Joseph A. Mindell, M.D., Ph.D. - US grants

This project is designed to attack questions regarding the basic biophysical properties of the ClC family of Cl- channels and the roles they play in salt-secreting epithelia. ClC channels are represented in all tissues, and are known to play a prominent part in ionic homeostasis by virtue of their contributions to human genetic disorders of salt transport, such as Bartter's syndrome and Dent's disease. Despite their importance, we remain remarkably ignorant of even the most basic features of the ClC family. In this proposal, I present a powerful new system, the shark rectal gland (SRG), to probe the architecture of ClC-type channels and their roles in electrolyte physiology. The SRG is perhaps the best-studied example of a salt-secreting epithelium, yet no one has previously looked for ClC-type channel genes in this tissue. My hypothesis is that ClC-type chloride channels contribute to NaCl secretion in SRG, and that this is an ideal system to study the role of these channels in salt-secreting epithelia. In preliminary work, l have cloned from the SRG four full length cDNA's encodinghomologs of ClC-type Cl channels. Here I propose to study two of these channels in detail: shark ClC-6 and ClC-7. I will take a new approach to functional expression (for these channels), preparing membrane vesicles from HEK 293 cells expressing sClC-6 and sClC-7 and fusing them into lipid bilayer membranes. Similar methodology has been successful in this lab for several other ion channels. Specific Aim 1. I propose to determine the single-channel properties of sClC-6 and sClC-7. These experiments will resolve several outstanding controversies regarding the molecular architecture of the ClC family. In particular, they will resolve the current dispute as to whether some ClC's have only one conduction pathway (in contrast to ClC-0, which has two). Such studies are a prelude to detail structure-function analysis of the ClC family. In Specific Aim 2, I present experiments designed to determine the subcellular localization o f sClC-6 and sClC-7 in their native tissue. Current data suggest that these channels may be in the apical membrane of SRG; I will test this theory using immunochemical staining. Whatever the distribution, the subcellular localization of these channels will yield insight into their physiological function. I will also test the effects on this localization of in vivo and in vitro rectal gland stimuli. Finally, in Specific Aim 3, I will perform coimmunoprecipiation experiments to determine whether the channels form heteromeric complexes, both in vivo and in vitro. Understanding the subunit composition of the functional channel is a critical step in structural analysis. These studies will dramatically improve our understanding of the role played by ClC channels in normal salt-secretion and will establish a paradigm for the eventual development of drugs to modulate these processes.

This was the third year for this project, which is using biochemical and biophysical methods to examine conformational changes related to transport in glutamate transporters. These proteins are of critical importance in the central nervous system, where they play important roles in clearing neurotransmitters from synapses and in shaping the electrical activity of post-synaptic neurons. These transporters have been implicated as playing roles in a variety of diseases, including ALS, Alzheimers disease, and exitotoxicity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model glutamate transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. Last year we discovered that the bacterial glutamate transporters display a chloride transport activity which is stoichiometrically uncoupled from glutamate uptake. This chloride transport activity is similar to one which is important in the mammalian transporters and suggests that the bacterial homologs provide an excellent structural model in which to study the process of chloride transport in these proteins. This year we completed our characterization of this chloride conductance demonstrating that it operates using similar parts of the protein as the eukaryotic glutamate transporters. We also found that, in contrast to the EAATs, no other ions besides Na+, Cl-, and the substrate are involved in the transport cycle. Our studies on conformational changes in these proteins began this year; using limited proteolysis we found evidence of a significant substrate-induced conformational change and performed preliminary experiments using cysteine-scanning mutagenesis and site-directed fluorescence labeling to localize the protein movement.

This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Disorders of lysosome function lead to a variety of diseases including neurological dysfunction (lysosomal storage diseases) and osteopetrosis (overcalcification of bone). Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase. Genetic evidence suggests that this additional ion pathway a ClC-type anion transporter, but functional experiments have not yet demonstrated such a pathway. We identified the ion transport pathways in lysosomes, characterized their properties, and to identified the responsible proteins. Last year we began examining endosomes to determine the effects of Cl transport on their acidification processes and began constructing a ClC-7 knockout mouse to further understand the effects of this transporter. In the past year we have focused our efforts on developing methods to measure pH in lysosomes in living cultured cells to examine the influence of ClC-7 on these organelles. We have also continued to develop our knockout mice. A recent publication suggested that a cation flux may also be important in lysosomal acidification and we are using our methods to test this hypothesis.

This project continued studying conformational changes in ClC-type chloride channel proteins. The ClC family of chloride-conducting ion channels is involved in a host of biological processes;these channels maintain the resting membrane potential in skeletal muscle, modulate excitability in central neurons, and are involved in the homeostasis of pH in a variety of intracellular compartments. Despite their physiological importance, the mechanisms by which these channels function are poorly understood. We are attempting to understand the functional properties of these proteins by examining several family members, including both eukaryotic and prokaryotic homologs. In this project, we are using a combination of methods to analyze the functional mechanisms of these proteins. Previously, we used fluorescence methods to demonstrate the existence a transport-related conformational change in a bacterial ClC antiporter. Currently, in collaboration with Henning Stahlberg in Switzerland, we are forming 2d crystals of this protein under a series of conditions to reveal the structural changes underlying this conformational change. We are currently studying the transport mechanism in two eukaryotic ClC antiporters, ClC-4 and ClC-7. The endosomal ClC, ClC-4, is sent to the plasma membrane when heterologously expressed in Xenopus oocytes allowing analysis using electrophyiological methods. We searched for molecular tools that might be useful in probing the transport process and found that Zn2+ and Cd2+ both inhibit ClC-4 currents. We used site-directed mutagenesis to locate the Zn2+ binding site and found intruiging interactions between Zn2+ binding and the ion transport process. These finding reveal movements in a part of the protein that has not been previously implicated in transport-related conformational changes. We are now following up on these studies to understand the detailed interaction between divalent metal ions and the ClC-4 transporter and to use this interaction as a tool to probe the transport mechanism. Recent reports demonstrate that another mammalian ClC, ClC-7, though usually targeted to lysosomes, can be retargeted to the plasma membrane by mutating a dileucine sorting motif. We have used this method to retarget ClC-7 and are studying its tranport properties using electrophysiology.

This year we expanded this project to include both GltPh, a model for the EAAT family of glutamate transporters and a new protein, vcINDY, a succinate transproter which has been implicated in longevity and obesity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We have continued our work using EPR spectroscopy to monitor conformational changes in GltPh. This work has identified local changes in the protein that may be important for coupling between the driving ion, Na+, and the substrate, aspartate. We are continuing work to identify the nature of this change. We recently reported that a extracellular loop of gltPH must be intact for effective transport. This year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. This year we also found that only the translocation of the substrate-bound form of the protein is affected--the apo, substrate-free transporter is unaffected by 34 loop cleavage. We have performed important controls eliminating alternative explanations for these effects and a paper describing this work is under review. This year we also began work on a new transporter, vcINDY, which is important for longevity in flies and is involved in obesity and insulin resistance in mammals. We performed the first successful functional reconstitution of vcINDY and directly demonstrated that it is a Na+ coupled succinate transporter. We have performed an extensive screen for substrates and identified that the protein carries three Na ions per succinate transpoted, that it is electrogenic (but does not have an uncoupled Cl- conductance) and that it primarily transports the doubly charged form of succinate. We are currently writing this work up for publication.