Cell, Vol.

64, 671-674, March 6, 1991, Copyright 0 1991 by Cell Press

Excitation-Contraction Coupling Minireview

in Vertebrate Skeletal Muscle:
A Tale of Two Calcium Channels
William A. Catterall
Department of Pharmacology
Unliversity of Washington
Seattle, Washington 98195
In all muscle cells, the cytosolic free calcium concentration
exerts primary control over the initiation, time course, and
force of contraction. The process of coupling chemical
and electrical signals at the cell surface to the intracellular
release of calcium and ultimate contraction of muscle fi-
bers is termed excitation-contraction coupling. Coupling
of cell surface signals to intracellular calcium release pro-
ceeds by different mechanisms in smooth, cardiac, and
skeletal muscle cells. However, in each of them, both volt-
age-gated calcium channels in the cell surface mem-
branes and intracellular calcium-release channels in the
sarcoplasmic reticulum play key roles.
In skeletal muscle, action potentials mediated by volt-
age-gated sodium and potassium channels in the plasma
membrane are conducted down the transverse tubules
toward the center of the muscle fiber (Figure 1). This depo-
larization of the transverse tubule membrane results in
ad:ivation of voltage-gated calcium channels in the trans-
verse tubules and influx of calcium into the muscle fiber.
However, unlike cardiac muscle and most smooth muscle,
in ,which calcium influx can initiate contraction, calcium
purified using their high-affinity binding of dihydropyridine
calcium channel antagonists, as a specific label. These
channels are a complex of five protein subunits (Figure 2;
reviewed in Catterall, 1988).
The al subunit is the central functional component of
the complex. Its mRNA encodes a polypeptide of 212 kd
having four homologous domains with six proposed trans-
membrane segments in each, like other members of the
voltage-gated ion-channel gene family (Figure 3; Tanabe
et al., 1987). The al subunit contains the receptor sites
for calcium channel antagonists and can function as a
voltage-gated ion channel when expressed in mammalian
cells (Perez-Reyes et al., 1989). Two forms of the al sub-
unit, having apparent molecular masses of 212 kd and 175
kd, have been identified (De Jongh et al., 1989). The 175
kd form accounts for more than 90% of al subunits in
purified preparations and transverse tubule membranes;
both forms are present in skeletal muscle cells in cell cul-
ture (Lai et al., 1990).
al subunits are present in a complex with an intracellu-
larly disposed p subunit of 55 kd and a glycosylated trans-
membrane y subunit of 30 kd that are encoded by separate
Plasma
TT , Membrane
\

dk5
entering skeletal muscle cells from the exterior through
voltage-gated calcium channels is not required for initiating
excitation-contraction coupling. Instead, the depolarization ‘, * tc Isr
SR
of the transverse tubule membrane initiates the release of
calcium by the calcium-release channels in the sarcoplas-
mic reticulum via an unknown mechanism. This process
is accompanied by a movement of membrane-bound elec-
Ca++ Ca+:
trical charge, termed gating charge, from the cytosolic sur-
face to the extracellular surface of the transverse tubule Figure 1. Calcium Release and Re-uptake at the Transverse Tubule-
Sarcoplasmic Reficulum Junction
membrane. This gating current is thought to represent
TT, transverse tubule; SR, sarcoplasmic reticulum; t, terminal cister-
the movement of voltage sensors in the transverse tubule nae of sarcoplasmic reticulum; IL,, longitudinal sarcoplasmic reticulum;
membrane that initiate excitation-contraction coupling F, feet.
(Schneider and Chandler, 1973).
This brief review focuses on recent experiments that
have shown that voltage-gated calcium channels are re-
sponsible for the movement of the gating charge in the
transverse tubule membrane. These experiments have led
to the proposal that protein-protein interactions between
the voltage-gated calcium channels in the transverse tu-
bule membrane and the calcium-release channels in the lTlTN
sarcoplasmic reticulum membrane are responsible for ex-
citation-contraction coupling in skeletal muscle. llllulu
Transverse Tubule Voltage-Gated
Calcium Channels
The principal voltage-gated calcium channels in muscle
cells are L-type and mediate long-lasting calcium cur-
rents. They are inhibited by a variety of pharmacological
agents used in the treatment of cardiovascular disorders, Figure 2. Subunit Structure of Transverse Tubule Voltage Sensitive
including the dihydropyridines and the phenylalkylamines. Calcium Channel
The transverse tubule L-type calcium channels have been Redrawn from Catterall (1968).
Cell
872
P
Extracellular
'6 i6 l7
Cytoplasmic
i
\ nr
Figure 3. al Subunit of Transverse Tubule Voltage-Sensitive
Channel
Redrawn from Catterall (1988) and Tanabe et al. (1987).
genes (Ruth et al., 1989; Jay et al., 1990). The al, 6, and
y subunits also interact with a disulfide-linked glycoprotein
complex of a2 and S subunits. These two subunits are
encoded by the same gene (Ellis et al., 1988) whose pro-
tein product is proteolytically processed to yield the
disulfide-linked a2 and 6 polypeptides (De Jongh et al.,
1990).
Although the entry of extracellular calcium into muscle
fibers through voltage-gated calcium channels is not re-
quired for excitation-contraction coupling, both contrac-
tion of skeletal muscle and transmembrane movement of
gating charge for excitation-contraction coupling are re-
duced by dihydropyridines and phenylalkylamines (Rios
and Brum, 1987). These results led to the hypothesis that
the transverse tubule calcium channel serves a dual role
as a voltage-gated ion channel mediating slow calcium
currents and as a voltage sensor in excitation-contraction
coupling. Direct evidence for this dual functional role has
come from an elegant series of experiments on cultured
muscle cells from mice with the mosculardysgenesis mu-
tation. These cells are unable to contract and have slow
calcium currents that are dramatically reduced. Microin-
jection of a mammalian expression vector encoding the
al subunit into multiple muscle cell nuclei results in recov-
ery of slow calcium currents, gating-charge movement,
and excitation-contraction coupling (Tanabe et al., 1988;
Adams et al., 1990). These results show that the a7 gene
is necessary for both slow calcium currents and voltage
sensing in excitation-contraction coupling.
Sarcoplasmic Reticulum Calcium-Release Channel
In excitation-contraction coupling, release of calcium from
the junctional cisternae of the sarcoplasmic reticulum is
mediated by a calcium channel whose ion conductance
activity is regulated by ATP and the drugs ryanodine and
caffeine, but not by membrane voltage. Purification of this
protein, using high-affinity ryanodine binding as a specific
assay, yields a complex with an apparent molecular mass
of over 2000 kd consisting of a tetramer of identical 565
kd subunits (Lai et al., 1988; lmagawa et al., 1987). The
primary structure of these subunits reveals four proposed
transmembrane segments with a large amino-terminal cy-
toplasmic domain containing a consensus site for ATP
Foot region
Membrane
CO-Z
L.
Calcium
Figure 4. Sarcoplasmic Reticulum Calcium Release Channel
Redrawn from Takeshima et al. (1989).
binding (Takeshima et al., 1989; Figure 4). This huge tetra-
merit protein has a rosette structure suggesting four trans-
membrane pores and a cytoplasmic lid (Wagenknecht et
al., 1989).
The sarcoplasmic reticulum calcium-release channel is
not strongly voltage gated and therefore cannot be acti-
vated directly by membrane potential changes in the trans-
verse tubule or sarcoplasmic reticulum membranes. The
arrival of the action potential at the transverse tubule-
sarcoplasmic reticulum junction must generate a chemical
or physical signal that activates the sarcoplasmic reticu-
lum calcium-release channel. Many low molecular weight
compounds, including calcium, inositol phosphates, and
adenine nucleotides, have been considered as second
messengers in this process, but none has been found to
have the correct physiological effects and kinetics of ac-
tion. Recent studies have focused on an alternative mech-
anism in which protein-protein interactions form the func-
tional link between the transverse tubule voltage-sensitive
calcium channel and the sarcoplasmic reticulum calcium-
release channel.
Allosteric Coupling of Excitation and
Calcium Release
The initiation of calcium release by the arrival of the action
potential at the transverse tubule-sarcoplasmic reticulum
junction is accompanied by an outward movement of posi-
tive gating charge across the transverse tubule membrane
(Schneider and Chandler, 1973). This gating-charge
movement is the electrical signature of voltage-driven con-
formational changes in the transverse tubule calcium
channel and, as for other voltage-gated ion channels (re-
viewed in Catterall, 1988), likely results from the trans-
membrane movement of positively charged amino acid
residues in the S4 transmembrane segments; these resi-
dues are thought to serve as voltage sensors in these
molecules. The earliest measurements of these gating-
charge movements led to the hypothesis that this trans-
membrane conformational change might be directly linked
to activation of calcium release through protein-protein
interactions (Schneider and Chandler, 1973). Experimen-
tal support for this idea is steadily accumulating.
Both transverse tubule calcium channels and sarcoplas-
Minireview
073
mic reticulum calcium-release channels are concentrated
in the junctions between these two membrane systems
(Block et al., 1988; Flucher et al., 1990). Freeze-fracture
or negatively stained images of the junctions show regular
checkerboard arrays of junctional “feet” that protrude into
the cytosol from the sarcoplasmic reticulum membrane
(Block et al., 1988). These “feet” structures have been identi-
fied as the sarcoplasmic reticulum calcium-release chan-
nel (Wagenknecht et al., 1989), and it is assumed that they
represent the cytoplasmic domain of the sarcoplasmic re-
ticulum channel (Figure 4). lntramembrane particles are
observed in a similar checkerboard array of square clus-
ters in the transverse tubule membrane (Block et al., 1988).
Considered together, the biochemical and morphological
results argue that transverse tubule calcium channels and
sa.rcoplasmic reticulum calcium-release channels are lo-
ca.ted primarily in the transverse tubule-sarcoplasmic re-
ticulum junction and may be organized in complementary
arrays in the two membrane systems. Protein-protein in-
teractions between the two channels may therefore serve
to transmit the signal across the junction.
In contrast to this mechanism of excitation-contraction
coupling in skeletal muscle, in cardiac muscle the entry of
extracellular calcium through voltage-gated calcium chan-
nels is required to induce calcium release. A similar re-
quirement for extracellular calcium is observed when the
al subunit of the cardiac calcium channel is expressed
in skeletal muscle myocytes from muscular dysgenesis
mice to restore excitation-contraction coupling (Tanabe
et al., 1990). Evidently, the mode of excitation-contraction
coupling is determined by the al subunit. To determine
which intracellular domain of the al subunit is responsible
for specifying the direct, calcium-independent, skeletal
muscle mode of excitation-contraction coupling, Tanabe
et al. (1990) produced a series of chimeric al subunits
containing each of the major intracellular domains from
the skeletal muscle subunit inserted into the cardiac al
subunit. The large intracellular loop connecting homolo-
gous domains II and Ill (Figure 3) was found to be sufficient
to induce direct excitation-contraction coupling in skeletal
muscle. This segment of the al subunit seems likely to be
responsible for interaction with the sarcoplasmic reticulum
calcium-release channel. It may interact directly with the
sarcoplasmic reticulum channel or may be coupled
through an additional 95 kd protein that has been recently
described (Kim et al., 1990).
Regulation of the Dual Functions of the Transverse
Tubule Calcium Channel
A single gene encodes al subunits serving two distinct
functions: voltage-gated slow calcium conductance and
voltage sensing in excitation-contraction coupling. Com-
parison of numbers of dihydropyridine binding sites with
calcium currents or fluxes suggests that no more than
5% of skeletal muscle calcium channels in muscle fibers
or in purified preparations are active in ion conductance
(Schwartz et al., 1985; Curtis and Catterall, 1988). Two
different mechanisms may contribute to regulation of the
ion conductance activity of these channels.
Analysis of calcium channel al subunits with anti-
peptide antibodies reveals two different size forms in puri-
fied preparations and in intact membranes and cells: a
minor 212 kd form containing the full sequence encoded
by the mRNA, and a major 175 kd form missing up to 300
amino acid residues from the carboxyl terminus (De Jongh
et al., 1989). Since only a single mRNA encoding al 212has
been characterized to date (Tanabe et al., 1987; Ellis et
al., 1988; Perez-Reyes et al., 1989), the two size forms of
the al subunit may arise from posttranslational proteolytic
processing. Because less than 5% of skeletal muscle cal-
cium channels are active in ion conductance and less than
5% of purified calcium channels have full-length al sub-
units, it has been proposed that a1212 may be specialized
for calcium conductance while al 175may serve as the volt-
age sensor for excitation-contraction coupling (De Jongh
et al., 1989). Thus, two protein forms encoded by the same
a 7 gene may be specialized for these two separate physio-
logical functions.
L-type calcium channels are also regulated by CAMP-
dependent protein phosphorylation. L-type calcium cur-
rents in skeletal muscle cells are increased by agents that
increase intracellular CAMP concomitant with a slow in-
crease in contractile force (Arreola et al., 1987). This regu-
lation of slow calcium conductance may be important in
replenishment of intracellular calcium during periods of
sustained muscle activity. Phosphorylation of purified cal-
cium channels increases the number that are active in ion
conductance (Flockerzi et al., 1986; Nunoki et al., 1989;
Hymel et al., 1988). Thus, phosphorylationldephosphory-
lation can also regulate the ability of al subunits to serve
as functional ion channels.
These two regulatory mechanisms may also interact. All
six of the conserved consensus sites for CAMP-dependent
protein phosphorylation on the al subunit are located in
the carboxy-terminal tail (Figure 3). At least three of these
are not present in a1175 (De Jongh et al., 1989). Thus, a
change in the relative amount of alp,2 vs. alIT also has
the potential to profoundly modify the regulation of the al
subunit by protein phosphorylation. This interactive regu-
latory mechanism may have an important role in modulat-
ing skeletal muscle contraction and perhaps other aspects
of calcium channel function.
Conclusion
These recent results shed new light on two major para-
doxes concerning calcium channels and excitation-con-
traction coupling in skeletal muscle: Why is there no re-
quirement for extracellular calcium for skeletal muscle
contraction, and why are there so many transverse tubule
calcium channel molecules when extracellular calcium is
not necessary for excitation-contraction coupling and
transverse tubule calcium currents are small and slow?
Apparently, rapid contraction of vertebrateskeletal muscle
is better served by a direct coupling mechanism that does
not involve a soluble second messenger like calcium but
uses two molecular components that are similar to those
of other muscle tissues: the transverse tubule and sarco-
plasmic reticulum calcium channels. The intrinsic voltage
sensitivity of the transverse tubule calcium channel under-
lies its role as avoltage sensor; the activation of the sarco-
plasmic reticulum calcium channel proceeds through pro-
tein-protein interactions rather than through binding of
 Cell
874

calcium or other small molecules. The assembly and func

tion of this system may be controlled by production of tw
forms of the transverse tubule calcium channel and b
protein phosphorylation in an interactive way. The moleci
lar mechanism for transmission of the activation sign:
between the transverse tubule and sarcoplasmic reticl
lum calcium channels and the physiological regulation c
these processes are interesting areas for further study.

References

Adams, 8. A., Tanabe, T., Mikami, A., Numa, S., and Beam, K. E
(1990). Nature 346, 569-572.
Arreola, J., Calvo, J., Garcia, M. C., and Sdnchez, J. A. (1987). .
Physiol. 393, 307-330.
Block, B. A., Imagawa, T., Campbell, K. P., and Franzini-Armstroq
C. (1988). J. Cell Biol. 707, 2587-2600.
Catterall, W. A. (1988). Science 242, 50-61.
Curtis, 8. M., andcatterall, W. A. (1986). Biochemistry25,3077-308:
De Jongh, K. S., Merrick, D. K., and Catterall, W. A. (1989). Proc. Nat
Acad. Sci. USA 86,8585-8589.
De Jongh, K. S., Warner, C., and Catterall, W. A. (1990). J. Biol. Cheer
265, 14738-14741.
Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H
Leung, A. T., Campbell, K. P., McKenna, E., Koch, W. J., Hui, A
Schwartz, A., and Harpold, M. M. (1988). Science 241, 1661-1664
Flockerzi, V., Oeken, H.J., Hofmann, F., Pelzer, D., Cavalie, A., an,
Trautwein, W. (1986). Nature 323, 66-68.
Flucher, B. E., Morton, M. E., Froehner, S. C., and Daniels, M. F
(1990). Neuron 5, 339-351.
Hymel, L., Striessnig, J., Glossmann, H., and Schindler, H. (1988;
Proc. Natl. Acad. Sci. USA 65, 4290-4294.
Imagawa, T., Smith, J. S., Coronado, R., and Campbell, K. P. (1987)
J. Biol. Chem. 262, 16636-16643.
Jay, S. D., Ellis, S. B.. McCue, A. F., Williams, M. E., Vedvick, T. S.
Harpold, M. M., and Campbell, K. P. (1990). Science 248, 490-492
Kim, K.C., Caswell, A. H.,Talvenheimo, J. A., and Brandt, N. R. (1990)
Biochemistry 29, 9281-9289.
Lai, F. A., Erickson, H. P., Rousseau, E., Liu, Q.-Y., and Meissner, G
(1988). Nature 337, 315-319.
Lai, Y., Seagar, M. J., Takahashi, M., and Catterall, W. A. (1990). J
Biol. Chem. 265, 20839-20848.
Nunoki, K., Florio, V., and Catterall, W. A. (1989). Proc. Natl. Acad
Sci. USA 86, 6816-6820.
Perez-Reyes, E., Kim, Ii. S., Lacerda, A. E., Horne, W., Wei, X. Y.
Rampe, D., Campbell, K. P., Brown, A. M., and Birnbaumer, L. (1989)
Nature 340, 233-236.
Rios, E., and Brum, G. (1987). Nature 325, 717-720.
Ruth, P., Rohrkasten, A., Biel, M., Bosse, E., Regulla, S., Meyer
H. E., Flockerzi,V.,andHofmann,F.(1989). Science245,1115-1118
Schneider, M. F., and Chandler, W. K. (1973). Nature 242, 244-246.
Schwartz, L. M., McCleskey, E. W., and Almers, W. (1985). Nature
314,747-751,
Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa.
K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and
Numa. S. (1989). Nature 339, 439-445.
Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H.,
Kangawa, K., Kojima, M., Matsuo, H., Hirose. T., and Numa, S. (1987).
Nature 328, 313-318.
Tanabe, T., Beam, K. G., Powell, J. A.. and Numa, S. (1988). Nature
336, 134-l 39.
Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S.
(1990). Nature 346, 567-569.
Wagenknecht, T., Grassucci, R., Frank, J.. Saito. A., Inui, M., and
Fleischer. S. (1989). Nature 338, 167-170.