at the end of the period of exposure to Ca2? and CPA-

containing external solution

[Ca2?]REST (lM)

Int. sol., Ext. sol. Mg CPA Ca CPA Fiber

High EGTA, Na 0.05 0.04 110909

0.03 0.20 *030810a

0.11 0.21 *130810

0.06 0.13 *171011

0.10 0.11 181011

0.08 0.06 191011a

0.03 0.06 *191011b

0.07 0.06 201011a

0.07 0.07 201011b

High EGTA, TEA 0.04 0.02 040809

0.10 0.15 *050809a

0.04 0.05 050809b

0.13 0.09 060809

0.04 0.04 070809

0.10 0.10 150909

0.12 0.09 030610

0.05 0.10 *100211

0.20 0.19 030810b

Mean 0.08 0.10

SEM 0.01 0.01

Low EGTA, TEA 0.65 1.02 *230610

0.37 0.36 220211

0.18 0.16 240211

0.19 0.10 230211

Low EGTA, Na 0.48 0.79 *010910

0.12 0.26 *030910

0.81 0.79 040910

0.30 0.24 090910

0.08 0.08 100910

Mean 0.35 0.42

SEM 0.08 0.12

Internal and external conditions are listed on the left, fiber identifier

on the right hand column. The means and SEM for high and low

[EGTA] are given. t = -1.574, p = 0.134, degrees of freedom = 17

in high [EGTA] and t = -1.2393, p = 0.2504, degrees of free-

dom = 8 in low [EGTA]. The cells judged to show SOCE are indi-

cated with an asterisk


123

significantly different between both groups independently

of the activation of SOCE. There was a statistically sig-

nificant difference in the percentage of recovery between

fibers in high and low intracellular [EGTA], with the for-

mer having higher recovery. As the myoplasmic [Ca2?]

went higher in the second group this difference is consis-

tent with Ca2?-dependent uncoupling (Verburg et al.

2005).

Mn2? entry was seldom increased as consequence

of SR depletion

The store operated Ca2? pathway in the plasma membrane

is also permeable to Mn2? among other ions. Mn2? is

known to quench the fluorescence of many dyes upon

binding. This property is used to monitor the activation of

Ca2? influx after store depletion by means of fluorescence

quenching using Mn2? as the permeant ion. The dye usu-

ally employed to this end was Fura-2 but Fura red was also

reported to exhibit Mn2? dependent fluorescence quench-

ing. We used the latter dye taken advantage of its visible

light excitation range. Ideally the dye should be excited at

the wavelength in which it is isosbestic for Ca2?. In cuvette

studies we found 462 nm to be the isosbestic wavelength.

Despite this, the 460 nm filters gave us a small Ca2? signal,

consisting in a reduction of fluorescence. The polarity of

the Ca2? signal was in the same direction as the effect of

Mn2?. Therefore we preferred to excite the dye at 450 nm,

in this way the increase in resting fluorescence as a con-

sequence of the increase in [Ca2?] due to SR depletion

should be reversed by Mn2? entry. The experimental pro-

tocol was as follows: after the dye loading period that was

carried out with Ca2? in the external solution, the fiber was

exposed to a Ca2?-free, 8 mM Mn2? external solution,

after equilibration in the new ionic condition CPA was

applied, always in Mn2?. In some fibers when exposed to

extracellular Mn2? and before the SR depleting treatment

an important reduction in fluorescence was observed

associated with a progressive reduction of the Ca2? tran-

sient. These fibers with spontaneous decrease in Fura red

fluorescence were discarded. Thus we used fibers in which

the resting fluorescence continued to rise in the presence of

Mn2? in the extracellular solution, albeit at a lower rate.

Upon the application of CPA a reduction of the voltage

elicited Ca2? transients was always observed. In a minority

of fibers after this reduction was more or less complete a

progressive decrease in resting fluorescence was observed.

This decrease was stopped but never reversed upon

washing out Mn2? from the central compartment.

In nine fibers studied only three gave this kind of

response. Figure 5A, B depicts one case in which there is

no clear change in the time course of the resting fluores-

cence upon SR depletion. Fluorescence roughly followed

the time course of the increase in dye concentration. The

other fiber shown (Fig. 5C, D) presented a drastic reduction

in resting fluorescence following SR depletion while the

concentration of the indicator continued to increase.

Ca2? entry secondary to SR depletion and changes

in membrane current were not correlated

In non muscle tissues the description of SOCE was quite

often based in its electrophysiological properties as Ca2?

carried a membrane current. This current, named ICRAC or

Fig. 2 Results from a fiber that lacked secondary Ca2? influx upon

SR depletion are shown. Same experimental conditions and protocol

as in Fig. 1 (high intracellular EGTA, TEA based external solutions)

were used. Ca2? transients records in response to voltage clamp

pulses to 0 mV and 100 ms duration obtained in different conditions

are shown. In the inset, the time course of the resting [Ca2?] under the

various conditions imposed is plotted. The vertical bars indicate the

change of bathing solutions. The records are numbered to indicate the

correspondence with the resting [Ca2?] measurements. Upon the

application of CPA in the Ca2?-free external a marked suppression of

the Ca2? transient is observed together with the increase in resting

[Ca2?]. Application of the Ca2?-containing external solution (with

the same [CPA]) did not further increase resting [Ca2?]. Wash out of

CPA maintaining the extracellular [Ca2?] reduced the resting [Ca2?]

as well as recovered the Ca2? transient. Fiber identifier 030810b,

[Fluo3] went from 218 to 310 lM, optical path = 60 lm,

diameter = 80 lm


123

ISOC, is an inwardly rectifying current that if activated

should contribute to the holding current needed to clamp

the cell to -90 mV. Therefore we selected those fibers in

which a SOCE like response and compared the change in

resting [Ca2?] with the effect on the holding current. The

effect of readmitting a Ca2?-containing solution in the

central compartment did not always increase the holding

current as expected. The collected results in this selected

group of fibers are shown in Table 3. The values of [Ca2?]

are repeated from Table 1. The values of holding current

are the average of the last five readings in each condition.

Trivially, the change in resting [Ca2?] was significant. On

the other hand the change in holding current was not (see

legend of Table 3). The change of extracellular solution

had little effect, no more than 5 %, and moreover was not

always in the same direction. To quantify this we per-

formed a linear regression between both quantities in the

cells with high [EGTA] (n = 6) yielding a Pearson

regression coefficient of 0.005 not significantly different of

0, confirming the poor correlation observed.

Discussion

The main finding in this study is the low frequency of

activation of Ca2? influx secondary to SR depletion in cut

frog skeletal muscle fibers. This was confirmed in various

combinations of extracellular and intracellular solutions.

Fig. 3 The figure shows two different outcomes of experiments in

Na-based external solutions with high intracellular EGTA. In A, a

fiber in which following depletion of the SR Ca2? influx occurred is

shown. The records of Ca2? transients elicited by clamp depolariza-

tion to 0 mV for 100 ms, in Ca2?-free external (1), after CPA

application in the same solution (2), in the presence of Ca2? and CPA

(3) and after washing out the SERCA pump blocker in Ca2?-

containing external solution (4), are shown. The inset shows the time

course of the resting [Ca2?], the numbers indicate the data points

corresponding to the plotted records. Fiber identifier 030810a, [Fluo3]

went from 213 to 359 lM, optical path = 90 lm, diameter = 80 lm.

In B a negative result is shown. A fiber subjected to the same protocol

where SR depletion did not produce an influx of Ca2?. The records

are voltage elicited transients in response to depolarization to 30 mV

for 50 ms. The inset shows the time course of the resting [Ca2?] under

the different conditions applied, the numbers indicate the data points

corresponding to the records shown. Fiber identifier 201011a, [Fluo3]

went from 143 to 309 lM, optical path = 80 lm, diameter = 98 lm


123

We initially studied cells in high intracellular EGTA and

our usual ‘‘charge movement’’ (devoid of the main per-

meant ions, except Ca2?) external solutions, conditions in

which the phenomenon was expected to occur. High

intracellular EGTA, in fact higher than our 5 mM con-

centration, was routinely used in many the original studies

of SOCE in non-excitable cells, particularly in electro-

physiological experiments where ICRAC or ISOC were

measured (Parekh and Putney 2005). In these studies the

high cytoplasmic Ca2? buffering did not preclude the

activation of SOCE upon depletion. Moreover, the added

buffer captured the released Ca2? and contributed to attain

the depletion of the store. The comparison of our condi-

tions with those of previous studies in skeletal muscle

showed that most of these studies were carried out in cells

bathed with physiological external perfusates and without

added Ca2? buffering to the myoplasm. We approximated

to these conditions as much as possible. In particular the

presence of physiological [Na?] appeared as an important

factor, for at least two reasons:(1) it might contribute to

outward Ca2? transport via Na?/Ca2? exchange and (2) in

one study it was reported that Na? entry during Ca2?-free

conditions might result in reverse Na?/Ca2? exchange

upon readmitting Ca2? in the extracellular medium

(Bolanos et al. 2009), who found a partial suppression of

post-depletion Ca2? entry by the Na?/Ca2? exchange

reverse mode blocker KB-R7943.

Consistent with the first possibility in previous studies

the myoplasmic [Ca2?] in Ca2?-free external solutions

declined after reaching a maximum during the treatment

with CPA, as the SR uptake was blocked this could be

explained only by Ca2? extrusion. Although it might occur

passively as CPA was applied in Ca2?-free external the

contribution of primary or secondary active transport

should be considered. In our experiments in high intra-

cellular EGTA the decay was not always apparent.

Therefore we studied fibers with 0.5 mM EGTA added, a

concentration that did not avoid contraction which was

blocked with BTS. Under this condition we did not find

substantial difference in the percentage of cells that show

SOCE-like response in Na?- containing when compared to

Na?-free external solution. Furthermore we did not find a

difference either in the amplitude or rate of the decay

between Na?-free and Na?-containing external solutions.

These findings also questioned that in frog muscle there is

a role played by reverse mode Na?/Ca2? exchange con-

tributing to Ca2? influx secondary to SR depletion as

described by Bolanos et al. (2009) in rat FDB muscle fibers.

The low rate of occurrence of SOCE-like response was

confirmed in Mn2? quenching experiments. Less than half

Fig. 4 In A records of voltage

triggered Ca2? transients from a

cell in Na based external and

low EGTA internal solutions are

shown. The inset plots the time

course of the resting [Ca2?]

during the part of the

experiment when the different

pharmacological and ionic

interventions were performed.

In this fiber following the

depletion of the SR the resting

[Ca2?] increased upon changing

from Ca2?-free to Ca2?-

containing extracellular

perfusate. Fiber identifier

010910, [Fluo3] went from 103

to 273 lM, optical

path = 80 lm,

diameter = 110 lm. B the

outcome of the application the

same protocol in a cell that did

not give a secondary Ca2? entry

upon SR depletion. The insetshows the time course of the

resting [Ca2?]. Fiber identifier

040910, [Fluo3] went from 124

to 315 lM, optical

path = 60 lm,

diameter = 80 lm


123

Table 2 Values of resting myoplasmic [Ca2?] (measured as in

Table 1), voltage triggered [Ca2?] transients (D [Ca2?], measured as

the difference between the maximum [Ca2?] attained during the

clamp pulse and resting [Ca2?]), linear capacitance (C), and holding

current per unit capacitance (IH) measured in Ca2?-containing

external salines for the initial (previous to the depletion protocol)

and final (after washing out CPA) conditions are given

Initial Final %D[Ca2?] Initial Final Fiber

[Ca2?]rest (lM) D[Ca2?] (lM) [Ca2?]rest (lM) D[Ca2?] (lM) C (nF) I (A/F) C (nF) I (A/F)

High EGTA

0.02 0.24 0.04 0.17 0.71 5.2 1.98 5.2 1.84 *030810a

0.06 0.92 0.07 0.45 0.49 2.6 4.20 2.4 3.98 *130810

0.02 0.77 0.14 0.41 0.53 6.2 3.02 5.6 3.28 *171011

0.05 0.25 0.07 0.09 0.36 3.8 3.66 3.9 3.76 *191011b

0.03 0.54 0.07 0.38 0.70 4.0 2.54 3.8 2.60 *050809a

0.03 0.34 0.04 0.13 0.38 7.1 2.46 7.2 2.32 *100211

Mean

0.03 0.51 0.07 0.27 0.53 4.8 2.98 4.7 2.96

SEM

0.01 0.12 0.01 0.06 0.06 0.7 0.34 0.7 0.34

0.02 0.35 0.01 0.15 0.43 4.0 2.56 4.1 2.33 110909

0.02 0.52 0.02 0.11 0.21 6.9 1.59 6.7 1.45 181011

0.03 0.52 0.04 0.21 0.40 5.2 3.65 5.0 3.56 191011a

0.01 0.30 0.03 0.14 0.47 6.9 1.70 6.8 1.68 201011a

0.05 0.90 0.08 0.15 0.17 5.3 2.08 5.2 2.03 201011b

0.04 0.85 0.08 0.16 0.19 7.3 2.74 7.7 2.54 040809

0.08 0.47 0.09 0.31 0.66 5.0 1.58 4.8 1.69 050809b

0.02 0.23 0.03 0.17 0.74 3.6 2.31 3.7 2.20 060809

0.05 0.36 0.07 0.20 0.56 5.3 3.33 5.0 3.61 070809

0.04 0.36 0.05 0.09 0.25 3.6 2.59 3.8 2.46 150909

0.01 0.63 0.06 0.27 0.43 3.6 1.05 3.4 1.12 030610

0.05 0.20 0.05 0.10 0.50 5.3 1.62 4.9 1.57 030810b

Mean

0.04 0.47 0.05 0.17 0.42 5.2 2.22 5.1 2.19

SEM

0.005 0.06 0.01 0.02 0.05 0.4 0.22 0.4 0.22

Low EGTA

0.10 3.00 0.40 0.20 0.07 3.9 1.35 4.1 1.41 *230610

0.09 1.70 0.15 0.85 0.50 6.2 1.47 6.3 1.60 *010910

0.09 1.80 0.10 0.34 0.19 3.6 1.50 3.5 1.39 *030910

Mean

0.09 2.17 0.22 0.46 0.25 4.6 1.44 4.6 1.47

SEM

0.003 0.42 0.09 0.20 0.13 0.8 0.04 0.8 0.07

0.07 2.80 0.14 0.40 0.14 3.4 2.14 3.6 1.94 220211

0.05 1.30 0.13 0.51 0.39 6.4 1.88 6.0 1.98 240211

0.03 0.80 0.05 0.13 0.16 4.3 1.43 4.6 1.30 230211

0.06 2.30 0.17 1.12 0.49 3.0 1.27 3.2 1.28 040910

0.06 3.10 0.20 0.20 0.06 4.6 3.34 4.8 3.11 090910

0.03 0.55 0.04 0.20 0.36 4.8 1.90 5.2 1.91 100910

Mean

0.05 1.81 0.12 0.43 0.27 4.4 1.98 4.5 1.92

SEM

0.01 0.44 0.03 0.15 0.07 0.5 0.30 0.4 0.27

Averages (Mean ± SEM) for the four groups of cells are given. Cells that showed SOCE are indicated with an asterisk. For a given intracellular solution the

initial parameters of cells with and without SOCE were compared by t test. Only the resting [Ca2?] in low intracellular [EGTA] was significantly different.

Also the fraction of recovery of D [Ca2?] was studied, finding no statistically significant difference


123

of the cells studied this way showed a decrease in Fura Red

fluorescence following SR depletion.

We do not have a clear explanation for the low fre-

quency at which the secondary Ca2? entry is observed in

frog skeletal muscle. The only other study in amphibian

muscle that we know (Launikonis et al. 2003) does not

mention anything in this regard. In this study carried out in

Australian cane toad skinned fibers the Ca2? sensitive dye

trapped in sealed over transverse tubules might have been a

more sensitive detection method compared to our

myoplasmic measurements. This difference in sensitivity is

supported by the fact that Launikonis and Rıos (2007)

successfully detected SOCE in depolarized cells with the

Ca2? probe in the source compartment (the sealed over

t-tubules) while Kurebayashi and Ogawa (2001) failed to

find it in intact cells measuring Ca2? in the myoplasm.

Launikonis and Rıos (2007) reported that the intensity of

SOCE in depolarized cells was about 20 % that in well

polarized cells. Thus, it is likely that in whole cell mea-

surements the changes in bulk myolasmic [Ca2?] produced

Fig. 5 Mn2? quenching of Fura Red fluorescence after SR depletion.

In A records of fluorescence of Fura Red excited at 450 nm are

shown. In response to voltage clamp depolarization (0 mV, 100 ms)

Ca2? was released from the SR and Fura Red gave positive going

transients. Upon changing the external solution to Ca2?-free 8 mM

Mn2? the voltage triggered transients persisted. After the application

30 lM CPA, dissolved in the Mn2? external solution, the transients

progressively decayed. The resting fluorescence increased continu-

ously, roughly in parallel with the [Fura red]. This is clearly shown in

B, where resting fluorescence readings (open circles) from the same

cell in a are plotted against time during the experiment. The numbers

identify resting fluorescence values corresponding to the records of

fluorescence transients shown in a, with the same numbers. Also

plotted, the time course of dye concentration (filled circles) estimated

from absorbance measurements at 460 nm. Fiber identifier 140311,

optical path = 110 lm, diameter = 117 lm. c and D a different

experiment in Mn2? external solution in which SR depletion was

followed by Mn2? influx. In C fluorescence transients elicited by

voltage clamp depolarization (1–3 are to -30 mV, 100 ms, 4 and 5 to

0 mV, same duration) under various conditions are shown. CPA

treatment reduced and eventually the transients. The resting fluores-

cence (open circles) started to decline despite the increasing dye

concentration (filled circles), as shown in D. Fiber identifier 010311,

optical path = 120 lm, diameter = 108 lm


123

by a low intensity Ca2? flux from the transverse tubules

might have been undetected. As will be discussed below,

SOCE in our frogs was generally of very low intensity,

therefore it might be the explanation for the high number of

cells that did not give a SOCE-related signal.

Launikonis et al. (2003) reported that in fibers in which

the SR was uncoupled from the t-tubules by previous

exposure to high intracellular [Ca2?] the store depletion

dependent Ca2? influx was drastically reduced. As

uncoupling by elevated levels of myoplasmic [Ca2?] could

occur even at relatively low values (a few hundred nM)

provided the exposure is long enough (Murphy et al. 2006)

this factor might be underlying the diminished SOCE we

observed. We specifically addressed this problem by

comparing the degree of recovery of the [Ca2?] transient in

cells that showed SOCE-like responses and those that

lacked it. The percentage of recovery can be taken as a

measure of uncoupling although other factors might con-

tribute importantly to the reduction of the Ca transient, for

instance incomplete reloading of the SR reticulum. The

average of percentage recovery was not statistically dif-

ferent between the two groups of cells. Although our cells

presented strong indications of uncoupling this did not

correlate simply with the presence or absence of the SOCE-

like response.

We cannot rule out species differences, both between

cane toads and rana catesbeiana as well as between the

latter and mammals, to account for our observations.

It has been reported a predominant role in Ca2? han-

dling by SERCA pump in frogs while there is an equal

contribution of SERCA and plasma membrane Ca2?

pumps in rats (Hemmings 2001). If the extrusion systems

were less developed this might correlate with an equally

less developed reloading mechanism.

Another systematic difference found in our study when

compared with previous ones is that both depletion of the

SR and the secondary Ca2? entry were much slower. The

speed of activation of SOCE is a unique property of skel-

etal muscle (Launikonis and Rıos 2007; Edwards et al.

2010b). We cannot fully separate slow gating from a slow

rise in [Ca2?] due to a low intensity flux (as will be dis-

cussed below). We do not have a conclusive explanation

but in part could be due to the lower temperature we used.

Also the bigger size of frog cells would make diffusion

times longer, consistent with the fact that the fastest acti-

vation was reported in skinned fibers where the delivery of

the depleting drugs might have been faster.

It is unclear whether SOCE activation and deactivation

is graded by the SR content (Collet and Ma 2004) or if it

occurs once a threshold is crossed, in any case the deple-

tion achieved in our experiments seems large enough to

activate Ca2? entry. Furthermore, the same degree of

depletion was followed by either an activation of secondary

Ca2? entry or the lack of it, therefore excluding this vari-

able as a cause of the different outcomes observed.

Given these differences we cannot rule out that the

secondary Ca2? entry we observed occurred through a

Ca2? pathway other than the SOCE complex (STIM1–

Orai1), not gated by store depletion but by an increase in

cytoplamsic [Ca2?].

Table 3 Values of resting myoplasmic [Ca2?] repeated from Table 1 for the fibers that had an increase in resting [Ca2?] upon extracellular

readmission of Ca2?

Int.sol, Ext. Sol. [Ca2?]Rest (lM) D[Ca2?]Rest (lM) IH (A/F) DIH (A/F) Fiber

Mg CPA Ca CPA Mg CPA Ca CPA

High EGTA, Na 0.03 0.20 0.17 2.12 2.15 0.03 030810a

0.11 0.21 0.10 3.50 3.31 -0.19 130810

0.06 0.13 0.07 3.75 3.57 -0.18 171011

0.03 0.06 0.03 4.00 4.10 0.10 191011b

High EGTA, TEA 0.10 0.15 0.05 2.03 1.89 -0.14 050809a

0.05 0.10 0.05 3.12 3.10 -0.02 100211

Mean 0.06 0.14 0.08 3.09 3.02 -0.07

SEM 0.01 0.02 0.02 0.34 0.35 0.05

Low EGTA, TEA 0.65 1.02 0.37 2.20 2.30 0.10 230610

Low EGTA, Na 0.48 0.79 0.31 2.20 2.10 -0.10 010910

0.12 0.26 0.14 1.85 1.85 0.00 030910

Mean 0.42 0.69 0.27 2.08 2.08 0.00

SEM 0.16 0.22 0.07 0.12 0.13 0.06

The corresponding values of the holding current (also measured as the average of the first 20 ms during the acquisition of voltage clamp pulses,

averaged over the last five readings in each condition) for the same experimental conditions. Also the respective differences (Ca2?-containing

minus Ca2?-free) are listed. Paired t-test on current values gave t = 1.18, p = 0.27, degrees of freedom = 8


123

As explained in results we compared the holding cur-

rents in Ca2?-free and Ca2?-containing solutions after

depletion of the SR was attained in the group of fibers that

showed SOCE-like response. As shown in Table 2 there

was no significant change in the holding current associated

with the secondary elevation of myoplasmic [Ca2?]. This

observation is consistent with previous reports in mam-

malian cells (Allard et al. 2006; Berbey and Allard 2009).

It is possible to put a lower limit in the change in holding

current expected from the measured change in the resting

[Ca2?]. Considering the fibers in 5 mM EGTA the lower

limit for the increase in total intracellular [Ca2?] is the

change in the Ca2? bound to EGTA which is the pre-

dominant buffer. The average in these six fibers is

0.64 mM (using a KD = 0.37 lM for EGTA, see Meth-

ods). The average time in which the change in total con-

centration occurred was 12 min giving a mean current of

0.17 A/l, an estimate one order of magnitude smaller than

the one given by Launikonis and Rıos (2007) in rat skinned

fibers. Considering the geometry and capacitance of our

fibers the average expected increase in holding current

would be 0.05 A/F, about 2 % of the holding current before

the application of Ca2? CPA external solution. Given its

small size it could have been undetected, obscured by other

superimposing currents.

In summary, our failure to detect consistent and robust

SOCE signals does not necessarily mean that this mecha-

nism is irrelevant for frog muscle. As discussed, many

factors might contribute to the poor signals we report here.

Based on phylogenetic arguments (Cai, 2007a, b) we

expect the molecular machinery to be present in the frog

species used in this study although this should be demon-

strated by direct methods and expression levels quantified.

Since uncoupling due to exposure to high myoplasmic

[Ca2?] does not fully explain the lack of SOCE we favor

the idea that this mechanism is a labile one and that our

methodology with prolonged dialysis of intracellular

components partially inhibit it, although the diffusible

regulatory factors that might be involved (i.e. Inositol tri-

phosphate (Launikonis et al. 2003); Calmodulin (Moreau

et al. 2005)) remain to be established.

Acknowledgments This study was supported by grant FCE07 258

(ANII) to GP.

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a study of store dependent ca2+ influx in frog skeletal muscle

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