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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 Santiago  2004 



Biol Res 37: 507-512, 2004


Functional implications of RyR-DHPR relationships in skeletal and cardiac muscles


Dept. Cell Developmental Biology, University of Pennsylvania School of Medicine, Anatomy/Chemistry Building B42, Philadelphia, PA 19104-6058, USA

Dirección para Correspondencia



Dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) interact during EC coupling within calcium release units, CRUs. The location of the two channels and their positioning are related to their role in EC coupling. als DHPR and RyR1 of skeletal muscle form interlocked arrays. Groups of four DHPRs (forming a tetrad) are located on alternate RyR1s. This association provides the structural framework for reciprocal signaling between the two channels. RyR3 are present in some skeletal muscles in association with RyR1 and in ratios up to 1:1. RyR3 neither induce formation of tetrads by DHPRs nor sustain EC coupling. RyR3 are located in a parajunctional position, in proximity of the RyR1-DHPR complexes, and they may be indirectly activated by calcium liberated via the RyR1 channels. RyR2 have two locations in cardiac muscle. One is at CRUs that contain DHPRs and RyRs. In these cardiac CRUs, RyR2 and a1c DHPR are in proximity of each other, but not closely linked, so that they may not have a direct molecular interaction. A second location of RyR2 is on SR cisternae that are not attached to surface membrane/T tubules. The RyR2 in these cisternae, which are often several microns away from any DHPRs, must necessarily be activated indirectly.

Key words: Calcium release units, dihydropyridine receptors, ryanodine receptors, transverse tubules, sarcoplasmic reticulum.



The functional link between depolarization of the plasmalemma (including its invaginations, the transverse T tubules) and the release of calcium from the sarcoplasmic reticulum (SR) in all types of muscle cells involves two calcium channels. The L-type calcium channels of the plasmalemma, also called dihydropyridine receptors (DHPRs), act as voltage sensors and initiate the cascade of events leading to excitation-contraction (EC) coupling (see also Rios and Zhou, 2004; Schneider and Rodney, 2004; Farrell et al., 2004, all in this issue). The calcium release channels of the sarcoplasmic reticulum, also called ryanodine receptors (RyRs), have a high permeability to calcium, and when they are open, they allow a rapid efflux of calcium from the SR lumen to the myofibrils, driven by the large lumenal-cytoplasmic concentration gradient.

The two channels can be detected by immunolabeling with specific antibodies and more directly by various electron microscopy techniques. These approaches have established the fact that DHPRs and RyRs are components of stable macromolecular complexes that have been named calcium release units (CRUs) for obvious reasons (Flucher and Franzini-Armstrong, 1996). CRUs are formed at sites where one or two SR cisternae dock on the plasmalemma/T tubules forming special intracellular junctions named triads, dyads and peripheral couplings (Fig. 1). Regardless of their shape, CRUs of skeletal and cardiac muscle in vivo and in vitro always contain a common complement of major components. These include the SR docking protein junctophilin (Takeshima et al., 2000); the two calcium channels defined above; the internal calcium binding protein calsequestrin (CSQ); the two proteins that mediate CSQ-RyR link, triadin and junctin (Jones et al., 1995; Guo et al., 1996); and a large number of proteins associated with and regulating the cytoplasmic domains of RyRs.

Figure 1. Sketch of a triad, the calcium release unit of most adult skeletal muscles, formed by the junctions of two cisternae of the sarcoplasmic reticulum (SR) with a central T tubule. Two proteins of the CRU complex are shown: the ryanodine receptors (RyRs) or calcium release channels of the SR, and the dihydropyridine receptors (DHPRs) or voltage sensors of the T tubules. Two of the four subunits (each with a smaller intramembrane and a larger cytoplasmic domain) are shown for each RyR. There are two types of RyRs in skeletal muscle. RyR1 is located within the junctional gap connecting SR and T tubules and is closely associated with DHPRs as shown. The other RyR, RyR3, is located parajunctionally, and it does not interact closely with DHPRs. Some muscles have only RyR1, and thus, their CRUs look like the lower half of the figure. Other muscles have equal amounts of RyR1 and RyR3, as shown in the upper half. In the absence of RyR1, RyR3 does not sustain EC coupling.


The structure, location and disposition of RyRs in situ are detected by electron microscopy of thin sections (Fig. 1) of freeze-fracture replicas and of replicas from shadowed, isolated SR vesicles. In thin sections, the large cytoplasmic domains of RyRs appear as electron dense masses that bridge the gap between apposed SR and plasmalemma/ T tubule membranes, called feet. In grazing views of the gap, each cytoplasmic domain (or foot) has an approximately square shape and is closely associated with those of the neighboring channels to form extensive arrays. Feet arrays have a handedness, that is, they appear different when viewed from the cytoplasmic or from the lumenal side of the SR membrane with which they are associated. This is due to the fact that the cytoplasmic domains of RyRs are themselves mirror symmetric. Also, they do not abut at the corners but overlap with each other by about a third of their sides. In the resultant array, RyR profiles are skewed relative to the lines connecting the centers of the tetrameric RyR feet, which are parallel to the long axis of the T tubules. RyRs have an inherent ability to organize themselves into arrays even in the absence of all other proteins of the sarcoplasmic reticulum (Takekura et al., 1995a; Yin and Lai, 2000), and they also are targeted to CRUs in the absence of DHPRs (Takekura et al., 1995b).

DHPRs are detected in freeze-fracture replicas of the plasmalemma/T tubule membranes. Each DHPR appears as a large intramembranous particle mostly associated with the cytoplasmic leaflet. In skeletal muscle, the DHPRs are arranged into arrays that are closely related to the arrays of RyRs (Fig. 2). In particular, four DHPRs, constituting a tetrad, are grouped at the position of alternate RyRs (Block et al., 1988), and the tetrad is skewed relative to the long axis of the T tubules. CRUs are formed in skeletal muscle in the absence of the a1 subunit of DHPRs (Flucher et at, 1993; Powell et al., 1996) and in the absence of RyRs (Takekura et al., 1995b; Takekura and Franzini-Armstrong, 1999), but DHPRs require an association with skeletal-type RyRs (RyR1) in order to form tetrads and tetrad arrays (Protasi et al., 2000). The alpha 2 subunit of DHPR also needs the alpha 1 for appropriate targeting (Powell et al., 1996). The positioning of DHPR tetrads and their size clearly indicate that a tetrad is due to the association of four DHPRs with the four equal subunits of the RyR. However, as noted above, tetrads are always associated with alternate feet along the arrays.

Figure 2. RyRs form precise arrays within the junctional SR membrane. This is due to the fact that their cytoplasmic domains (shown here as empty circles) interlock with each other. In skeletal muscles of all vertebrates, DHPRs (each shown as a black sphere) are located in precise apposition to the four identical subunits of RyRs, thus forming a group called a tetrad. Tetrads are located opposite alternate feet in the array, probably due to steric hindrance that does not allow DHPRs to be on the subunits of adjacent RyRs.

The recent availability of 3-D reconstructions of both RyR and DHPR (Radermacher et al., 1994; Serysheva et al., 1999, 2002; Wolf et al., 2003), is bringing us close to a final understanding of the structural interaction between these two molecules. In order to take the next step in this understanding, it is necessary to know exactly how DHPRs and RyRs fit together, and this, in turn, requires knowledge of the relative orientation of the arrays of the two molecules. The orientation of the arrays relative to each other can be deduced by examining arrays of DHPR tetrads as seen in freeze-fracture and arrays of feet as seen in rotary shadowed replicas of isolated heavy SR vesicles. Both images contain orientation clues, and if care is taken with mounting the grids in the electron microscope, images that have the same orientation can be obtained (Paolini et al., 2004). Superimposition of an oriented array of tetrads over a similarly oriented array of feet, shows that each of the four DHPR freeze-fracture particles is in the same relationship with the four RyR subunits and that each is close to, but not quite halfway, along the side of the square outline defining the foot (Fig. 2). In addition, it is clear that the DHPR tetrad is larger than the outline of the foot, and this in part explains why tetrads are associated with alternate feet. These images define specific restrictions on the location of DHPRs, which will acquire importance in the near future, once higher resolution images are available.

The specific positioning of skeletal-type DHPRs in relation to RyR1 molecules is at the basis of the proposed bidirectional interaction that allows the two channels to control each other's function during excitation-contraction coupling (Nakai et al., 1996). More details on this structural interaction are given below.

Many skeletal muscles contain type 3 ryanodine receptors (or beta in the lower vertebrates) in addition to type one (or alpha), some at equal molar concentrations (Sutko and Airey, 1996; Murayama and Ogawa, 2002). RyR3 fails to sustain EC coupling in vitro and in vivo (Takeshima et al., 1994; Ivanenko et al., 1995; Ward et al., 2001; Buck et al., 1997) or to induce tetrad formation by DHPRs when expressed in dyspedic (RyR1 null) cells that have skeletal DHPR (Protasi et al., 2000). Structural observations give some clues to the possible role of RyR3. Comparison of muscles that contain either none, or little or a relatively high proportion of RyR3, show that presence of RyR3 correlates well with the presence of parajunctional feet that are located not within the area of SR membrane that associated with T tubules, but immediately adjacent to it (Felder and Franzini-Armstrong, 2002). Identification of these parajunctional feet with RyR3 (Fig. 1) suggests that activation of RyR3 is not directly via an interaction with DHPRs, but may be associated indirectly, perhaps by the calcium that is released by the RyR1, in keeping with the physiology.

Comparison of RyR and DHPR dispositions in skeletal and cardiac muscle also yields results that are significant in functional terms. Unlike those in skeletal muscle, the cardiac isoforms of the two channels (RyR2 and a1c DHPR) do not interact directly. As a result, while EC coupling in skeletal muscle is independent of extracellular calcium and thus does not require calcium permeation through the DHPR channel, cardiac EC coupling depends on extracellular calcium (Dirksen and Beam, 1999; Proenza et al., 2002: Cleeman and Morad, 1991). In cardiac muscles, DHPRs and RyRs are colocalized at CRUs (Carl et al., 1995; Sun et al., 1995; Protasi et al., 1996), but the DHPRs are not organized into tetrads, an indication that they are not specifically linked to the RyR subunits (Fig. 3). One may infer from this that as in the case of RyR3 in skeletal muscle, the cardiac RyR is not directly activated by a molecular interaction with DHPRs, in keeping with the physiology. Expression of RyR2 in dyspedic (RyR null, or lacking feet) cells fails to restore EC coupling (either of the skeletal or cardiac type) and DHPR tetrads.

The availability of null mutations for RyR1 in mouse skeletal muscle where RyR3 plays a minor role (Takeshima et al., 1994) and of a cell line carrying the RyR1 mutation (Buck et al., 1997) opened the possibility of exploring the functional and structural requirement for the skeletal-type, or direct, DHPR-RyR interaction. RyR1-RyR2 chimerae and an effective virus-based infection mechanism were engineered in P. D. Allen's laboratory, and the structural observations follow the functional experiments performed in K.G. Beam's laboratory (Nakai et al., 1996). An initial observation is that DHPRs are targeted to CRUs in the absence of RyR, but they require an interaction with RyR1 in order to assemble into tetrads (Fig. 3). Thus, the presence of tetrads is indicative of a link between DHPRs and RyRs (Protasi et al., 1998). The first major functional observation from the work involving dyspedic cells is that the communication between skeletal type DHPR and RyR goes in both directions: DHPR activity directly affects the open probability of the RyR channel, but interaction with RyR is also necessary for effective calcium permeation through the DHPR (Nakai et al., 1996). Specific regions of RyR1 necessary for these interactions have been identified. The immediate question is whether there is a relationship between the requirements for a functional communication and those for the formation of DHPR tetrads, indicative of a specific molecular link with the RyR subunits. Interestingly, while the general answer is that indeed those RyR1-RyR2 chimerae that restore skeletal-type EC coupling also restore the presence of tetrads, the effectiveness of specific chimerae to restore tetrads is not exactly the same as that in restoring the functional link (Protasi et al., 2002). It appears that several regions of the RyR sequence may be involved in both the functional and structural interaction, but the regions that are required for holding the two molecules in the appropriate relative position are not exactly the same as those required for the functional interaction during EC coupling. This is an interesting, if not unexpected, result.

Thus in skeletal muscle DHPRs and RyRs are held in close physical proximity by a molecular connection that may be direct, and this proximity is a requirement for their functional interaction. The story is obviously considerably more complicated than presented here, but this is a good beginning,


Figure 3. Diagramatic views of the relationship between RyRs and DHPRs in skeletal and cardiac muscles of vertebrates and in dyspedic (RyR null) skeletal muscle. In skeletal muscle, as also shown in Fig. 2, DHPRs occupy a specific position relative to the four equal RyR subunits. In cardiac muscle, DHPRs are in proximity of RyRs but not locked into them, predicting a direct interaction, perhaps via a messenger, such as calcium. In the dyspedic muscles, DHPRs are targeted to CRUs, where they cluster in proximity of the SR, but they are not organized into tetrads because they are not linked to RyRs (which are missing).



The work presented was performed in collaboration with Drs. PD Allen and Kurt Beam and is indebted to the contributions of Drs. Edward Felder, Feliciano Protasi, Cecilia Paolini and Hiroaki Takekura.


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Corresponding author: Clara Franzini-Armstrong, Department of Cell Developmental Biology, University of Pennsylvania School of Medicine Anatomy/Chemistry Building B42 Philadelphia, PA 19104-6058, USA. Phone: (1-215) 8798 3345, Fax: (1-215) 573-2170, E-mail:

Received: January 23, 2004. Accepted: June 7, 2004.


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