1 Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago 7; 3 Centro de Estudios Científicos, Valdivia, Chile; and 2 Department of Physiology, University of California Los Angeles, Los Angeles, California 90024
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The
Na+/Ca2+ exchanger participates in
Ca2+ homeostasis in a variety of cells and has a key role
in cardiac muscle physiology. We studied in this work the exchanger of
amphibian skeletal muscle, using both isolated inside-out transverse
tubule vesicles and single muscle fibers. In vesicles, increasing
extravesicular (intracellular) Na+ concentration
cooperatively stimulated Ca2+ efflux (reverse mode), with
the Hill number equal to 2.8. In contrast to the stimulation of the
cardiac exchanger, increasing extravesicular (cytoplasmic)
Ca2+ concentration ([Ca2+]) inhibited this
reverse activity with an IC50 of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at
90 mV. Photolysis of a cytoplasmic caged Ca2+ compound
activated an inward current (forward mode) of 23 ± 10 nA
(n = 3), with an average current density of 0.6 µA/µF. External Na+ withdrawal generated an outward
current (reverse mode) with an average current density of 0.36 ± 0.17 µA/µF (n = 6) but produced a minimal increase
in cytosolic [Ca2+]. These results suggest that, in
skeletal muscle, the main function of the exchanger is to remove
Ca2+ from the cells after stimulation.
intracellular calcium regulation; electrogenic ion transport; calcium fluxes; calcium permeability; plasma membrane transporters
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE SODIUM/CALCIUM EXCHANGER (NCX) is an electrogenic and reversible countertransport system with a well-established role in Ca2+ homeostasis in a variety of cells (6). In its forward mode, the exchanger transports Ca2+ against its transmembrane electrochemical gradient, making use of the Na+ electrochemical gradient. Given the right balance between the respective electrochemical gradients, the NCX also operates in the reverse mode, transporting Ca2+ into cells and Na+ out (6).
Three mammalian isoforms of the NCX protein, products of three different genes, have been cloned (28, 31, 32) and appear to have very similar properties (29). Mammalian cardiac muscle expresses high levels of the NCX1 isoform (31), whereas amphibian cardiac muscle has an NCX that presents a novel molecular determinant that causes different regulation by cAMP from that observed in mammalian cardiac muscle (37). The mammalian NCX1 isoform has been found in varying amounts in most other tissues, including kidney, brain, pancreas, liver, placenta, and skeletal muscle, where it is present both in transverse tubule (T tubule) and surface plasma membranes (35). In addition, the mammalian isoforms NCX2 (28) and NCX3 (32) are present in skeletal muscle and brain.
Most of the current knowledge of NCX properties has been obtained from the many studies performed on mammalian cardiac muscle, where the NCX plays a central role in transporting Ca2+ out of the cells during cardiac muscle relaxation (6, 34). By allowing Ca2+ entry during membrane depolarization, the exchanger might also participate in cardiac excitation-contraction coupling, but this alleged role remains controversial (6).
The NCX of skeletal muscle has been less studied than its cardiac counterpart, and a clear-cut physiological role has not been defined yet. Small bundles of muscle fibers from amphibian skeletal muscle (7), as well as single muscle fibers (22), exhibit Na+-dependent Ca2+ fluxes. This trait is shared by sarcolemmal fractions isolated from mammalian muscle (14, 30) and by T tubules isolated from amphibian muscle (10, 18). The NCX present in T tubules from amphibian muscle displays in the forward mode a Michaelis constant of 2.7 µM for intracellular Ca2+ (10). In agreement with this rather low Ca2+ affinity, to detect Na+-dependent Ca2+ efflux in amphibian single fibers, it is necessary to increase the intracellular Ca2+ concentration ([Ca2+]) well above its resting level (22). The reverse mode of the exchanger may cause the enhancement of contraction that takes place after external Na+ withdrawal in phasic (8, 13, 17, 26) or tonic (24) amphibian skeletal muscle fibers.
In mammalian skeletal muscle, exchanger reverse currents, associated with net Na+ efflux, have been measured in giant inside-out excised sarcolemmal patches (17). However, these currents are of a lower magnitude than their cardiac counterparts measured under similar conditions (20). Recent experiments suggest that intact single skeletal muscle fibers from the mouse have an NCX that becomes activated in its forward mode when cytoplasmic [Ca2+] is increased to levels similar to those produced by tetanic stimulation (3). Other observations, albeit of a more indirect nature, also support the presence of a functional NCX in mammalian skeletal muscle (4, 39).
The purpose of this work was to further characterize the NCX transporter of amphibian muscle, using both isolated sealed inside-out T tubule vesicles and single fibers. Vesicular experiments indicate that increasing extravesicular (intracellular) Na+ concentration ([Na+]) from 0 to 120 mM resulted in a marked and cooperative stimulation of Ca2+ efflux from the vesicles (reverse mode). Increasing extravesicular (intracellular) [Ca2+] inhibited the reverse skeletal NCX activity in contrast to the stimulation reported in cardiac muscle. In single fibers, photolysis of a cytoplasmic caged Ca2+ compound activated an inward current, as expected from the exchanger operating in its forward mode. Na+ withdrawal from the external solution activated an outward current, indicative of the reverse mode, but produced only a very modest increase in cytosolic [Ca2+].
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and Characterization of Amphibian Membrane Fractions
T tubule vesicles were isolated from the skeletal muscle of the Chilean frog Caudiverbera caudiverbera using a procedure described in detail elsewhere (19). After isolation, vesicles were resuspended in 0.3 M sucrose and 20 mM Tris-maleate, pH 7.0, frozen in liquid nitrogen, and stored atVesicular Ca2+ Fluxes
To determine Ca2+ influx, T tubule vesicles in 0.3 M sucrose and 20 mM Tris-maleate, pH 7.0, were diluted 5- to 10-fold to 0.1 mg protein/ml in loading solution. The loading solution contained variable 45CaCl2 at a specific activity of 15-20 mCi/mmol, 10 mM potassium gluconate, 150 mM HEPES-Tris, pH 7.4, plus 1 µM valinomycin. After dilution, vesicles were incubated for 3 h at the temperatures specified in text. To determine exchanger-mediated Ca2+ influx, the vesicles were incubated for 3 h as above, except that the loading solution contained 2 mM nonradioactive CaCl2; after this time, an aliquot of 45CaCl2 was added, and the incubation was continued for up to 15 min. To stop Ca2+ influx, a 0.01-ml fraction of the incubation solution was mixed with 1 ml of an ice-cold solution (quench solution) containing (in mM) 5 MgCl2, 10 EGTA-Tris, 10 LaCl3, and 20 HEPES-Tris, pH 7.4. A 0.9-ml fraction was immediately filtered through Millipore filters (HA 0.45 µm) previously soaked with (in mM) 0.1 CaCl2, 5 MgCl2, and 20 HEPES-Tris, pH 7.4. The filters were washed three times with 3 ml of quench solution, and the radioactivity retained in the dried filters was determined by liquid scintillation counting. For Ca2+ efflux determinations, T tubule vesicles (1 mg/ml) were first equilibrated for 3 h at the temperatures specified in the text in a loading solution containing (in mM) 10 potassium gluconate, 2 CaCl2 plus 45CaCl2 to a specific activity of 15-20 mCi/mmol, and 150 HEPES-Tris, pH 7.4. All efflux experiments were carried out in the presence of 1 µM valinomycin and equal K+ concentrations inside and outside the vesicles to maintain the membrane potential clamped at 0 mV. To determine passive Ca2+ efflux, vesicles were diluted 100-fold in a solution containing (in mM) 10 potassium gluconate, 10 EGTA, and 160 HEPES-Tris, pH 7.4. To measure Na+-dependent Ca2+ efflux, vesicles were diluted 100-fold in a solution that contained (in mM) 140 sodium gluconate, 10 potassium gluconate, 10 EGTA, and 20 HEPES-Tris, pH 7.4. To study the effect of extravesicular [Na+] or [Ca2+] on Na+-dependent Ca2+ efflux, vesicles were diluted in solutions with varying [Na+], replacing Na+ with choline, or at different free [Ca2+], calculated using published values of binding constants (16). Ca2+ efflux was stopped by rapid filtration of 1 ml of each dilution through Millipore filters (HA 0.45 µm) previously soaked in the solution described above. The filters were washed three times with 3 ml of ice-cold quench solution and dried, and their radioactivity was measured in a liquid scintillation counter.Electrophysiological Measurements
The experiments were carried out using short segments of single muscle fibers dissected from the semitendinosus muscle of Rana catesbeiana. Individual fibers were mounted on a triple Vaseline gap chamber as described (2, 21).Fibers, first dissected in normal Ringer solution (in mM: 115 NaCl, 2.5 KCl, 1.8 CaCl2, and 10 Na-MOPS, pH 7.0), were bathed with a relaxing solution containing (in mM) 94 K2SO4 and 10 K-MOPS, pH 7.0. Fibers were then transferred to the Vaseline gap chamber and laid across the three grease seals that divided the chamber into four pools. The end pool solutions were exchanged for an internal solution and allowed to equilibrate for 45-90 min before the experiment was initiated. The composition of the internal solution was (in mM) 110 potassium aspartate or 110 cesium aspartate, 20 MOPS, pH 7.0, adjusted with KOH or CsOH, 2 MgCl2, 5 K2-ATP, 0.1 mg/ml creatine phosphokinase, 5 Na2-phosphocreatine, and 0.2 EGTA. One of the Ca2+ indicators (calcium orange-5N, fluo 3, or rhod 2), alone or in combination with the cage-Ca2+ compound DM-nitrophen (Molecular Probes), was added to the end pool solution. In some experiments, rhodamine B (Sigma) was also added to the end pool solution to monitor non-Ca2+-specific changes in fluorescence. The [Ca2+] of the internal solution was measured using a Ca2+-sensitive microelectrode (calibrated with a commercial kit from WPI, Sarasota, FL) and was adjusted to pCa 7.0. A segment of the fiber lying in one of the two central pools (pool A) was voltage clamped, and the external solution bathing this segment was changed by a steady flow of Ringer solution into the pool while removing the excess. Membrane currents were normalized by fiber capacitance rather than by current density and are expressed as microampere per microfarad. This normalization eliminates possible errors in estimating the area of outer membrane in the central pool A, which might occur due to irregularities in the fiber cross-sectional area and in the grease seal boundaries. Fiber capacity (µF) was calculated from the integral of the transient currents elicited in response to hyperpolarizing pulses. The voltage clamp chamber was mounted on the stage of a modified compound fluorescence microscope used as a vertical optical bench. This setting was designed to allow epi-illumination of the voltage-clamped segment of muscle fiber by focusing the luminous field diaphragm on the preparation with a ×20 objective (Fluo-20, numerical aperture 0.75; Nikon). Fluorescent light from the preparation was focused on a low-noise photodiode, converted to voltage, and amplified (27, 38).
Flash Photolysis
Ultraviolet (UV) flashes from an argon laser were delivered to the muscle fiber segment in pool A to elicit photolysis of DM-nitrophen, following a similar procedure to that described by Sanchez and Vergara (36) but adapted for Ca2+ detection, as illustrated by Escobar et al. (11, 12). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vesicular Experiments
The amphibian T tubule vesicles used in this study had a high density of dihydropyridine binding sites. DensitiesCa2+ influx in native T tubule vesicles.
As shown in Fig. 1, isolated T tubule
vesicles incubated at 25°C in a loading solution containing 2 mM
45CaCl2 accumulated Ca2+
exponentially as a function of time, with a rate constant of 0.048 min1. Lowering the temperature to 5°C decreased the
rate constant of Ca2+ accumulation threefold to 0.015 min
1 (Fig. 1), indicating that Ca2+ influx
had a higher temperature dependence than a passive diffusion process.
On average and regardless of incubation temperature, vesicles incubated
in 2 mM CaCl2, and extensively washed with 10 mM EGTA to
remove contaminating external Ca2+, accumulated at
equilibrium 32 ± 10 (SD) nmol/mg protein (n = 16). These values are higher than expected for simple equilibration. Previous evidence indicating that T tubule vesicles have luminal (extracellular) Ca2+ binding sites (18), with
a dissociation constant (Kd) of 2.3 ± 0.3 mM and a maximal binding (Bmax) of 70 ± 2 nmol/mg,
was confirmed in the present work. Isolated T tubule vesicles incubated
to equilibrium in solutions with varying CaCl2
concentration displayed a single class of intravesicular low-affinity
Ca2+ binding sites with a Kd of
3.5 ± 1.1 mM and a Bmax of 73 ± 10 nmol/mg
(mean ± SD, n = 3).
|
|
Passive Ca2+ efflux.
To study passive Ca2+ efflux, T tubule vesicles were first
incubated in 2 mM 45CaCl2 for 3 h and were
then diluted in Na+-free solution plus 10 mM EGTA. These
experimental conditions were chosen to prevent exchange of vesicular
Ca2+ with external (cytoplasmic) Na+ or
Ca2+. After dilution, the vesicular Ca2+
content decreased exponentially with time, with a rate constant of
0.009 min1 at 15°C (Fig.
3).
|
Na+-dependent Ca2+ efflux.
To measure the effects of external Na+ on the reverse NCX
reaction, vesicles were passively equilibrated in 2 mM
CaCl2 and subsequently diluted in a solution containing 140 mM Na+ plus 10 mM EGTA. In the experiment shown in Fig.
4, vesicles exponentially lost 40% of
their vesicular Ca2+ after dilution, with a rate constant
of 2.8 min1. On average, at 5°C, the rate constant of
Na+-dependent Ca2+ efflux was 4.2 ± 1.8 (SD) min
1 (n = 4). Twofold higher rate
constant values were obtained at 25°C (data not shown), but the
process became too fast to collect accurate data manually
(half-time = 5 s). For this reason, subsequent experiments
were done at 5°C to improve the accuracy of data collection. At this
temperature, the values for Na+-dependent Ca2+
efflux were in the range of 42 to 63 nmol · mg
1 · min
1. Because
by definition Na+-dependent Ca2+ fluxes
represent Na+/Ca2+ exchange, these results
confirm previous observations (18) indicating that the
reverse mode of the exchanger operates in inside-out T tubule vesicles
isolated from amphibian skeletal muscle.
|
Effect of extravesicular Na+ on
Na+-dependent Ca2+ efflux.
To study the effect of cytoplasmic Na+ on the reverse mode
of the exchanger, Na+-dependent Ca2+ efflux was
determined at 5°C at different extravesicular [Na+] and
10 mM EGTA. In the absence of extravesicular Na+,
Ca2+ efflux had a rate constant of 0.012 ± 0.006 (SD)
min1 (n = 3). Increasing extravesicular
[Na+] produced a significant and nonlinear increase in
the rate constant of exchange (Fig. 5),
which reached its maximal value at
120 mM [Na+].
|
Effect of extravesicular [Ca2+] on
Na+-dependent Ca2+ efflux.
To investigate the effect of extravesicular Ca2+, reverse
Na+/Ca2+ exchange was measured in
extravesicular solutions containing 140 mM [Na+] and
different [Ca2+]. As shown in Fig.
6, increasing extravesicular
[Ca2+] from pCa 9 to pCa 5 produced a threefold decrease
in the rate constants of Na+-dependent Ca2+
efflux without affecting the total amount of Ca2+
exchanged. The IC50 for [Ca2+] was 91 nM
(pCa0.5 = 7.04).
|
Fiber Experiments
Single frog fibers were used to investigate the operation of NCX in muscle cells. Ionic currents and intracellular [Ca2+] were measured simultaneously after either removing Na+ from the external solution or increasing intracellular [Ca2+] by flash photolysis of a cytoplasmic caged Ca2+ compound. To eliminate contributions from changes in membrane potential, all experiments were done in muscle fibers voltage clamped atEffect of Na+ removal from the external solution.
In zero external Na+ and at 90 mV a functional NCX system
should operate in the reverse mode, exchanging intracellular
Na+ for extracellular Ca2+. This mode of
operation should generate an outward Na+ current and a
concurrent increase in intracellular [Ca2+]. As shown in
Fig. 7, replacement of extracellular
Na+ by tetramethylammonium produced a net change in outward
current of 10 nA. Replacement of external Na+ with
Li+ or N-methylglucamine produced similar
results (data not shown). Normalized current changes ranged from 0.18 to 0.65 µA/µF, giving an average value of 0.36 ± 0.17 (SD)
µA/µF (n = 6). The outward current lasted the
entire period while the fiber was perfused with zero Na+
solution. On reperfusing the fiber with normal Ringer, the current returned to the basal level (Fig. 7).
|
Effect of increasing intracellular [Ca2+] by photolysis of caged Ca2+. To study the forward operation of NCX in skeletal muscle fibers, the intracellular [Ca2+] was suddenly increased in the cytoplasm by flash photolysis of the caged Ca2+ compound DM-nitrophen.
As shown in Fig. 8, photolysis of DM-nitrophen produced at 15°C an immediate increase in intracellular [Ca2+] that was detected with the Ca2+ indicator rhod 2 and that had the concomitant appearance of an inward ionic current with a magnitude of 20 nA. Both the current and the intracellular [Ca2+] increase lasted the 50 ms of recording time. The same immediate increase in intracellular [Ca2+] was observed when using fluo 3 or calcium orange-5N instead of rhod 2 as Ca2+ indicators (data not shown). After photolysis of DM-nitrophen, three independent experiments gave on average an inward ionic current of 23 ± 10 (SD) nA. The corresponding current density measured at 15°C and
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present results provide a characterization of the effects of varying cytoplasmic [Na+] and [Ca2+] on the reverse NCX reaction in T tubule vesicles isolated from amphibian skeletal muscle. In addition, this is to our knowledge the first description of the currents associated with the forward and reverse mode of the NCX in whole skeletal muscle fibers.
Vesicular Experiments
In the absence of external Na+ or Ca2+ to avoid operation of the NCX, T tubule vesicles equilibrated with millimolar [Ca2+] displayed very low passive Ca2+ efflux, despite the large chemical gradient for Ca2+ present in these experiments. These results indicate that the isolated T tubule vesicles were tightly sealed and thus maintained after isolation the low Ca2+ permeability of resting muscle fibers (5).The reverse NCX reaction engaged only a fraction of the luminal
Ca2+.
Of the total amount of Ca2+ equilibrated in the vesicles,
most of it was bound to low-affinity sites. Because the T tubules used
in this work were sealed only with the inside-out configuration, these
sites, whose nature remains to be characterized, should correspond in
vivo to extracellular Ca2+ binding sites present in the
lumen of the T tubules. Only a fraction of the total luminal
Ca2+, which varied from 25 to 43%, was available for fast
exchange with Na+. Partial dissipation of the
Na+ gradient may explain this limited exchange. The amount
of Ca2+ exchanged, which on average was 12 nmol/mg, should
produce a net Na+ entry of 36 nmol/mg. With a T tubule
luminal volume of 0.5 µl/mg protein (P. Donoso and C. Hidalgo,
unpublished observations), this Na+ entry should increase
luminal [Na+] and may produce a decrease in driving
force, limiting the amount of Ca2+ exchanged for
Na+. In addition, the NCX may be present in only a fraction
of the vesicles, or all vesicles would have the exchanger, but a
fraction of their luminal Ca2+ might be bound to sites that
are not readily available for fast exchange with Na+. The
present results do not allow a distinction between these last two options.
Effects of external [Na+] on the reverse NCX
reaction.
Increasing extravesicular [Na+] produced a significant
and nonlinear increase in the rate constant of reverse exchange that reached its maximal value at 120 mM [Na+]. Assuming a
cooperative model for Na+ activation of the reverse mode of
the exchanger, a Hill equation fitted to the data yielded
nHill = 2.8 and
kHill = 55.9 mM Na+. Although
there are no other data available for amphibian muscle, similar values,
with nHill = 2.4 and
kHill = 55 mM Na+, were
reported for Na+ activation of the reverse NCX currents in
excised membrane patches from mammalian skeletal muscle
(17). If this Na+ dependence mirrors the
physiological situation, at the resting cytoplasmic [Na+]
of 9 mM (15), the exchanger would operate in the reverse
mode at ~1% of its maximal rate. Furthermore, the inhibition by
cytoplasmic [Ca2+] of the amphibian skeletal NCX (see
below) should further decrease the reverse operation of this
transporter in resting muscle.
Effects of extravesicular [Ca2+] on the reverse NCX reaction. Cytoplasmic [Ca2+] influences the activity of all native NCX transporters examined to date. Increasing cytoplasmic [Ca2+] stimulates the NCX of cardiac muscle, squid axons, and other cells (6, 34) but inhibits the exchanger present in Drosophila (23). Our previous experiments in amphibian T tubules indicated that addition of 20 µM [Ca2+] to the extravesicular solution containing 140 mM Na+ decreased the reverse rate of the exchanger (18). This finding suggested that cytoplasmic Ca2+ had an inhibitory effect on the amphibian NCX, but a detailed characterization was not carried out.
In the present work, we found an IC50 for inhibition of the reverse NCX by [Ca2+] ofFiber Experiments
This is the first description, to our knowledge, of NCX-mediated currents in whole muscle fibers from skeletal muscle. To measure NCX-mediated currents in skeletal muscle cells under voltage conditions that mimic those prevailing in whole muscle cells at rest, a constant membrane potential ofReverse NCX currents. After Na+ withdrawal from the external solution, a reverse NCX current of 0.36 µA/µF, equivalent to 0.36 µA/cm2, was measured in amphibian fibers containing 10 mM internal [Na+]. For comparison, in 90 mM [Na+] and at 0 mV, reverse NCX currents of the order of 5 pA have been measured in isolated inside-out patches from mammalian skeletal muscle (17). The reverse currents measured in similar conditions in patches from mammalian cardiac muscle are about 10-fold higher (20).
To compare measurements, it is necessary first to correct for the differences in the [Na+] used in patch-clamp experiments and our measurements. If the reverse NCX activity in whole fibers behaves toward Na+ as in isolated vesicles (see Fig. 5), the reverse NCX current of 0.36 µA/µF measured in 10 mM [Na+] would correspond to 1% of the maximal current. In 90 mM [Na+], the reverse current would be 80% of maximal, originating a density ofThe reverse NCX reaction produced only a limited increase in cytoplasmic [Ca2+]. In cardiac muscle, removal of external [Na+] produces a significant increase of cytoplasmic [Ca2+] that can reach the micromolar range in only a few seconds (1). In contrast, only a marginal increase in cytoplasmic [Ca2+] was observed in single mammalian fibers after replacing external Na+ (3). Inhibition of the sarcoplasmic reticulum (SR) Ca2+ pump or of mitochondrial Ca2+ uptake did not increase further cytoplasmic [Ca2+], raising the possibility of a very limited reverse NCX operation in skeletal muscle (3).
The present results contribute to clarify this issue, since a significant reverse NCX current was recorded in amphibian skeletal muscle in zero external [Na+], indicating that the NCX was effectively activated, yet [Ca2+] increased only marginally, as observed in mammalian muscle (3), despite the fact that the increase in intracellular [Ca2+] due to the measured reverse NCX current should have been much higher. Because the NCX moves three Na+ per one Ca2+, the reverse current of 0.36 µA/µF represents a coupled Ca2+ influx of 3.6 pmol · cmForward NCX current. To study the forward operation of NCX in skeletal muscle fibers, the intracellular [Ca2+] was suddenly increased in the cytoplasm by flash photolysis of the caged Ca2+ compound DM-nitrophen. This procedure has been successfully used in cardiac muscle to activate the direct mode of NCX, producing a net inward Na+ current density of 0.7 µA/µF when measured at 20°C and 0 mV (33).
The results shown in Fig. 8 demonstrate that flash photolysis of DM-nitrophen is a robust method to force a change in [Ca2+]. Theoretical calculations from a model of the flash photolysis reactions (12) suggest that 30% conversion of DM-nitrophen by a single UV flash would generate the fluorescence transient observed. In that case, the free [Ca2+] is predicted to jump from the resting value of 0.1 µM to ~0.4 µM. At the same time, the predicted jump in free Mg2+ concentration ([Mg2+]) is from ~5 µM at rest to ~13 µM after the flash. Thus the current elicited in response to the UV flash is more likely associated with the change in [Ca2+] than with the very small change in free [Mg2+]. The inward current density of amphibian skeletal muscle measured at 15°C and ![]() |
ACKNOWLEDGEMENTS |
---|
We appreciate the helpful discussions with Drs. Paulina Donoso and
Ariel Escobar during the course of this work. The institutional support
to the Centro de Estudios Científicos by Fuerza Aerea de Chile,
Municipalidad de Las Condes, and a group of Chilean companies
(Administradora de Fondos de Pensiones Provida, Compaía Manufacturera de Papelas y Cartones, Codelco, and Minera Collahuasi) is
also recognized.
![]() |
FOOTNOTES |
---|
This work was supported by Fondo Nacional de Ciencia Technologia Grants 1970914 and 8980009 and by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-25201 to J. Vergara.
Present address for F. Cifuentes: Instituto de Fisiología Celular, UNAM, DF 04510, México.
Address for reprint requests and other correspondence: C. Hidalgo, ICBM, Facultad de Medicina, Univ. de Chile, Casilla 70005, Santiago 7, Chile (E-mail: chidalgo{at}machi.med.uchile.cl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 23 July 1999; accepted in final form 20 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, DG,
Eisner DA,
Lab MJ,
and
Orchard C.
The effects of low sodium solutions on intracellular calcium concentration and tension in ferret ventricular muscle.
J Physiol (Lond)
345:
391-407,
1983[Abstract].
2.
Ashcroft, FM,
Heiny J,
and
Vergara J.
Inward rectification in the transverse tubular system of frog skeletal muscle studied with potentiometric dyes.
J Physiol (Lond)
359:
269-291,
1985[Abstract].
3.
Balnave, CD,
and
Allen DG.
Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers of the mouse.
Am J Physiol Cell Physiol
274:
C940-C946,
1998
4.
Benders, AAGM,
Li J,
Lock RAC,
Bindles RJM,
Bonga SEW,
and
Veerkamp JH.
Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger.
Pflügers Arch
428:
461-467,
1994[ISI][Medline].
5.
Bianchi, CP,
and
Shanes AM.
Calcium influx in skeletal muscle at rest, during activity and during potassium contracture.
J Gen Physiol
42:
803-815,
1959
6.
Blaustein, MP,
and
Lederer WJ.
Sodium/calcium exchange: its physiological implications.
Physiol Rev
79:
783-854,
1999.
7.
Caputo, C,
and
Bolaños P.
Effect of external sodium and calcium on calcium efflux in frog striated muscle.
J Membr Biol
41:
1-14,
1978[ISI][Medline].
8.
Castillo, E,
Gonzalez-Serratos H,
Rasgado-Flores H,
and
Rozycka M.
Na-Ca exchange studies in frog phasic muscle cells.
Ann NY Acad Sci
639:
554-557,
1991[ISI][Medline].
9.
Damiani, E,
Barillari A,
Tobaldin G,
Pierobon S,
and
Margreth A.
Biochemical characteristics of free and junctional sarcoplasmic reticulum and of transverse tubules in human skeletal muscle.
Muscle Nerve
12:
323-331,
1989[ISI][Medline].
10.
Donoso, P,
and
Hidalgo C.
Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle.
Biochim Biophys Acta
978:
8-16,
1989[ISI][Medline].
11.
Escobar, AL,
Cifuentes F,
and
Vergara JL.
Detection of Ca2+-transients elicited by flash photolysis of DM-nitrophen with a fast calcium indicator.
FEBS Lett
364:
335-338,
1995[ISI][Medline].
12.
Escobar, AL,
Velez P,
Kim AM,
Cifuentes F,
Fill M,
and
Vergara JL.
Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.
Pflügers Arch
434:
615-631,
1997[ISI][Medline].
13.
García, MC,
Diaz AF,
Godinez R,
and
Sánchez JA.
Effect of sodium deprivation on contraction and charge movement in frog skeletal muscle fibers.
J Muscle Res Cell Motil
13:
354-365,
1992[ISI][Medline].
14.
Gilbert, JR,
and
Meissner G.
Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles.
J Membr Biol
69:
77-84,
1982[ISI][Medline].
15.
Godt, RE,
and
Maugham DW.
On the composition of the cytosol of relaxed skeletal muscle of the frog.
Am J Physiol Cell Physiol
254:
C591-C604,
1988
16.
Goldstein, DA.
Calculation of the concentration of free cations and cation-ligand complexes in solutions containing multiple divalent cations and ligands.
Biophys J
26:
235-242,
1979[Abstract].
17.
Gonzalez-Serratos, H,
Hilgemann DW,
Rozycka M,
Gauthier A,
and
Rasgado-Flores H.
Na-Ca exchange studies in sarcolemmal skeletal muscle.
Ann NY Acad Sci
779:
556-560,
1996[ISI][Medline].
18.
Hidalgo, C,
Cifuentes F,
and
Donoso P.
Sodium-calcium exchange in transverse tubule vesicles isolated from amphibian skeletal muscle.
Ann NY Acad Sci
639:
483-497,
1991[ISI][Medline].
19.
Hidalgo, C,
Parra C,
Riquelme G,
and
Jaimovich E.
Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation.
Biochim Biophys Acta
855:
279-286,
1986.
20.
Hilgemann, DW.
The cardiac Na-Ca exchanger in giant membrane patches.
Ann NY Acad Sci
779:
136-158,
1996[ISI][Medline].
21.
Hille, B,
and
Campbell DT.
An improved vaseline gap voltage clamp for skeletal muscle fibers.
J Gen Physiol
67:
265-293,
1976[Abstract].
22.
Hoya, A,
and
Venosa RA.
Characteristics of Na+-Ca2 exchange in frog skeletal muscle.
J Physiol (Lond)
486:
615-627,
1995[Abstract].
23.
Hryshko, LV,
Matsuoka S,
Nicoll DA,
Weiss JN,
Schwarz EM,
Benzer S,
and
Philipson KD.
Anomalous regulation of the Drosophila Na+-Ca2+ exchanger by Ca2+.
J Gen Physiol
108:
67-74,
1996[Abstract].
24.
Huerta, M,
Muñiz J,
Vásquez C,
Marín JL,
and
Trujillo X.
Sodium/calcium exchange in tonic skeletal muscle fibers of the frog.
Jpn J Physiol
41:
933-944,
1991[ISI][Medline].
25.
Jaimovich, E,
Donoso P,
Liberona JL,
and
Hidalgo C.
Ion pathways in transverse tubules. Quantification of receptors in membranes isolated from frog and rabbit skeletal muscle.
Biochim Biophys Acta
855:
89-98,
1986[ISI][Medline].
26.
Kawata, H,
and
Fujishiro N.
Effects of sodium depletion on the caffeine-induced contraction of frog's skeletal muscle.
Jpn J Physiol
40:
243-251,
1990[ISI][Medline].
27.
Kim, AM,
and
Vergara JL.
Fast voltage gating of Ca2+ release in skeletal muscle revealed by supercharging pulses.
J Physiol (Lond)
511:
509-518,
1998
28.
Li, Z,
Matsuoka S,
Hryshko LV,
Nicoll DA,
and
Philipson KD.
Cloning of the NCX2 isoform of the plasma membrane Na+-Ca2+ exchanger.
J Biol Chem
269:
17434-17439,
1994
29.
Linck, B,
Qiu Z,
He Z,
Tong Q,
Hilgemann DW,
and
Philipson KD.
Functional comparison on the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3).
Am J Physiol Cell Physiol
274:
C415-C423,
1998
30.
Mickelson, JR,
Beaudry TM,
and
Louis CF.
Regulation of skeletal muscle sarcolemmal ATP-dependent calcium transport by calmodulin and cAMP-dependent protein kinase.
Arch Biochem Biophys
242:
127-136,
1985[ISI][Medline].
31.
Nicoll, DA,
Longoni S,
and
Philipson KD.
Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger.
Science
250:
562-565,
1990[ISI][Medline].
32.
Nicoll, DA,
Quednau BD,
Qui Z,
Xia Y-R,
Lusis AJ,
and
Philipson KD.
Cloning of a third mammalian Na+-Ca2+ exchanger, NCX3.
J Biol Chem
271:
24914-24921,
1996
33.
Niggli, E,
and
Lederer J.
Molecular operations of the sodium-calcium exchanger revealed by conformation currents.
Nature
348:
621-624,
1991[ISI].
34.
Reeves, JP.
Na+/Ca2+ exchange and cellular Ca2+ homeostasis.
J Bioenerg Biomembr
30:
151-160,
1998[ISI][Medline].
35.
Sacchetto, R,
Margreth A,
Pelosi M,
and
Carafoli E.
Colocalization of the dihydropyridine receptor, the plasma membrane calcium ATPase isoform I and the sodium/calcium exchanger to the junctional membrane domain of transverse tubules of rabbit skeletal muscle.
Eur J Biochem
237:
483-488,
1996[Abstract].
36.
Sanchez, JA,
and
Vergara J.
Modulation of action potential and Ca2+ transients by flash photolysis of caged cAMP in single skeletal muscle fibres.
Am J Physiol Cell Physiol
266:
C1291-C1300,
1994
37.
Shuba, YM,
Iwata T,
Naidenov VG,
Oz M,
Sandberg K,
Kraev A,
Carafoli E,
and
Morad M.
A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger.
J Biol Chem
273:
18819-18825,
1998
38.
Vergara, J,
Di Franco M,
Compagnon D,
and
Suarez-Isla B.
Imaging of calcium transients in skeletal muscle fibers.
Biophys J
59:
12-24,
1991[Abstract].
39.
Welsh, DG,
and
Lindinger MI.
L-type Ca2+ channel and Na+/Ca2+exchange inhibitors reduce calcium accumulation in reperfused skeletal muscle.
J Appl Physiol
80:
1263-1269,
1996
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |