ARTICLE
ATP Signaling in Skeletal Muscle: From Fiber
Plasticity to Regulation of Metabolism
Mariana Casas 1, Sonja Buvinic 2, and Enrique Jaimovich 1
1
Center for Molecular Studies of the Cell, Biomedical Sciences Institute, Faculty of Medicine, Universidad de
Chile; and 2Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
CASAS, M., S. BUVINIC, and E. JAIMOVICH. ATP signaling in skeletal muscle: from fiber plasticity to regulation of
metabolism. Exerc. Sport Sci. Rev., Vol. 42, No. 3, pp. 110Y116, 2014. Tetanic electrical stimulation releases adenosine triphosphate
(ATP) from muscle fibers through pannexin-1 channels in a frequency-dependent manner; extracellular ATP activates signals that
ultimately regulate gene expression and is able to increase glucose transport through activation of P2Y receptors, phosphatidylinositol 3-kinase,
Akt, and AS160. We hypothesize that this mechanism is an important link between exercise and the regulation of muscle fiber plasticity and
metabolism. Key Words: gene expression, purinergic, pannexin channels, frequency dependency, glucose uptake, G proteins(
calcium signals
INTRODUCTION
skeletal muscle to regulate a variety of processes, among them
gene expression. ATP release is regulated by a direct interaction between Cav1.1 and Panx1 channels.
During the past several years, our laboratory has searched
for the mechanisms that link changes in membrane potential
to regulation of gene expression in skeletal muscle, a work
performed first in cultured myotubes and later on in isolated
adult muscle fibers. After a series of publications, a mechanism involving a new protein complex in the transverse tubule
(T-tubule) membrane that includes Cav1.1 (dihydropyridine
receptor (DHPR)), pannexin-1 (Panx1), and P2Y purinergic
receptors was proposed in 2009 (10). We evidenced that tetanic electrical stimulation releases adenosine triphosphate
(ATP) from muscle cells through Panx1 channels and that
extracellular ATP activates a signaling cascade that ultimately
regulates gene expression. This mechanism was shown to be
present in adult skeletal muscle fibers isolated from mice (14)
and to depend on the frequency of stimulus. In a recent article
(24), we show evidence of the colocalization of Cav1.1 and
Panx1 in the T-tubules, and that ATP release through Panx1
channels, regulated by Cav1.1, is frequency dependent, occurring only at low frequencies and being the trigger for expression
of slow muscle fiber genes. Our hypothesis based on these results is that extracellularly released ATP is a master signal in
On a Mechanism for Excitation-Transcription Coupling
Studies on voltage-gated ion channels in excitable cells have
focused naturally on their ion permeation and gating properties,
but we focused on a new property for an L-type Ca+2 voltagegated channel as a voltage sensor for a G proteinYregulated
signal involving activation of phosphatidylinositol 3-kinase
(PI3K) and phospholipase C (PLC). At present, little information exists about signaling pathways activated by conformational changes of voltage-gated channels. One example is
the skeletal muscle excitation-contraction coupling (EC coupling) model, where there is a bidirectional cross-talk between
the Cav1.1 and the ryanodine receptor (RyR), independently of the Ca+2 channel function of the former (6). Our
laboratory has shown that membrane depolarization obtained
by either high potassium or tetanic electrical stimulation induces PLC activation followed by an inositol 1,4,5-trisphosphate
(IP3)Ydependent slow Ca+2 signal (19,23). Absence of extracellular Ca+2 does not affect slow Ca+2 signal progression,
whereas treatment with an agonist dihydropyridine (Bay K 8644)
in the absence of external Ca+2 accelerates it. Transfection
with the >1S subunit cDNA of the Cav1.1 (the voltage sensor
and pore-forming subunit of the channel) in myotubes that
lack >1S rescues the slow Ca+2 signal (3,19). These results
suggest that action potentials activated by electrical stimulation may induce conformational transitions of Cav1.1 that,
independently of its Ca+2 channel properties, set in motion
signaling pathways involved in PLC activation.
Address for correspondence: Enrique Jaimovich, Ph.D., ICBM, Facultad de Medicina,
Universidad de Chile, Independencia 1027, Santiago, Chile (E-mail: [email protected]).
Accepted for publication: March 25, 2014.
Associate Editor: Stephen E. Alway, Ph.D., FACSM
0091-6331/4203/110Y116
Exercise and Sport Sciences Reviews
Copyright * 2014 by the American College of Sports Medicine
110
Copyright © 2014 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
We used an external electrical field to generate action potentials in myotubes so it allows us to cycle the Cav1.1 through
its voltage-dependent conformational states. The action potential as stimulus for the slow Ca+2 signals was confirmed by
blocking the voltage-gated sodium channels with tetrodotoxin
(19). The onset of the slow calcium signal, induced by tetanic
stimulation in myotubes (400 1ms pulses at 45 Hz), has a 30- to
40-s delay after the end of stimulation (19). This observation
suggests that a signaling pathway must be activated during this
period, although there is scant evidence in the literature for
G protein activation induced by cell depolarization. In our
system, we have demonstrated that >1S is critical for the
depolarization-induced PLC activation, involving the participation of a pertussis toxin (PTX)Ysensitive G protein (3,18,19).
Some works suggest a modulation of Cav1.1 by G proteinY
coupled receptors (GCPR) in adult muscle fibers (31). The
phenomenon that we described (18) suggests that the Cav1.1
conformational transitions may be activating the G protein. In
particular, we showed evidence involving a GAF from a PTXsensitive G protein in PLC activation.
The common subcellular location (T-tubule) of Cav1.1 and
G protein is in agreement with their possible role in the signaling pathway outlined in this work, where Cav1.1 acts as a
voltage sensor and the G protein as the transductor involved in
effector (PLC) activation (18).
At least two ways how GAF subunit may mediate PLC activation have been described in the literature. Either GAF interacts with the pleckstrin homology (PH) domain of PLCA
acting as an allosteric site for the activation of the catalytic core
of the protein or GAF may activate the PI3KF, which catalyzes
the phosphorylation of phosphatidylinositol-4,5-bisphosphate
(PIP2) in the D3 position of the inositol ring yielding phosphatidylinositol 3,4,5-trisphosphate (PIP3). The latter is a known
second messenger involved in membrane recruitment of PLCF,
where it is activated after tyrosine phosphorylation. Within this
context, we evaluated the participation of PI3K as part of this
transduction mechanism and we measured PIP3 production after
tetanus. Indeed we found an increase in PIP3 40 s after tetanic
electrical stimulation. In addition, viral transfection of carboxyl
terminus of the A-adrenergic receptor kinase (ctAARK) abolished
PIP3 production. To further evaluate whether PI3K is involved
in PLC activation induced by electrical stimulation, we exposed myotubes to PI3K inhibitors. Both LY294002 and
wortmannin completely blocked both the slow Ca+2 signals
and the transient increase in IP3 mass induced by the electrical stimulation. These results, together with the fact that
PI3KF is known to be activated by the GAF subunit, suggested
that PI3KF was a good candidate for PIP3 production.
Although the presence of PI3KF mRNA in skeletal muscle
was reported more than 10 years ago, its physiological role has
not been elucidated. We confirmed its presence in myotubes
by Western blot using two different antibodies, and immunofluorescence localization indicates a distribution of PI3KF consistent with location near the T-tubule (18).
Signaling by Extracellular Nucleotides
ATP for a long time was considered as an intracellular molecule that was involved only with energy and metabolism of
many cells. Nevertheless, in the last decades, ATP has been
considered as an extracellular messenger for autocrine and
Volume 42 & Number 3 & July 2014
paracrine signaling (1). Several mechanisms for ATP release
have been proposed: 1. Exocytosis, 2. ABC transporters, 3.
Release through stretch- or voltage-activated channels, and
4. Release through connexin or pannexin hemichannels (25).
There are two families of receptors for extracellular nucleotides: P2X and P2Y. P2X receptors are ion channels activated by ATP, which induce fast and nonselective inward
currents of Na+ and Ca2+. P2Y receptors are GPCR activated
by ATP, adenosine diphosphate (ADP), uridine triphospahte
(UTP), or uridine diphospahte (UDP) (1). To date, seven
mammalian P2X subtypes (P2X1-7) and eight mammalian P2Y
subtypes (P2Y1,2,4,6,11,12,13,14) have been cloned and characterized pharmacologically.
In physiological conditions, the skeletal muscle simultaneously is subject of many stimuli, including membrane depolarization as well as hormones, metabolites, and extracellular
molecules that can activate G proteinYcoupled receptors and
control cellular processes. During the past years, we have demonstrated that extracellular ATP is a relevant mediator between
membrane depolarization and Ca+2 transients in skeletal muscle (10). In several tissues, it has been described that extracellular ATP is the main autocrine-paracrine mediator for
cell signaling evoked by hormones, growth factors, neurotransmitters, mechanical stimuli, and inflammation (25), so
we propose that ATP could be the central integrator of all the
stimuli received for skeletal muscle cells to allow and coordinate events for muscle gene expression.
Extracellular Nucleotides as Good Candidates for
Regulation of Skeletal Muscle Activity and
Gene Expression
ATP is released from skeletal muscle cells in response to
muscle contraction. Adenine nucleotides are released from both
the nerve terminals and the muscle fibers on electrical stimulation of innervated skeletal muscles. Electrical stimulation evokes
ATP release from rat primary myotubes (10,27) and mouse skeletal fibers (24,28) through a Panx1 channel. Ectonucleotidases
activities have been described in skeletal muscle to metabolize
extracellular nucleotides. In chicken skeletal muscle coexist
ecto-ATPase, ecto-ADPase, and 5¶-nucleotidase; an association
of these proteins forming functional complexes in the T-tubule
membranes has been suggested.
A relevant role for extracellular ATP in glucose uptake has
been described in rat primary myotubes (27), and electrical
stimulation of rat myotubes evokes ATP release (10,27).
Functional nucleotide receptors (P2X4, P2X5, P2X7, P2Y1,
and P2Y4) have been described in mice myotubes, which evoke
Ca+2 transients when are stimulated with exogenous nucleotides. In differentiated cells derived from human skeletal muscle,
activation of P2Y1 receptors by exogenous agonists promotes
Ca+2 transients dependent on the IP3 pathway but independent of RyRs, which leads to extracellular signalYregulated
kinase 1/2 (ERK1/2) activation (26).
We have demonstrated expression of P2Y1, P2Y2, P2Y13,
and P2Y14 receptors in skeletal fibers from mouse flexor digitorum
brevis (FDB) muscle. Voluntary physical activity of mice during
5 weeks modifies the expression pattern of these receptors
(P2Y2 is downregulated and P2Y14 is overexpressed) and increases fiber sensitivity to ATP for IL-6 expression induction
(20). Purinergic signaling also has been involved with skeletal
ATP Signaling in Skeletal Muscle
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111
pathophysiological conditions. P2X7R mRNA and protein is
upregulated in a model of Duchenne muscular dystrophy (DMD),
the mdx mouse. P2X7 activation has an increased response in
cytoplasmic Ca2+ rise and ERK phosphorylation in mdx.
Considering all this background, extracellular ATP and its
metabolites could be proposed as muscle plasticity regulators and
open very interesting questions about the interaction of nucleotide receptors with other proteins, such as Cav1.1, relevant
for the excitation-contraction and excitation-transcription events.
ATP Released Through Panx1 Channel as a New
Player in E-T Coupling Process
We have evidenced that ATP is released by physiological
activity of skeletal muscle cells, and it is metabolized extracellularly; that P2Y/P2X functional receptors are expressed in
this system and that Ca+2 transients can be evoked on nucleotide exposure. Moreover, calcium transients evoked by tetanic electrical activity can be inhibited by interfering with
extracellular nucleotide pathways. We have postulated (10)
the hypothesis that extracellular nucleotides can be crucial
mediators between electrical stimulation and both the fast
and slow Ca+2 transients respectively involved in muscle activity and plasticity.
ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In
skeletal primary myotubes, depolarizing stimuli induce both a
fast calcium signal associated with contraction and a slow signal
that regulates gene expression. We have shown that nucleotides, extracellularly released after electrical stimulation, are
involved partly in the fast component and largely are responsible for the slow signals. In rat skeletal myotubes, a tetanic
stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and adenosine monophosphate
(AMP) after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50
value of 7.8 T 3.1 HM. Exogenous ADP, UTP, and UDP also
promoted calcium transients. Both fast and slow Ca+2 signals
evoked by tetanic stimulation were inhibited by either 10 to
100 HM suramin or 2 U mL-1 apyrase. Apyrase also reduced fast
and slow Ca+2 signals evoked by tetanus (45 Hz, 400 0.3-ms
pulses) in isolated mouse adult skeletal fibers. A likely candidate for ATP release pathway was the Panx1 channel, whose
blocker inhibited both Ca+2 transients and ATP release. In
protein extracts from myotubes, Cav1.1 coimmunoprecipitated
both with P2Y2 receptor and Panx1.
Moreover, ATP by itself, independently of electrical stimulus, is able to activate some genes previously shown to depend on IP3-dependent calcium signals. These results suggest
that nucleotides released during skeletal muscle activity through
Panx1 channels act through P2X and P2Y receptors to modulate the process of E-T coupling (10). In adult muscle fibers,
we also have demonstrated that ATP is released through Panx1
in a frequency-dependent manner (24).
Slow Ca+2 Signals in Adult Muscle Fibers
Although we have characterized slow Ca+2 signals in cultured muscle cells extensively, these signals have proven difficult to see in adult skeletal muscle fibers.
We searched for slow Ca+2 signals in adult muscle fibers
using isolated adult FDB fibers from 5- to 7-wk-old mice,
112 Exercise and Sport Sciences Reviews
loaded with fluo-3AM (12). When stimulated with trains of
0.3-ms pulses at various frequencies, cells responded with a
fast calcium signal associated with muscle contraction, followed
by a slower signal similar to one previously described in cultured
myotubes. Nifedipine inhibited the slow signal more effectively
than the fast one, suggesting a role for the Cav1.1 in its onset.
The IP3 receptor (IP3R) inhibitors Xestospongin-B or C (5 KM)
also inhibited it. The amplitude of posttetanic Ca+2 transients
depend on both tetanus frequency and duration, having a
maximum at 10 to 20 Hz. At this stimulation frequency, an
increase of the slow isoform of troponin I mRNA was detected,
whereas the fast isoform of this gene was inhibited. All three
IP3R isoforms were present in adult muscle. IP3R-1 was expressed differentially in different types of muscle fibers, being
higher in a subset of fast-type fibers. These results support the
idea that IP3R-dependent slow Ca+2 signals may participate in
the activation of specific transcriptional programs of slow and
fast muscle fiber phenotype (24).
ATP Release and IP3 Production Are Regulated by the
Frequency of Electrical Stimulation
As we have seen previously that amplitudes of the posttetanic
Ca2+ transients were dependent on the frequency of stimulation, being lower at high frequencies (90 Hz) and higher at
low frequencies (20 Hz), we looked for the frequency dependence of molecular events upstream this IP3-dependent Ca2+
signals: ATP release and IP3 production. For that, FDB muscle
fibers were electrically stimulated at 20 or 90 Hz, and we
measured ATP release and IP3 production at different times
after the end of the stimulus. We found that ATP release takes
place in fibers stimulated at 20 Hz but not at 90 Hz. IP3 production also showed the same frequency dependence: an increase in intracellular IP3 levels was observed after 20-Hz
stimulation, but no changes were observed after 90-Hz stimulation. We also show that ATP release plays a key role in the
transcription of the TnI genes induced by 20-Hz stimulation
because fibers treated with apyrase to hydrolyze ATP, or
carbenoxolone (CBX) to inhibit ATP release by inhibition of
Panx1 channels, did not exhibit the changes in mRNA levels
produced at this stimulation frequency. Moreover, extracellular
addition of 30 HM ATP induced the same transcriptional
changes in TnI genes as electrical stimulation at 20 Hz. The
fact that external ATP increased IP3 levels suggests that these
transcriptional events occur in response to the same signaling
pathways elicited by 20-Hz electrical stimulation or by external ATP addition (24).
These data constitute the first evidence of molecular events
controlled by frequency of stimulation that participate in activation of plasticity-related transcriptional changes in adult
skeletal muscle.
Cav1.1 as a Sensor for Different
Voltage-Dependent Processes
As previously exposed, Cav1.1 acts as a voltage sensor for
excitation-transcription (E-T) coupling as well as for E-C
coupling, and in addition, it is a voltage-gated Ca2+ channel
(driving an L-type current that is not needed to activate E-C
coupling). The dependence on membrane depolarization relies
on conformational changes induced in Cav1.1 on depolarization, producing the activation of all these different functions.
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At the structural level, it is a hetero-oligomeric complex
protein composed of four subunits: >1S, >2-C, A1a, and F1
subunits (13). All subunits have intramembrane domains, except for the A1a subunit that is entirely cytosolic. The most
studied subunit corresponds to the >1s, which is responsible
for the formation of the conduction pore, the voltage sensing
and the gating apparatus (13). This subunit is organized in four
homologous domains (IYIV), with six transmembrane segments
(S1YS6) in each. S4 segments have four to five charged amino
acid residues conferring the voltage-sensing property to Cav1.1.
Because of the high homology between the Cav1.1 and K+
channels S4 segments, it is accepted widely that the movement
of this charged residues is responsible for the intramembrane
charge movements induced by plasma membrane depolarization. The >1s is the subunit present in the Cav1.1. Related
isoforms as those in Cav1.2 (cardiac) and Cav1.3 (neuronal)
are present in tissues other than skeletal muscle.
As told, the roles of Cav1.1 as voltage sensor in E-C coupling
and E-T signaling, both activated by membrane depolarization,
are independent of the function of Cav1.1 as a Ca2+ current
carrier. Although a possible role for Ca+2 currents in E-C
coupling has not been ruled out completely (17) and the molecular mechanisms behind these different functions still are
not established completely, several mutations in Cav1.1 differentially affect Ca2+ currents and E-C coupling, suggesting
that different parts of the Cav1.1 molecule would be responsible for each function (6). This also could be the case for the
activation of the signaling cascade related to E-T coupling
and dependent on ATP release.
Panx1 Channels Function and Regulation
In the last years, ATP has been considered increasingly as
an extracellular messenger for autocrine and paracrine signaling (15). Several studies have demonstrated recently that
ATP can be released by Panx channels in a variety of cell
types that include myotubes. The widespread distribution of
Panx1 has been confirmed in several tissues, with the highest
levels in skeletal muscle (5).
Panx1 is an integral membrane glycoprotein that belongs
to a family of three genes in vertebrates (Panx1, Panx2, and
Panx3). Pannexins have been proposed to be the homologs of
the invertebrate gap junction forming proteins, innexins, and
they also present similar topology but little homology to a
family of connexins (7). Connexins and pannexins are predicted to present similar protein structures consisting of four
>-helical transmembrane domains, two extracellular loops,
and one intracellular loop, with their amino and carboxyl
termini exposed to the cytoplasm. Six subunits are required
to form a functional channel that presents a large conductance
(550 pS) (4) that allows the passage of relatively large molecules
such as ATP, arachidonic acid derivatives, and fluorescent dyes.
Even if the opening of Panx1 is essential to assure a number
of different functions, a deregulation in its activity leading to
an uncontrolled opening may cause cell death (29). So, a robust mechanism controlling the opening and closure of Panx1
is of vital importance to the cells.
Little information exists on the mechanism controlling the
closure of the Panx1 channel. It has been shown that ATP
released by the Panx1 itself may inhibit the channel activity
as a way to prevent further release. Evidence also exists showing
Volume 42 & Number 3 & July 2014
that the interaction between Panx1 and the K+ channel auxiliary subunit KvA3 attenuated its sensitivity to changes in redox potentials (9). Recently, Zhan et al. (34) showed that the
protein stomatin physically interacts with Panx1 in astrocytes
and could play an important role to maintain the closed state
of Panx1 channels under physiological conditions.
It seems then that interaction of Panx1 with functional
proteins is a way to regulate its activity in a context- and
function-dependent manner. This could represent an advantage for a widespread expressing protein as Panx1 that plays
such multiple physiological roles.
Cav1.1 Controls the First Step of Frequency
Decoding
In our recent work (24), we have shown that Cav1.1 participates in the mechanism regulating ATP release after electrical stimulation of muscle cells. Indeed, ATP release practically
is absent in myotubes lacking the >1s subunit of this channel
(impairing the assembly of the whole channel at the plasma
membrane), whereas in adult muscle fibers, the Cav1.1 blocker
nifedipine completely abolished ATP release and the transcriptional changes produced by 20-Hz stimulation. Interestingly, (-)S-BayK 8644 also inhibited these two processes but
having the opposite effect on Ca2+ current through the Cav1.1
channel than nifedipine. We have shown previously that IP3dependent Ca2+ signals are independent from extracellular
Ca2+ entry (12). The fact that these two dihydropyridines,
nifedipine and (-)S-BayK 8644, have the same inhibitory effects on ATP release and gene expression indicates that the
Ca2+ current trough Cav1.1 is not relevant to activation of the
E-T coupling process, as is the case for E-C coupling. More
importantly, it suggests the existence of function of Cav1.1
independent of the Ca2+ current and related to activation of
E-T coupling that is blocked by these two drugs (24).
The mechanisms of frequency decoding by Cav1.1 could
be important for other excitable cells as well. In neurons, it
has been shown that intracellular Ca2+ increases depending
on Cav1.3 (the homologous of Cav1.1 in neurons) occur with
stimulations at 10 Hz but not at 100 Hz, and this Ca2+ increase
has an important role in cAMP response element-binding
(CREB) activation-dependent gene expression (32).
The Panx1YCav1.1 Interaction in E-T Coupling
To have a deeper understanding of the mechanism by
which Cav1.1 could control ATP release via Panx1 channels,
we looked for a possible protein-protein interaction between
these two molecules. We found that Cav1.1 colocalizes with
Panx1 channels in the T-tubule of adult muscle fibers by in
situ proximity ligation assay (PLA) and by coimmunoprecipitation approaches (24). Interestingly, after stimulation
of fibers at 90 Hz, the intensity of PLA labeling in fibers appears weaker than after stimulation at 20 Hz, suggesting that
their interaction can be in some way being altered by frequency of electrical stimulation (24).
Interestingly, in a mouse model of DMD, the mdx mice,
increased levels of ATP release are also observed and are related to activation of proapoptotic pathways (30). In this work,
we observed an ‘‘uncoupling’’ between electrical stimulation
and ATP release that could be explained by a loss of control
of Panx1 channels by Cav1.1, supported by the data showing
ATP Signaling in Skeletal Muscle
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113
Figure. Schematic description of the adenosine triphosphate (ATP)Ydependent mechanism of excitation-transcription coupling in skeletal muscle. ATP
is released through pannexin-1 (PnX1) channels after low-frequency stimulation; the voltage sensor being Cav1.1 calcium channels or dihydropyridine
receptors (DHPR). ATP is fast degraded to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine by action of ectonucleotidases.
Both ATP and ADP can act on P2Y, G proteinYcoupled receptors that in turn activate PI3 kinase in the T-tubule membrane, which catalyzes phosphorylation
of phosphatidylinositol biphosphate (PIP2), yielding PIP3, a highly charged residue that recruits phospholipase C (PLC) to the membrane, triggering PIP2
hydrolysis and yielding both diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3) as products. IP3 will bind to IP3 receptors present both in the
nuclear envelope and in the sarcoplasmic reticulum, eliciting a faint calcium release signal both cytosolic and nucleoplasmic, which contributes (possibly
with other signaling cascades) to activation of transcription factors (TF) that leads to expression or repression of genes involved in muscle cell phenotype.
As examples, the slow and fast isoforms of troponin I (TnIs and TnIf) were drawn. At the left-hand side of the figure, the excitation-contraction coupling
machinery that also depends on DHPR acting on ryanodine receptors (RyR) is depicted.
a reduction in the interaction between Cav1.1 and Panx1
observed in mdx fibers. This could be caused by destabilization
of the plasma membrane by the absence of dystrophin or by
the change in the relative amounts of Cav1.1 and Panx1.
It has been suggested that Cav1.1 can interact with dystrophin (21), and Cav1.1 function is altered in mdx muscle
fibers (11,14,33). So it seems possible that, in the absence of
dystrophin, the function of Cav1.1 could be altered, in particular, its function as activator of Panx1 channels after lowfrequency electrical stimulation to induce ATP release but
also, and importantly, as a basal negative regulator to maintain low levels of basal ATP release in resting conditions.
These results suggest that Cav1.1-Panx1 interaction is essential for activation of gene transcription in muscle cells, in
particular, for processes related to muscle plasticity.
Regarding the mechanism by which Cav1.1 could control
ATP release via Panx1 channels, we propose that a direct
physical interaction between Cav1.1 and Panx1 channels exists, as discussed (24). During electrical stimulation, conformational changes in Cav1.1 could activate the opening
of Panx1 to allow ATP release. Panx1 has been proposed to
114 Exercise and Sport Sciences Reviews
interact with other membrane proteins and in particular with a
beta subunit of a voltage-dependent potassium channel. Importantly, this interaction can alter Panx1 function (8).
From the Cav1.1 side, it has been shown that associated
proteins also can alter their function. The most studied case is
the retrograde control of Cav1.1 by RyR1 (2). Nevertheless,
other proteins interacting with Cav1.1 also can alter its function
as is the case among others of caveolin-3 (16), Junctophilins 1
and 2 (22), and G-protein A1F2 dimer (31). As already mentioned in the previous section, dystrophin also has been proposed to interact and modulate Cav1.1 function.
All these data suggest that we are in the presence of a new
functional protein-protein interaction able to regulate the
functions of Cav1.1 and Panx1, and that this regulation is related to E-T coupling (see illustration in the Figure).
A Role for Extracellular ATP in Glucose Uptake
In a recent work, we found that, in cultured skeletal muscle
cells, electrical stimulation, ATP, and insulin each increased
fluorescent 2-NBD-glucose (2-NBDG) uptake. Importantly,
insulin action was not inhibited by adenosine phosphate
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phosphatase or by purinergic receptor blocker suramin. As
told, electrical stimulation transiently elevates extracellular
ATP and, in this work, we showed that it caused Akt phosphorylation that was inhibited by suramin. Moreover, exogenous ATP transiently activated Akt whereas inhibition
of either PI3K or Akt or the use of a dominant-negative Akt
mutant reduced ATP-dependent 2-NBDG uptake and Akt
phosphorylation. ATP-dependent 2-NBDG uptake was
inhibited by G-protein AF subunitYinteracting peptide
ctAARK and by PI3KF inhibitor AS605240. We found an
ATP-caused translocation of the recombinant glucose
transporter GLUT4myc-eGFP to the cell surface, mediated
by increased exocytosis involving AS160/Rab8. Finally, we
found that ATP stimulated 2-NBDG uptake in both normal
and insulin-resistant adult muscle fibers, mimicking the
reported effect of exercise (27).
CONCLUSIONS
ATP-mediated signals seem to be an important part of the
mechanisms leading to skeletal muscle growth and regeneration as well to the exquisite interplay that takes place between
nerve and muscle cells during this process. In particular, these
mechanisms could be a fundamental step in the process of
muscle adaptation to exercise. How calcium signals generated
this way are decoded finally by the muscle cell and how are
they distinguished from the large background Ca+2 oscillations
required for normal muscle contractions is part of the exciting
developments that we surely will witness during the next few
years. Detailed knowledge of the mechanisms for excitationtranscription coupling in muscle cells, the localized nuclear
Ca+2 signals, and their role in transcription control and the
Ca+2-mediated signaling processes that regulate events as important as the stability of the neuromuscular junction and the
activation of muscle satellite cells certainly will help design
therapies for a vast variety of conditions leading to muscle atrophy and wasting.
Acknowledgments
This work was financed by CONICYT ACT 1111 (E. Jaimovich, M. Casas,
S. Buvinic), FONDECYT no. 1110467 (E. Jaimovich, S. Buvinic, M. Casas),
11100454 (S. Buvinic), and U-INICIA VID 2011, U-INICIA 02/12M; University of Chile (M. Casas). All three authors M. Casas, S. Buvinic, and
E. Jaimovich declare to have no conflict of interest.
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