Immunolocalization and Structural Configuration of Membrane and Cytoskeletal Proteins Involved in Excitation-Contraction Coupling of Cardiac Muscle

Joy S. Frank; Alan Garfinkel

I INTRODUCTION

The focus of this chapter is on the structures involved in excitation–contraction (E−C) coupling. This is not intended to be a comprehensive review but does cover areas that have been of particular interest to the Cardiovascular Research Laboratory at UCLA School of Medicine and that are particularly related to the other chapters in this book. The structure of key functional units in E−C coupling is covered, especially the transverse (T) tubules, junctional and corbular sarcoplasmic reticulum (SR), and the diadic space formed by the close apposition of the sarcolemma (SL) and the junctional sarcoplasmic reticulum (jSR). In addition, the subcellular localization of proteins involved in the regulation of intracellular Ca2 + are discussed. This includes the distribution of the Na+ − Ca2 + exchanger, Na+K+ATPase, the Ca2 + release channel/ryanodine receptor (CRC/RR), and the Ca2 + channel (DHP receptor). The organization of proteins within regions of the SL or SR results in domains of functional significance. Thus a discussion on the role of cytoskeletal proteins and their relationship to Ca regulatory proteins completes the chapter.

II Ca RECEPTORS, CHANNELS, RELEASERS, AND STORAGE

A DIADIC CLEFT

The region where the SL and the SR appose each other contains the most important functional units for the coupling of excitation to contraction. The close apposition of the lateral cistern of the jSR to the SL defines a space (~ 15 nm) containing channels, receptors, and exchangers whose function revolves around the regulation of Ca flux into and out of the myocyte. For all its importance, this space and the membranes that restrict it have lacked an all-encompassing name. Lederer et al. (1990) coined the term fuzzy space for this region. Langer and Peskoff (1996) have designated this space the diadic cleft and have produced a working model of its structural and functional characteristics (see Chapter 5).

1 Ca Release Channel/Ryanodine Receptor

The membrane of the jSR facing the SL has regularly arranged projections or “foot” processes (Franzini-Armstrong, 1980) that extend ~ 12 nm into the diadic cleft to contact the membrane of the T tubule forming an interior coupling or to contact the surface SL to form a peripheral coupling. The structure and function of these junctional spanning proteins or “feet” have been studied in great detail (Franzini-Armstrong, 1980; Kelly and Kuda, 1979; Lai et al, 1988; Anderson et al, 1989). It is well accepted that the feet that are periodically arrayed in the diadic cleft contain the ryanodine receptor (RR), a high-conductance calcium channel that provides for the release of calcium ions from the SR during contraction (Ogawa, 1994; Anderson et al, 1989).

Figure 1 contains two useful images of the diadic cleft. Figure 1A is a thin-section electron micrograph from rabbit papillary muscle. It is from this conventional type of preparation that the feet were first visible, due partly to the fact that they are equally spaced along the jSR. Figure 1B presents a more three-dimensional (3-D) perspective of the structure of the diadic cleft. The tissue is prepared here with freeze-fracture/deep-etching on cells that are not chemically preserved but ultrarapidly frozen. An “end-on view” of the feet/calcium release channel (CRC) can easily be seen to span the cleft bridging the jSR and the T tubular membrane. Immunolabeling studies with antibodies against the RR have localized the CRC/RR in adult cardiac muscle predominantly at the level of the T tubules, in clearly defined banding pattern at the Z lines [Figs. 2A and B].

The large tetrameric CRC/RR ion channel complex has been isolated from skeletal muscle. With the use of the isolated channels, Radermacher et al. (1994) were able to obtain frozen hydrated samples that were used for cryo-electron microscopy to produce excellent preservation of the macromolecular ultrastructure of the channels. With 3-D reconstruction techniques, the most detailed view to date of the CRC structure was produced. The “foot” portion of the CRC is a large assembly (29 × 29 × 12 nm) linked to a smaller transmembrane component. The resolution afforded by the 3-D reconstruction is such that a cylindrical, low-density region extending down the center of the foot assembly could be discerned and most likely corresponds to the Ca2 + conducting pathway (Fig. 3).

In skeletal muscle, at least, there is good structural evidence for a direct interaction between the CRC/RR and the Ca2 + channels (DHP receptors) located within the T tubular membrane. This is an important point because the mechanism of excitation—contraction (E−C) coupling in skeletal muscle is believed to occur by sarcolemmal depolarization acting on the DHP receptors, which in turn act as voltage sensors (Leung et al., 1988; Rios and Brum, 1987) that undergo a confirmation change. It is this direct molecular interaction that is believed to induce the opening of the CRC/RR and thus the release of calcium from the jSR. Block et al. (1988) identified large intramembrane particles (IMPs) in the P face (membrane face adjacent to the cytoplasm) of the T tubular membrane in skeletal muscle. These are the only intramembraneous particles seen in the T tubular membrane, and they have the characteristics of the DHP receptors. In addition, skeletal muscle T tubular membranes are the richest source of DHP receptors. This adds additional support to the idea that these particles represent the DHP receptors. Interestingly, the particles are arrayed in clusters of four particles forming a tetrad. The axis of fourfold symmetry is rotated and, as a result, each tetrad of IMPs in the tubular membrane is opposite alternating foot structures of the CRC/RR. This structural arrangement between the CRC/ RR in the jSR and the DHP receptors in the T tubular membrane provides direct support for the...