Ernest M. Wright, PhD DSc - US grants

The goal of this project is to obtain a molecular understanding of solute and water transport across the intestinal epithelium, and to elucidate how these processes are controlled by hormones and toxins. Membrane vesicles isolated from the brush border and basolateral surfaces of epithelial cells will be used to study the mechanisms of solute transport. Transport across the plasma membranes of vesicles will be measured using radioactive tracers and optical probes. Tracers will yield information about diffusive and carrier mediated transport, while optical probes will be used to obtain membrane potentials, ion permeability ratios, and net fluxes. These methods will be used to examine in detail ion conductive pathways (channels), KC1 symport, and C1/OH antiport in basolateral membranes, and Na-cotransport systems (Na-lactate) in brush border membranes. In the long term we propose to study these processes by electrophysiological techniques in planar bilayers ("patch-clamp" preparations). Regulation of ion transport systems in plasma membranes by hormones (e.g., VIP) will also be explored by these techniques, and interactions between hormones, receptors and cyclases will be evaluated by radiation inactivation analysis. This project should provide a clear understanding of ion and solute transport across the epithelial cells of the small intestine in health and disease. In particular, our studies will provide insight into the molecular mechanisms of secretory diarrhea caused by hormones (e.g., VIP) and toxins (e.g., cholera toxin).

The long-term objective is to identify the cellular and molecular mechanisms responsible for cerebrospinal fluid (CSF) secretion and the maintenance of the blood-CSF barrier. In vitro preparations of the choroid plexus will be used to study i) the secretion of the CSF, ii) the transport of solutes (amino acids) across the blood-CSF barrier, iii) the control of these processes by hormones and neurotransmitters. Electrophysiological techniques including, ion-specific microelectrodes and patch-clamp recordings will be utilized to define the transport properties of the intact epithelium and membrane patches. Biochemical and biophysical techniques will be used to identify and characterize specific transport processes in isolated plasma membrane vesicles. In particular, fluorescent probes will be used to label specific membrane proteins, and the functional topology of these proteins will be measured by energy transfer and quenching techniques. During this phase of this long-standing project, two very specific questions will be addressed: the first is to test our hypothesis that brush border HCO-3 channels are involved in the regulation of CSF secretion, and the second is to identify and characterize the Imino carrier in brush border membranes. Understandably, the physiology of the blood-CSF barrier has important implications in the clinical management of the central nervous system in such diverse cases as trauma, hydrocephalus, and organic mental disorders. Our studies will pave the way towards therapeutic management of CNS fluid disorders using pharmacological procedures. In addition, our work has unique relevance to basic concepts of transport in neurons and epithelial cells.

It has long been established that the "active transport" of sugars, amino acids, and other metabolites is due to the presence of Na gradients across plasma membranes and Na-cotransport systems in the membranes. Recently, the intestinal brush border Na/glucosecotransporter has been identified using fluorescent covalent probes bound to the glucose and/or Na-active sites. Our long term goal is to determine the structure of the Na/glucose cotransporter and to correlate this structure with the transport properties of the polypeptide(s). The specific aims are: 1) isolate and purify the functional cotransporter to homogeneity. This will be accomplished by fluorescently labeling the glucose carrier covalently at the glucose and/or Na-binding sites, and following the enrichment of the protein through chromatofocusing and affinity chromatography; 2) determine the structure of the purified carrier (sugar content, amino acid composition, and amino acid sequences of the Na and glucose active sites); 3) determine the molecular topography of the active sites using fluorescent probes (fluorescent quenching, energy transfer, and native trypophan fluorescence); and 4) develop a structural and kinetic model of Na/glucose cotransport. Transport activity in highly purified brush border vesicles, soluble protein, and reconstituted systems will be measured using Na-dependent a) glucose transport, b) phlorizin binding, c) potential changes, and d) quenching of fluorescent probes on the glucose-site. These studies will have general impact on our understanding of the mechanism of sugar, amino acid, metabolic intermediate transport across plasma membranes of the intestine, liver, kidney and brain, and particular relevance to the absorption of sugars by the small intestine.

Glucose-galactose malabsorption (GGM) is an autosomal recessive disease which we have shown to be due to a defect in the intestinal brush border Na+/glucose cotransporter. The goal of this investigation is to identify the mutations in the gene (SGLT1) coding for this membrane transport protein. Our principle interests include the use of these new data to (1) develop diagnostic genetic methods to identify afflicted patients as well as heterozygote carriers, and (2) exploit the experiments of nature represented in deleterious missense mutations to more effectively address the structure/function relationships of the Na+/glucose cotransporter protein. To achieve these ends we propose to (1) identify mutations in GGM-diagnosed patients by examining PCR-amplified DNA fragment for single strand conformational polymorphisms (SSCPs) followed by sequencing the SSCP fragments and (2) map the SGLT1 gene. We will PCR-amplify a) cDNA isolated from intestinal mucosal biopsies, b) cDNA prepared from illegitimately expressed RNA from the gene in lymphocytes and c) genomic DNA coding for SGLT1 exons. Gene mapping will yield sequence data flanking each exon permitting simple, rapid analysis of homo- or heterozygote carriers by PCR followed by SSCP, sequencing, or, where appropriate, restriction analysis. The functional significance of the mutations will be examined using in vitro and in vivo expression systems. In oocytes we will measure the ability of the mutant RNA to express functional Na+/glucose cotransporters in the plasma membrane. After injection of cRNA into oocytes we will measure Na+-dependent sugar transport by tracer by tracer and electrophysiological techniques. in vitro translation experiments will allow us to examine translation, membrane insertion and glycosylation. Quantitative PCR, in situ hybridization and immunocytochemistry will be employed to examine the effect of mutations on transcription, translation, membrane insertion and processing. This study will enable us to develop tools to aid in diagnosis of glucose- galactose malabsorption, to identify carriers of the mutations. The results will also provide unique information about the structure and function of the Na+/glucose cotransporter.

Glucose-Galactose Malabsorption (GGM) is characterized by a neonatal onset of a several diarrhea that results in death unless the offending sugars are removed from the diet. It is caused by a defect in the brush border Na/glucose cotransporter (SGLT1). We have identified the transporter, cloned and mapped the gene, and found the mutations responsible for the disease in 33 patients. The goal now is to understand the molecular and cell biology of GGM. We plan to: continue screening new patients for mutations; determine how the mutations cause the malabsorption; devise new therapies to treat patients; and resolve whether or not carriers of GGM mutations have impaired glucose absorption. New patients will be screened using genomic DNA, PCR-amplification of exons, single strand conformational polymorphism (SSCP) gels, and sequencing. 90 percent of the mutations are in the gene coding region and result in the production of mutant and truncated proteins. Our hypothesis is that the truncated proteins are defective and that mutant proteins are not inserted into the plasma membrane. We will test this hypothesis by expressing mutants in Xenopus laevis oocytes an using electrophysiological, immunological, biochemical and microscopic techniques. Once the reason for impaired sugar transport is determined in the model expression system, we will test this in biopsy samples from the patients. In those cases where mutations cause a defect in trafficking SGLT1 protein between the endoplasmic reticulum and the plasma membrane, we will devise strategies to increase the delivery of SGLT1 to the plasma membrane. Finally, in kindreds where we have identified GGM mutations, we will screen family members for heterozygotes and normal homozygotes. Sugar absorption will be measured using H-breath tests to determine if carriers exhibit any symptoms of malabsorption. This study will enable us to identify and treat patients with sugar malabsorption due to mutations in the SGLT1 gene, and will provide unique information about the synthesis, trafficking and function of the cotransporter.

Co-transporters are an important class of membrane proteins responsible for the accumulation of sugars, amino acids, peptides, neurotransmitters and ions in cells. These proteins are now known to also co-transport water (200-500 molecules per cycle) and small polar molecules such as urea. The energy for the substrate transport comes from the Na+ (or H=) electrochemical potential gradient across the plasma membrane. Our goal is to understand how co-transporters use the free energy released from downhill Na+ transport to drive uphill substrate and water transport. Using the intestinal Na+/glucose co-transporter (SGLT1), and a bacterial homolog (Vibrio SGLT), we plan to identify and determine the architecture of the Na+ and substrate transport pathways, and to determine how Na+ changes the conformation of SGLTs to couple Na+ transport to sugar and water transport. The experimental strategy is to express truncated parts of the co-transporters in Xenopus laevis oocytes and bacteria, and to determine which parts of the protein retain partial reactions, e.g. Na+ uniport, sugar uniport, Na+/sugar co-transport, and water or urea transport. Once parts of the protein are found that exhibit the partial transport reactions, we will use a combination of molecular, biophysical and biochemical techniques to determine their structure and how they interact. Preliminary studies indicate that the C-terminal part of SGLT1 can function as a glucose uniporter, and we have identified several residues in this domain that interact with sugar during Na+/glucose co- transport. The plan is to locate the other residues in the sugar transport pathway, and carry out similar studies on the Na+ transport pathway. These studies will provide a low resolution topological map of the co- transporter and structural information about Na+/sugar/water coupling, and lay the ground work for obtaining higher resolution structures.

The long term goal of this project is to clone, sequence, express and map the distribution of nutrient transporters in the blood-brain and blood- cerebrospinal-fluid barriers. The endothelial cells of the brain capillaries regulate the uptake of nutrients into the brain, and the epithelial cells of the choroid plexus clear the cerebrospinal fluid of many metabolites and drugs. Relatively little is known about the transporters involved in these vital processes. We will clone blood- brain barrier transporters using two different strategies. The first is to determine if the transporters already cloned from other tissues are expressed at the blood-brain-barrier. A candidate clone for brain capillaries is the renal Na+/myo-mositol cotransporter, since isolated brain microvessels transport this important phospholipid precursor, and a candidate for the choroid plexus is the renal Na+/nucleoside cotransporter. Northern and Western blots, in situ hybridization, and immunocytochemical techniques will be used to map the distribution of candidate clones at the blood-brain interface. The second strategy is to use expression cloning techniques to isolate specific brain capillary and choroid plexus transporters, e.g. The L-type amino acid and Na+- dependent proline transporters. The objective is to use the tools of cell and molecular biology to obtain a detailed understanding of nutrient and metabolite transport between the blood and brain.

DESCRIPTION (provided by applicant): Glucose is the primary fuel for the body and adequate supplies must be maintained to support the normal function of organs and tissues. Over the long term an excess of glucose intake results in weight gain and added risk for developing diabetes. Blood glucose is held within narrow limits to balance the supply and demand for energy and a major factor in this control is the transport of excess glucose into muscle and fat for storage. There are two classes of glucose transporters, GLUTs and SGLTs, but research on glucose transport in health and disease has focused on the GLUTs. SGLTs genes are expressed throughout the body indicating a larger role of these transporters in the transport of glucose into cells than anticipated. The goal is to elucidate the importance of SGLTs (sodium/glucose cotransporters) in the human physiology of glucose homeostasis and metabolism using Positron Emission Tomography (PET). The Introduction of the glucose probe 2-FDG and PET has revolutionized studies of glucose uptake and metabolism in organs and tissues in the human body. Clinically this has had an enormous impact in cancer detection, staging and therapy. However, 2-FDG is not a substrate for SGLTs and this has limited our understanding of glucose transport and metabolism in normal cells and tumors. The specific aims of this proposal are to: 1). Design PET probes specific for SGLTs;2). Synthesize [F-18]-labeled probes for SGLTs;and 3). Use PET imaging with these new glucose probes to examine the physiological role of SGLTs in animal and human subjects. The role of SGLT1 and SGLT2 in glucose uptakes will be tested by including in the study patients with mutations in the SGLTs genes, patients with Glucose- Galactose-Malabsorption and Familial Renal Glucosuria. In parallel, we will conduct studies using 2-FDG to determine the relative importance of GLUTs and SGLTs in glucose uptakes in organs of interest. Studies will also be carried out with both SGLT and GLUT probes on available rodents and SGLT and GLUT knockout mice to compliment the studies on patients. Clearly, identification of all the pathways for glucose uptake into cells is required for a full understanding of energy balance in health and disease. This project is designed to test the sorely neglected role of sodium glucose cotransporters in glucose homeostasis.Relevance Statement: This study of designed to determine the role of sodium/glucose cotransporters (SGLTs) in glucose homeostasis. SGLTs are expressed throughout the body but until now their role outside the intestinal kidneys has been neglected. The results of this study may provide new drug targets for diabetes and obesity and develop new PET tracers to detect and stage cancer. PUBLIC HEALTH RELEVANCE: This study is designed to determine the role of sodium/glucose cotransporters (SGLTs) in glucose homeostasis. SGLTs are expressed throughout the body but until now their role outside the intestine and kidneys has been neglected. The results of this study may provide new drug targets for diabetes and obesity and develop new PET tracers to detect and stage cancer.