An Introduction

The mammalian brain is composed of electrically excitable cells (neurons) that communicate with each other via specialized cell-cell junctions called synapses. Effective transmission of nerve impulses between neurons requires the precise localization and high concentration of neurotransmitter receptors and ion channels in the membrane of the receiving, or postsynaptic cell. In the central nervous system, a single neuron can receive thousands of different synaptic inputs, each of which can use a variety of neurotransmitter receptors. Coordinating the localization of the appropriate receptors and ion channels for each different synapse is a formidable task. A fundamental goal in neurobiology is to determine the molecular mechanisms involved in targeting and clustering of neurotransmitter receptors and ion channels at synaptic sites. Importantly, the regulation of receptor localization at synapses is thought to form the basis of certain kinds of learning and memory. Our focus is on a family of proteins called syntrophins, intracellular membrane-associated proteins that interact with both ion channels and signaling proteins. As such, syntrophins appear to be important for linking ion channels to intracellular signaling pathways.

 The importance of syntrophins also stems from their direct association with dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin, together with other transmembrane, extracellular, and cytosolic proteins (including syntrophins) form a transmembrane link between the cytoskeleton and the extracellular matrix. Collectively, this group of proteins is referred to as the dystrophin-associated protein (DAP) complex. It has been proposed that the DAP complex provides mechanical reinforcement to the plasma membrane of muscle fibers and protects them from potentially damaging stresses developed during contraction. The absence of dystrophin in DMD leads to the loss of syntrophins and other components of the DAP complex from the muscle membrane, and renders muscle fibers susceptible to the effects of mechanical stress. As a result, most DMD patients die in their late teens or early twenties due to involvement of the diaphragm and other respiratory muscles by the disease. These observations highlight the critical importance of the DAP complex for normal muscle stability. It is now recognized that several different types of muscular dystrophy arise from defects in the genes encoding proteins of the DAP complex. DMD, an X-chromosome linked disease, is by far the most severe and most common of these dystrophies, affecting approximately 1 in 3500 boys.

 

 

 

 

 

 

 

 

 

 

An alternative (but not necessarily competing) view holds that the DAP complex functions as a scaffold for assembling ion channels and signaling molecules into multi-component signal transduction networks. In support of this hypothesis dystrophin and DAPs, including syntrophins, are expressed in many non-contractile tissues such as the brain where presumably, they have non-structural roles. In agreement with the latter view, DMD is also associated with a variable but often significant degree of mental retardation. This cognitive impairment is non-progressive and usually not related to the severity of muscle symptoms. Nevertheless, all DMD patients are affected, especially at the level of verbal skills, with approximately 30% of individuals being clearly below the norm for the unaffected population. Although moderate cognitive impairment is a consistent feature of DMD, no specific histopathological abnormalities have been demonstrated in the brains of DMD-affected individuals. This points to subtle functional abnormalities associated with the absence of dystrophin in specific neurons. Consistent with this idea, dystrophin is localized to synaptic specializations of several types of neurons in areas associated with cognitive function and learning. Its absence from DMD brain suggests that abnormalities in synaptic organization may be the underlying mechanism of cognitive dysfunction. Thus, an important aspect of our research program is to understand the molecular mechanisms that contribute to synaptic organization in the brain. This is essential if we are to understand the basis of the cognitive defects in DMD and to design suitable therapies for the disease.

Syntrophins (a1, b1, b2, g) are a family of proteins that bind to the carboxy (C) terminus of dystrophin and the related gene products utrophin and dystrobrevin. The tight association of syntrophins with the C-terminus of dystrophin places them in close proximity to the inner face of the plasma membrane. As we will see below, syntrophins are thus ideally situated to interact with transmembrane receptors and ion channels, as well as with cytoplasmic signal transduction molecules. The existence of multiple syntrophin isoforms raises several important questions. Most importantly, which isoforms are responsible for localizing different ion channels or signal transduction proteins? And how is the localization of each isoform regulated? One of our goals is to decipher the molecular interactions that control the selective targeting of different isoforms.

  A key to understanding syntrophin function lies in the structural organization of this family of proteins. Analysis of the syntrophin amino acid sequence reveals an absence of known enzymatic or catalytic domains but an abundance of motifs that are likely involved in binding to other proteins. The four known isoforms share a common domain organization which includes two tandem pleckstrin homology (PH) domains, a single PDZ domain (named for the first three proteins recognized to contain this domain: the post-synaptic density protein PSD-95, the drosophila Discs-large tumor suppressor protein, and the mammalian tight junction protein ZO-1), and a C-terminal domain of ~ 70 amino acids that is unique to syntrophins (syntrophin unique, SU). As described below, this domain organization allows syntrophins to participate in a variety of protein-protein and protein-lipid interactions. In this regard, they are unique among the proteins of the dystrophin complex. To understand the function of syntrophins and the DAP complex in synaptic organization, we have focused on the role of these domains, and in particular of the PDZ domain.

 

Recent studies have highlighted the importance of a newly recognized modular protein-interaction domain (the PDZ domain) in the molecular organization of cellular junctions. Proteins containing PDZ domains are involved in the clustering of ion channels and transmembrane receptors at specific subcellular sites and have an especially important role in the spatial organization of voltage- and ligand-gated ion channels at synapses. PDZ domains bind to other PDZ domains or to specific amino acid sequences located at the C-terminus of membrane proteins. Our previous studies in collaboration with David Bredt at the University of California, San Francisco demonstrated that the PDZ domain of a1-syntrophin binds directly to the PDZ domain of the neuronal isoform of nitric oxide synthase (nNOS), an enzyme that produces NO at synaptic junctions in brain and at motor endplates in skeletal muscle. The association of nNOS with a1-syntrophin links the enzyme to the dystrophin complex in muscle and accounts for the absence of nNOS from the membrane of DMD muscle. Our results suggest that the absence of this key enzyme may be a contributing factor to the pathogenesis of DMD. In addition, we have identified interactions between syntrophin PDZ domains and skeletal muscle sodium channels as well as voltage-gated potassium channels. Our working hypothesis is that syntrophins are modular adapter proteins that link ion channels and signaling proteins to DAP complexes. These interactions provide a mechanism for anchoring membrane proteins to the extracellular matrix, the cytoskeleton and to intracellular signaling pathways.

Last Updated April 10, 2001