Chapter 4 RYR-DHPR RELATIONSHIPS IN SKELETAL AND CARDIAC MUSCLES

Chapter 4

RYR-DHPR RELATIONSHIPS IN SKELETAL AND CARDIAC MUSCLES

Clara Franzini-Armstrong

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

INTRODUCTION

The functional link between depolarization of the plasmalemma 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 and transverse (T) tubule, also called dihydropyridine receptors (DHPRs), act as voltage sensors and initiate the cascade of events linking excitation to contraction (EC coupling). The calcium release channels of the sarcoplasmic reticulum, also called ryanodine receptors (RyRs), have a high permeability to calcium and 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 within calcium release

units (CRUs)

at sites where one or two SR cisternae dock on the

plasmalemma/T tubules forming intracellular junctions named triads, dyads

and peripheral couplings (Fig. 4-1). Regardless of their shape, CRUs of

skeletal and cardiac muscle in vivo and in vitro contain a common

complement of major components. These include the SR docking protein

junctophilin;

the two calcium channels defined above; the internal calcium

binding protein calsequestrin (CSQ);

the two proteins that mediate CSQ-

RyR link, triadin and junctin;

and a large number of proteins associated with and regulating the cytoplasmic domains of RyRs (Fig. 4-1).

Figure 4-1. Calcium release units (usually in the form of triads in skeletal muscle) contain L-type calcium channels (DHPRs), the sarcoplasmic reticulum calcium release channels (RyRs) and other SR proteins. In these false-color confocal images, the position of hot spots of DHPRs (A), RyRs (B) and triadin (C), labeled by the correspondent antibodies, mark the positions of the triads at the A-I junction. In the electron microscope (D) triads are composed of a central transverse tubule flanked by two SR cisternae.

RYR DISPOSITION

The structure, location and disposition of RyRs in situ is detected by

electron microscopy of thin sections (Fig. 4-1), of freeze-fracture replicas

37 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. The cytoplasmic domains of RyRs are mirror symmetric and also they do not abut at the corners within the arrays, but they overlap with each other by about a third of their sides.

Thus in the array, RyR profiles are skewed relative to the lines connecting the centers of the tetrameric RyR feet, which is parallel to the long axis of the T tubules (Fig. 4-2). RyRs have an inherent ability to organize themselves into arrays even in the absence of all other proteins of the sarcoplasmic reticulum

and they are also targeted to CRUs in the absence of DHPRs.

Figure 4-2. RyRs (shown as four white circles) form precise arrays within the junctional SR membrane. This is due to the fact that their cytoplasmic domains 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 associated with subunits of adjacent RyRs.

RYR-DHPR RELATIONSHIP

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. In particular, four DHPRs, constituting a tetrad, are associated with the four equal subunits of the RyR (or feet), but are positioned over alternate feet (Fig. 4-2).

Dysfunctional CRUs containing RyR, but not DHPRs are formed in skeletal muscle in the absence of the subunit of DHPRs

and units containing DHPRs are formed in the absence of RyRs.

This indicates that the two proteins are independently targeted to and retained within CRUs. However, DHPRs require an association with skeletal type RyRs (RyR1) in order to form tetrads and tetrad arrays.

The alpha 2 subunit of DHPR also needs the alpha1 for appropriate targeting.

The recent availability of 3-D reconstructions of both RyR and DHPR is bringing us close to a final understanding of the structural interaction between these two molecules.

We have recently reexamined the relative positions of DHPR and RyRs arrays relative to each other by superimposing 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. 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. 4-2). In addition it is clear that overall 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 will be 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.

More on this below.

RYR3 LOCATION

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.

RyR3 fails to sustain EC-coupling in vitro and in vivo

and to induce tetrad formation by DHPRs when expressed in

dyspedic (RyR1 null) cells that have skeletal DHPR.

Structural

observations give some clues to the possible role of RyR3. Comparison of

39 muscles that contain, alternatively, none, few or a relatively high proportion of RyR3, show that presence of RyR3 correlates well with the presence of parajunctional feet, located not within the area of SR membrane that associate with T tubules, but immediately adjacent to it.

Identification of these parajunctional feet with RyR3 (Fig. 4-3) suggests that activation of RyR3 is not directly via an interaction with DHPRs, but indirectly, perhaps by calcium released from the RyR1, as also indicated by the known properties of the RyR3 channels.

Comparison of RyR and DHPR dispositions in skeletal and cardiac muscles also yields results that are significant in functional terms.

Differently from those in skeletal muscle, the cardiac isoforms of the two channels (RyR2 and DHPR) do not interact directly. As a result, while in skeletal muscle EC-coupling is independent of extracellular calcium, and thus it does not require calcium permeation through the DHPR channel, cardiac EC-coupling depends on extracellular calcium.

In cardiac muscles, DHPRs and RyRs are colocalized at CRUs,

but the DHPRs are not organized into tetrads, an indication that they are not specifically linked to the RyR subunits. One may infer from this that like 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.

EXPERIMENTAL APPROACHES

The availability of null mutations for RyR1 in mouse skeletal muscle where RyR3 plays a minor role

and of a cell line carrying the RyR1 mutation

opened the possibility of exploring the functional and structural requirement for the skeletal type, or direct, DHPR-RyR interaction. The functional experiments performed in K.G. Beam’s laboratory

clearly led the way. Nakai et al.

showed that the talk 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. RyR1-RyR2 chimerae and an effective virus based infection mechanism were engineered in P. D.

Allen’s laboratory, allowing the following structural observations. An initial

result 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. Thus the

presence of tetrads is indicative of a specific link between and

RyR1.

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 talk 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.

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. An interesting, if not unexpected, result.

Figure 4-3. Diagrammatic view of the relationship between type 1 and type 3 RyRs in the triads of skeletal muscles (e.g., from the frog, the tail of some fish and some birds) that contain a 1:1 ratio of RyR1 and RyR3. RyR1 are positioned in the junctional gap between SR and T tubules, so that they can associate directly with DHPRs. RyR3 are located in a parajunctional region, where they are in proximity of RyR1, but cannot interact with DHPRs.

It is assumed that they are activated independently. Courtesy of E. Felder.

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.

41 Further interesting confirmation of the specificity of the DHPR-RyR link in skeletal muscle comes from recent experiments exploring the possibility that conformational changes in the RyR may be revealed by an alteration of DHPR tetrads.

Ryanodine induces substantial and persistent conformational changes in RyR. At relatively high concentrations (above ryanodine locks the channel in a closed state by binding to low affinity sites. Two cells lines (BC3H1 and RyR1-infected 1B5) that have extensive clusters of associated and RyR1 at the cell surface were treated with ryanodine for 24 hrs, freeze-fractured and rotary shadowed. The center-to-center distances between individual DHPR within tetrads decreased by ~2 nm in the ryanodine-treated cells relative to the control cells. These results indicate that the DHPR-RyR complex acts as a single unit, confirming a specific interaction between the channels, and further suggest that ryanodine induces large conformational changes in the cytoplasmic RyR domain responsible for linking to DHPR.

CONCLUDING REMARKS

The specificity of the RyR-DHPR interaction in skeletal muscle is revealed by electron microscopy studies that define the special relationship between the two channels.

ACKNOWLEDGEMENTS

The work presented in this chapter was performed in collaboration with

Drs. P.D. Allen; Kurt Beam and Isaac Pessah and is due to the contributions

of Drs. Edward Felder, Feliciano Protasi, Cecilia Paolini, and Hiroaki

Takekura.