Molecular architecture of membranes involved in excitation-contraction coupling of cardiac muscle.

Abstract

Abstract. Peripheral couplings are junctions between the sarcoplasmic reticulum (SR) and the surface membrane (SM). Feet occupy the SR/SM junctional gap and are identified as the SR calcium release channels, or ryanodine receptors (RyRs). In cardiac muscle, the activation of RyRs during excitation-contraction (e-c) coupling is initiated by surface membrane depolarization, followed by the opening of surface membrane calcium channels, the dihydropyridine receptors (DHPRs). We have studied the disposition of DHPRs and RyRs, and the structure of peripheral couplings in chick myocardium, a muscle that has no transverse tubules. Immunolabeling shows colocalization of RyRs and DHPRs in clusters at the fiber's periphery. The positions of DHPR and RyR clusters change coincidentally during development. Freeze-fracture of the surface membrane reveals the presence of domains (junctional domains) occupied by clusters of large particles. Junctional domains in the surface membrane and arrays of feet in the junctional gap have similar sizes and corresponding positions during development, suggesting that both are components of peripheral couplings. As opposed to skeletal muscle, membrane particles in junctional domains of cardiac muscle do not form tetrads. Thus, despite their proximity to the feet, they do not appear to be specifically associated with them. Two observations establish the identity of the structurally identified feet arrays/junctional domain complexes with the immunocytochemically defined RyRs/DHPRs coclusters: the concomitant changes during development and the identification of feet as the cytoplasmic domains of RyRs. We suggest that the large particles in junctional domains of the surface membrane represent DHPRs. These observations have two important functional consequences. First, the apposition of DHPRs and RyRs indicates that most of the inward calcium current flows into the restricted space where feet are located. Secondly, contrary to skeletal muscle, presumptive DHPRs do not show a specific association with the feet, which is consistent with a less direct role of charge movement in cardiac than in skeletal e-c coupling. CALCIUM is an intracellular messenger in all cell types. In muscle fibers, cytosolic-free calcium concentration regulates contraction and relaxation. The endoplasmic reticulum is a storage compartment for calcium and the sarcoplasmic reticulum (SR) 1 of muscle cells cycles large amounts of calcium at each contraction and relaxation (for a review see Pozzan et al., 1994). Rapid release of calcium from ER and SR occurs through the ryanodine receptor (RyR), a channel with high permeability to calcium, encoded by three different genes with various tissue specificity

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle.

“…Complex (q~) charging waveforms appear specifically to occur in skeletal muscle in which excitation-contraction coupling may involve direct allosteric interactions between DHPR-voltage sensors and RyR-Ca 2+ release channels (for review see Huang, 1993). In contrast, the Ca 2+ channel gating currents that also initiate elevations of cytosolic Ca 2+ in arthropod skeletal muscle and mammalian cardiac muscle are simple monotonic decays whose associated charge nevertheless also exhibits steep steady-state voltage dependences similar to those of the q~ system (Gilly and Scheuer, 1984;Bean and Rios, 1989) these latter systems may be indirectly activated by a triggering entry of extracellular Ca 2 rather than through such direct receptor-receptor couplings (Beuckelman and Wier, 1988;Fabiato, 1985;Sun et al, 1995). The question then arises whether the complex qv kinetics in skeletal muscle specifically reflect allosteric interactions between the molecules that initiate excitationcontraction coupling.…”

“…Recent biochemical evidence (Anderson and Meissner, 1995) suggests that DHPRs are linked allosterically to RyR-I or the RyR~ isoforms in mammalian or amphibian skeletal muscle triads, respectively (Olivares et al, 1991), but not to the RyR-II isofonns in cardiac muscle (Sun et al, 1995). Such a scheme would still permit DHPRs to generate a steadystate q~ charge transfer driven by transverse tubular potential (cf.…”

“…They do not contribute to the monotonically decaying Ca 2+ channel gating currents in neurons (Kostyuk et al, 1981), arthropod skeletal muscle (Gilly and Scheuer, 1984), and mammalian cardiac muscle (Bean and Rios, 1989), even though all these systems are activated by elevations of cytosolic Ca 2+. However, the latter systems are differently triggered indirectly by extracellular Ca 2 entry (Beuckelman and Wier, 1988;Fabiato, 1985;LopezLopez et al, 1995;Cannell et al, 1995;Sun et al, 1995). Such comparisons would associate complex q~ charge movements specifically with the allosteric coupling proposed between tubular DHPRs and cisternal RyRs in skeletal muscle.…”

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“…Indeed, studies in chicken developing peripheral couplings have shown that there is a very close relationship between the size of arrays of feet in the SR and the area covered by DHPR in the junctional portion of the sarcolemma (25). While DHPRs are clearly clustered closely to RyRs, freeze-fracture studies have shown no evidence of organization of DHPRs into tetrads (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). DHPRs are randomly clustered in junctional domains of exterior membranes and no spatial relationship with feet was detected ( Figure 3, A and C, and Figure 5, B and D).…”

“…The possible reason for this alternate tetrad/RyR1 association will be further discussed in section 6. In cardiac muscle, immunohistochemistry experiments and morphological studies have shown that DHPRs are also clustered in close correspondence with RyR domains (7,14). Indeed, studies in chicken developing peripheral couplings have shown that there is a very close relationship between the size of arrays of feet in the SR and the area covered by DHPR in the junctional portion of the sarcolemma (25).…”