The Muscle Research Group studies molecular interactions between two Ca2+ ion channels that underlie Ca2+ signalling in muscle. The channels are the dihydropyridine receptor (DHPR) Ca2+ channel in the surface membrane and the ryanodine receptor (RyR) Ca2+ release channel in the sarcoplasmic reticulum (SR) Ca2+ store. These proteins are essential for movement. Excitation-contraction (EC) coupling is broadly defined as the signal transduction process that links an action potential to contraction, but more narrowly encapsulates the processes that intervene between depolarization of the surface membrane and Ca2+ release from the SR. EC coupling in the heart depends on RyR activation by Ca2+ ions that enter through the DHPR ion channel. In marked contrast, EC coupling in skeletal muscle does not depend on external Ca2+. Instead, a depolarisation-dependent signal is transmitted from the DHPR to the RyR by conformational coupling between the two proteins. Proper Ca2+ signalling depends (a) the activity of the RyR during EC coupling and (b) on the amount of Ca2+ available for release within the Ca2+ store. The factors that set RyR channel activity and the Ca2+ load within the store are the focus of our investigations.
Although many of our questions are very basic, the research is increasingly directed to understanding disease-related mutations in the proteins that are linked to debilitating skeletal myopathies and to fatal cardiomyopathies. The long-term goal in this regard is the rational design of drugs that might help alleviate the symptoms of these disorders.
The research of the Group is dedicated to understanding the cellular mechanisms that underlie changes in cytoplasmic calcium signalling in general, and more specifically those mechanisms which trigger contraction following an electrical signal on the surface membrane of skeletal and cardiac muscle fibres. Our goal is to understand the normal function of the proteins that regulate calcium signalling and changes in the function of the proteins in skeletal myopathies and in heart disease. Several different approaches are used to tackle this problem.
The Ryanodine Receptor Calcium Release Channel
The ubiquitous ryanodine receptor calcium release channel is found in the membranes of intracellular calcium stores and is the major calcium release pathway from these stores in many cell types. Although regulation of cytoplasmic calcium is basic to the function of all cells, especially in striated muscle, the mechanisms controlling ryanodine receptor activity are not well understood.
We are examining the regulation of ion flow through the ryanodine receptor by studying the currents through single channels incorporated into artificial lipid bilayers. Our specific interests are the modulation of channel activity by calcium and magnesium ions, following sulfhydryl reduction and oxidation (by oxidants such as NO), by FK-506 binding proteins (FKBPs), by co-proteins like triadin, junctin and calsequestrin and by protein-protein interactions with the skeletal muscle L-type calcium channel (an essential step in excitation-contraction coupling), which is also known as a dihydropyridine receptor (DHPR). We have identified basic mechanisms in (a) calcium magnesium regulation sites, (b) redox state, (c) FKBP and Homer in controlling the "gating" of the ion channel. Our studies have shown for the first time that small peptides, corresponding to a sequence in the DHPR, both activate and inhibit single ryanodine receptor channels and that the activation is modified by FKBP12. We have further shown how interactions between triadin, junctin and calsequestrin allow the environment in the lumen of the SR to regulate Ca2+ release through the RyR. We have also identified the triadin binding domain on the RyR and shown an essential role for triadin in EC coupling. These studies are continuing to identify regions of the RyR that bind to junctin and regions on triadin and junctin that bind to the RyR. The research on the RyR includes regions in the protein that are variably spliced, their role in development, myotonic dystrophy and in excitation-contraction coupling. Finally, we are looking at interactions between glutathione transferases and the cardiac ryanodine receptor and the possibility of using parts of the GST protein as a template for drugs that will target the cardiac ryanodine receptor in heart failure.
We will investigate the sequences in the ryanodine receptor and co-proteins and the structural constraints that allow regulatory interactions to proceed. We are also examining the effects of the ryanodine receptor mutations in various myopathies on single-channel activity.
The Muscle Research Group was largely responsible for much of the basic work on voltage-dependence of excitation-contraction coupling in mammalian skeletal muscle. However, the molecular mechanism of excitation-contraction coupling in skeletal muscle remains unnsolved mystery. We know that depolarization of the surface membrane activates a voltage sensor which is a part of the dihydropyridine receptor in the transverse tubule membrane. The loop between the second and third transmembrane segment of the alpha 1 subunit of the dihydropyridine receptor is thought to be involved in transmitting the depolarisation-evoked signal to the ryanodine receptor by physically interacting with the ryanodine receptor. We have recently published the first atomic-level structure of the II-III loop and we are examining the structure of a SPRY2 domain in the RyR that interacts with the II-III loop. We now believe that the beta subunit of the dihydropyridine receptor is a major player in excitation-contraction coupling and we are examining its interactions with both the ryanodine receptor and with the alpha 1 subunit of the dihydropyridine receptor.
In the near future will examine the interactions between the alpha 1S II-III loop and the beta1a subunit of the dihydropyridine receptor with the ryanodine receptor and with other co-proteins, especially the FKBPs, triadin and junctin, so that a model can be developed of the in vivo activation of the ryanodine receptor by the dihydropyridine receptor during excitation-contraction coupling.
Calsequestrin and cardiac disorders and heart failure
We have been conducted much of the recent work that has led to an understanding of how the calcium binding protein calsequestrin regulates the ryanodine receptor and calcium release from the sarcoplasmic reticulum in fast-twitch skeletal muscle. We are now focussing (a) on slow-twitch skeletal muscle and (b) the heart, where the role of calsequestrin is important but very different from that in fast-twitch skeletal muscle. The isoforms of calsequestrin differ in fast- and slow-twitch muscle and in the heart, while the isoforms of the ryanodine receptor differ in the heart and skeletal muscle. In all cases, calcium release is regulated by the calcium concentration in the sarcoplasmic reticulum calcium store and on the communication of this to the ryanodine receptor via calsequestrin. We are discovering how the isoforms of the proteins in each muscle type determine the effect of luminal calcium concentration on contraction and how this is distorted in myopathies and in heart failure.