Hereditary tubular transport disorders comprise a group of diseases that usually present in the neonatal period, lead to profound derangements in the homeostasis of electrolytes, minerals, or organic solutes in the body, and can be associated with significant morbidity. In the past two decades, remarkable progress has been made in our understanding of the molecular pathogenesis of hereditary tubulopathies. Molecular genetics and molecular biology studies have led to the identification of numerous tubular disease-causing mutations, have provided important insight into the defective molecular mechanisms underlying various tubulopathies, and have greatly increased our understanding of the physiology of renal tubular transport.This chapter summarizes the general characteristics of hereditary tubular transport disorders, reviews the molecular pathophysiology and genetic aspects of the diseases, describes the clinical feature of the tubulopathies, and briefly summarizes their therapy. The focus of this chapter is on isolated tubulopathies involving an impairment in a single tubular function, which results from primary gene defects in transporters or channels operating along the renal tubule.Despite the exciting progress made, numerous issues remain unsettled and warrant additional research on the molecular mechanisms and functional defects underlying the impaired transport in various tubulopathies. These studies may significantly improve our understanding of the mechanisms underlying renal salt homeostasis, urinary mineral excretion, and blood pressure regulation in health and disease. The identification of the molecular defects in inherited tubulopathies may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders.

Ongoing research continually uncovers surprises in the roles for membrane-bound proteins including transporters. Classical era studies of amino acid transporters focused on classifications by substrate selectivity, voltage, and ion dependency. Landmark studies by Stevens and colleagues, using intestinal brush-border and basolateral membrane vesicles, identified sodium-dependent transporters including the major neutral amino acid (NBB, now known as B0AT1) and proline selective (Imino) transporters.1 Following the expression cloning of intestinal sodium–glucose cotransporter SGLT1 in 1987,2 sodium-dependent transporters including B0AT1 and Imino (SIT1) were cloned and expressed in Xenopus laevis oocytes and cultured cells.3,4 B0AT1 (SLC6A19) and SIT1 (SLC6A20) are members of the large SLC6 family that includes sodium-dependent neurotransmitters, with a separate clade of nutrient amino acid transporters mainly expressed in the intestine, kidney, and/or brain.4–6 It was discovered that BoAT1 was regulated by tissuespecific apical membrane expression of two members of the Renin Angiotensin System (RAS): collectrin in the kidney; and angiotensin-converting enzyme 2 (ACE2) in the intestine.6 Collectrin is a 50% homologue of ACE2 sharing identity with the non-catalytic, transmembrane helix but lacks the ACE2 carboxypeptidase ectodomain. In the oocyte expression system, collectrin and ACE2 increase trafficking of B0AT1 to the plasma membrane and modulate transporter activity. The effect of intestinal ACE2 on transport is independent of its function as a carboxypeptidase. Much clinical interest in the regulation of B0AT1 in the intestine and kidney stems from mutations that cause Hartnup disease.3,6,7 Considerable biochemical evidence has been gathered showing that B0AT1 and ACE2 are co-expressed in intestinal brushborder membranes, but the nature of the interactions was not known. This changed dramatically in 2020 with the publication of the cryo-EM high resolution structure of B0AT1 complexed with ACE2 expressed and purified from HEK293F cells.8 The complex is assembled as a 2[ACE2: B0AT1] dimer-of-heterodimers (See, Figure 1 in reference 8; and Figure 4 in reference 9). Apart from ACE2’s function as the SARS-CoV-2 receptor, and the importance of co-expressing B0AT1 with ACE2 in the screening development of a COVID-19 mRNA vaccine, the 3D atomic coordinates raised provocative questions about the functional interaction of ACE2 and B0AT1. Such questions include how does ACE2 govern B0AT1 brush border expression and activity. Stevens and colleagues addressed this question directly in intestinal brush borders by deploying the well-established method of radiation inactivation, as reported in the literature to determine the functional unit of various enzymes, receptors, channels, and transporters.9 They assayed sodium-dependent alanine and serine transport in rabbit brush border membrane vesicles before and after exposing the membranes to varying doses of high energy electrons. Then using electron flux targeting theory developed by Ellis Kempner, they estimated the molecular size of the functional unit responsible for neutral amino acid transport activity and used molecular modelling to locate the residues involved in interface contact between subunits. The functional molecular size of the B0AT1: ACE2 complex was 184 kDa—the expected sum of B0AT1 and ACE2 subunits in a heterodimer, which was half the 345 kDa size of the dimerof-heterodimers complex (Figure 1A). Furthermore, their molecular modelling predicted the location, identity, and distance of pairs of resides involved in heterodimer interface contacts: three extracellular pairs between B0AT1 TM7 and the neck of the noncatalytic membrane-anchoring domain of ACE2; and one pair between B0AT1 TM4 and the transmembrane domain of ACE2 (Figure 1A).

SGLTs are sodium glucose transporters found on the luminal membrane of the proximal tubule, where they reabsorb some 180 g (1 mol) of glucose from the glomerular filtrate each day. The natural glucoside phlorizin completely blocks glucose reabsorption. Oral SGLT2 inhibitors are rapidly absorbed into the blood stream, where theyremain in the circulation for hours. On glomerular filtration, they bind specifically to SGLT2 in the luminal membrane of the early proximal tubule to reduce glucose reabsorption by 50%-60%. Because of glucose excretion, these drugs lower plasma glucose and glycosylated hemoglobin levels in patients with type 2 diabetes mellitus. The drugs also protect against heart and renal failure. The aim of this review is to summarize what is known about the physiology of renal SGLTs and the pharmacology of SGLT drugs.

Abstract Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Västerbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog α-methyl-D-glucopyranoside (αMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Västerbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015, and higher than the general European population, 0.000067. This explains the high number of GGM cases in this region of Sweden.

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1–5 and TM 6–10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

The transport of d-glucose, d-galactose, and d-fructose across epithelial cells is mediated by SGLTs, GLUT5, and GLUT2 in the apical and/or basolateral membrane. The SGLTs (sodium–glucose cotransporters) are responsible for active glucose transport, while the GLUTs (facilitative glucose transporters) are responsible for passive glucose and fructose transport. The structure and function of each of these transport proteins are summarized, and then we highlight the similarities and differences between these two classes of membrane transporters. We next discuss their roles in sugar absorption in the intestine and glucose reabsorption from the glomerular filtrate in the kidney. Reference is made to genetic disorders of glucose transport in the intestine, Glucose–Galactose Malabsorption, and in the kidney, Familial Renal Glucosuria, and the Fanconi–Bickel syndrome, and the use of specific SGLT2 inhibitors to treat Type II Diabetes Mellitus.

Model systems ranging from the frog skin to the fish gall bladder have played an indisputable role in advancing our understanding of epithelial physiology. Apart from the curiosity and talents of scientists such as Hans Ussing and Jared Diamond, a critical factor was the simplicity and viability of the epithelia chosen for study. Almost 50 years ago, we chose the frog choroid plexus to unravel the mysteries of cerebrospinal fluid secretion using the then state-of-the art physiological, biochemical and biophysical tools. Here we summarize ion transport across the frog choroidal epithelium and our interpretation of the transport mechanisms involved. The challenge now in the era of functional genomics is to identify the transporters and channels responsible, and to extend our understanding to CSF secretion in man.

George Sachs, Distinguished Professor of Medicine and Physiology at UCLA, passed away peacefully and unexpectedly at home on November 12, 2019. He was a dominant force in gastroenterology for over forty years, and was responsible for the development of PPIs, a therapy that revolutionized management of gastric acid and peptic disorders.1Modlin I.M. George Sachs-“I did it my way”.J Clin Gastroenterol. 2006; 40: 867-869Crossref PubMed Scopus (2) Google Scholar George was born on August 26, 1935 in Vienna, Austria to Arpad and Toni Sachs, both physicians. In those unsettled times, due to the rise of Nazism, his father had the foresight in 1936 to secure a UK visa and a clinical practice in Edinburgh, Scotland. George and his mother joined him in 1939 just prior to the outbreak of the Second World War. George was immediately enrolled in the George Watson College, from which he graduated in 1952 as head of his class (Dux). He read medicine at Edinburgh University, but took time out to obtain a B.Sc. in Biochemistry. An instructor in chemical biology who made a major impact on his subsequent career was Peter Mitchell, Nobel Laureate in 1978 for his far-sighted chemic-osmosis theory to explain ATP synthesis. George completed his medical training in 1960 with honors, and then undertook post-doctoral training at Albert Einstein College and Columbia University, before accepting a faculty position in Physiology and Medicine at the University of Alabama in Birmingham. Under the mentorship of Basil Hirschowitz, Chief of Gastroenterology, George turned his interest to gastric biology. Together they published three dozen papers on the mechanism and regulation of gastric acid secretion. He became increasingly drawn to the involvement of gastric ATPase activity in acid secretion. Using isolated gastric glands, parietal cells and membrane vesicles combined with elegant biochemical and biophysical tools, he determined that a gastric H+ - K+ ATPase was responsible for acid secretion. This proton pump carried out an electroneutral exchange of two protons for two potassium ions for each mole of ATP hydrolyzed. George recognized the limitations of cimetidine (Tagamet; GlaxoSmithKline, Brentford, London, UK) for treatment of acid-related diseases and hypothesized that the gastric ATPase would be a better target for acid inhibition. A chance encounter with the Swedish pharmaceutical company Hassle AB, later Astra and AstraZeneca, led him to develop the first Proton Pump Inhibitor (PPI). The initial compound, a substituted benzimidazole, was a weak base prodrug that required acid activation to function.2Fellenius E. Berglindh T. Sachs G. et al.Substituted benzimidazoles inhibit gastric acid secretion by blocking (H+ + K+)ATPase.Nature. 1981; 290: 159-161Crossref PubMed Scopus (597) Google Scholar George was recruited to UCLA in 1982 as Director of the Center for Ulcer Research and Education (CURE), and he established the Laboratory of Membrane Biology on the VA Greater Los Angeles Healthcare System campus. He held appointments as Distinguished Professor of Medicine and Physiology, and was a Senior Medical Investigator at the VA. He continued his work on H+, K+-ATPase biology, with a shift in focus from molecular mechanisms to secretory physiology.3Sachs G. Shin J.M. Munson K. et al.Gastric acid-dependent diseases: a twentieth-century revolution.Dig Dis Sci. 2014; 59: 1358-1369Crossref PubMed Scopus (19) Google Scholar His work on inhibitors led to FDA approval of the first PPI, Omeprazole (Prilosec; AstraZeneca, Cambridge, UK) in 1989. This drug had efficacy in acid inhibition to a degree not seen previously and revolutionized the treatment of gastric ulcers and GERD. He received numerous awards for his work on acid secretion and acid inhibition, including The Janssen Award for Special Achievement in Gastroenterology in 1998 and The Canadian Gairdner International Award in 2004. George’s interest in Helicobacter pylori naturally evolved from his background in gastric physiology and acid secretion. He dove into the world of microbiology with the mentality and toolkit of a physiologist, and his laboratory made significant contributions to our understanding of how this bacterium is able to survive in the stomach. He recognized the bacteria as a pathogen and the problems with the complex and multiple antibiotic treatment regimens in an era of rapidly emerging antibiotic resistance. His goal was to better understand the gastric biology of the bacteria, in order to discover gastric colonization mechanisms as potential targets to optimize treatment. His laboratory proved that the bacteria is a neutralophile and he adapted the term “acid acclimation” to explain periplasmic buffering as a means to colonize the acidic gastric surface rather than simply transit the stomach. He was convinced early on that the only membrane protein in the critical urease operon had to be a urea channel, and his laboratory demonstrated the proton gated channel physiology of UreI using oocyte injections, work that was published in Science.4Weeks D.L. Eskandari S. Scott D.R. et al.A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization.Science. 2000; 287: 482-485Crossref PubMed Scopus (379) Google Scholar Following this landmark discovery, his group was able to crystalize the channel and his recent research has been dedicated to unlocking the signaling pathways that work in concert with UreI to facilitate gastric colonization. In parallel, he hoped to develop mechanisms to optimize treatment through use of stronger acid suppression, which would lead to bacterial division and improved antibiotic efficacy. George has been involved in the development of newer acid suppressive medications, including the potassium competitive acid blockers and pro-PPIs, with a persistent desire to both improve treatment of acid-related disease and facilitate eradication of H pylori. Over his long career, he published over 600 papers and reviews, several books, served on editorial boards and review committees at the NIH and VA, and trained numerous students, post-doctoral fellows and visiting faculty, many of whom now hold leadership positions in academia and industry. His father Arpad played a dominant role in his education and intellectual development by encouraging him as a child to read five books a week, and he remained an avid reader throughout his life. He was particularly drawn to 20th century history, was a staunch devote of Winston Churchill, and was fascinated by his relationship to Franklin Roosevelt. George has touched the lives of many colleagues, mentees, and friends in a manner that will not be forgotten, and the clinical impact of his work is staggering. Most significantly, George was devoted to his beloved wife, Joyce, who he met in New York in 1963, their four children, Stephen Bennett Sachs (Margo Francis), Andrew Adam Sachs (Elizabeth Evans Sachs), Paula Sachs Grayson (Paul Grayson), Lara Day Sachs-Fishman (David Fishman), and six grandchildren, Oscan Hokin Sachs, Lucas Francis Sachs, Nicholas Arpad Grayson, Natalie Eve Grayson, Oliva Belle Fishman, and Teala Fishman.

A novel glucose transporter, the sodium glucose cotransporter 2 (SGLT2), has been demonstrated to contribute to the demand for glucose by pancreatic and prostate tumors, and its functional activity has been imaged using a SGLT specific PET imaging probe, α-methyl-4-[F-18]fluoro-4-deoxy- d -glucopyaranoside (Me-4FDG). In this study, Me-4FDG PET was extended to evaluate patients with high-grade astrocytic tumors. Me-4FDG PET scans were performed in four patients diagnosed with WHO Grade III or IV astrocytomas and control subjects, and compared with 2-deoxy-2-[F-18]fluoro- d -glucose (2-FDG) PET and magnetic resonance imaging (MRI) of the same subjects. Immunocytochemistry was carried out on Grade IV astrocytomas to determine the cellular location of SGLT proteins within the tumors. Me-4FDG retention was pronounced in astrocytomas in dramatic contrast to the lack of uptake into the normal brain, resulting in a high signal-to-noise ratio. Macroscopically, the distribution of Me-4FDG within the tumors overlapped with that of 2-FDG uptake and tumor definition using contrast-enhanced MRI images. Microscopically, the SGLT2 protein was found to be expressed in neoplastic glioblastoma cells and endothelial cells of the proliferating microvasculature. This preliminary study shows that Me-4FDG is a highly sensitive probe for visualization of high-grade astrocytomas by PET. The distribution of Me-4FDG within tumors overlapped that for 2-FDG, but the absence of background brain Me-4FDG resulted in superior imaging sensitivity. Furthermore, the presence of SGLT2 protein in astrocytoma cells and the proliferating microvasculature may offer a novel therapy using the SGLT2 inhibitors already approved by the FDA to treat type 2 diabetes mellitus.

Key Points The goal was to determine the importance of the sodium–glucose cotransporter SGLT1 and the glucose uniporter GLUT2 in intestinal glucose absorption during oral glucose tolerance tests (OGTTs) in mice. Glucose absorption was determined in mice using positron emission tomography and three non‐metabolizable glucose probes: one specific for SGLTs, one specific for GLUTs, and one a substrate for both SGLTs and GLUTs. Absorption was determined in wild‐type, Sglt1−/− and Glut2−/− mice. Gastric emptying was a rate‐limiting step in absorption. SGLT1, but not GLUT2, was important in fast glucose absorption. In the absence of SGLT1 or GLUT2, the oral glucose load delivered to the small intestine was slowly absorbed. Oral phlorizin only inhibited the fast component of glucose absorption, but it contributed to decreasing blood glucose levels by inhibiting renal reabsorption. AbstractThe current model of intestinal absorption is that SGLT1 is responsible for transport of glucose from the lumen into enterocytes across the brush border membrane, and GLUT2 for the downhill transport from the epithelium into blood across the basolateral membrane. Nevertheless, questions remain about the importance of these transporters in vivo. To address these questions, we have developed a non‐invasive imaging method, positron emission tomography (PET), to monitor intestinal absorption of three non‐metabolized glucose tracers during standard oral glucose tolerance tests (OGTTs) in mice. One tracer is specific for SGLTs (α‐methyl‐4‐[18F]fluoro‐4‐deoxy‐d‐glucopyranoside; Me‐4FDG), one is specific for GLUTs (2‐deoxy‐2‐[18F]fluoro‐d‐glucose; 2‐FDG), and one is a substrate for both SGLTs and GLUTs (4‐deoxy‐4‐[18F]fluoro‐d‐glucose; 4‐FDG). OGTTs were conducted on adult wild‐type, Sglt1−/− and Glut2−/− mice. In conscious mice, OGTTs resulted in the predictable increase in blood glucose that was blocked by phlorizin in both wild‐type and Glut2−/− animals. The blood activity of both Me‐4FDG and 4‐FDG, but not 2‐FDG, accompanied the changes in glucose concentration. PET imaging during OGTTs further shows that: (i) intestinal absorption of the glucose load depends on gastric emptying; (ii) SGLT1 is important for the fast absorption; (iii) GLUT2 is not important in absorption; and (iv) oral phlorizin reduces absorption by SGLT1, but is absorbed and blocks glucose reabsorption in the kidney. We conclude that in standard OGTTs in mice, SGLT1 is essential in fast absorption, GLUT2 does not play a significant role, and in the absence of SGLT1 the total load of glucose is slowly absorbed.

The concentration of glucose in plasma is held within narrow limits (4–10 mmol/l), primarily to ensure fuel supply to the brain. Kidneys play a role in glucose homeostasis in the body by ensuring that glucose is not lost in the urine. Three membrane proteins are responsible for glucose reabsorption from the glomerular filtrate in the proximal tubule: sodium−glucose cotransporters SGLT1 and SGLT2, in the apical membrane, and GLUT2, a uniporter in the basolateral membrane. ‘Knockout’ of these transporters in mice and men results in the excretion of filtered glucose in the urine. In humans, intravenous injection of the plant glucoside phlorizin also results in excretion of the full filtered glucose load. This outcome and the finding that, in an animal model, phlorizin reversed the symptoms of diabetes, has stimulated the development and successful introduction of SGLT2 inhibitors, gliflozins, in the treatment of type 2 diabetes mellitus. Here we summarise the current state of our knowledge about the physiology of renal glucose handling and provide background to the development of SGLT2 inhibitors for type 2 diabetes treatment.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

It has been 30 years since the intestinal sodium glucose cotransporter SGLT1 was cloned, and, in the intervening years, there have been many advances that have influenced physiology and medicine. Among the first was that SGLT1 is the founding member of the human gene family SLC5, containing 11 diverse transporters and a glucose sensor. Equally surprising was that SGLTs are members of a structural family of cotransporters and exchangers in different gene families. This led to the conclusion that these proteins operate by a mechanism where transport involves the opening and closing of external and internal gates. The mechanism is shared by a wide variety of transporters in different structural families, e.g., the human facilitated glucose transporters (SLC2) in the huge major facilitator superfamily (MFS). Not surprising is the finding that mutations in Sglt genes cause the rare diseases glucose-galactose-malabsorption (GGM) and familial renal glucosuria (FRG). However, it was not envisaged that SGLT inhibitors would be used to treat diabetes mellitus, and these drugs may be able to treat cancer. Finally, in 2017, we have just learned that SGLT1 may be required to resist infection and to avoid recurrent pregnancy loss.

Sodium-dependent glucose transporters (SGLTs) are members of the large solute carrier (SLC) family of proteins that exploit the sodium ion concentration gradient to transport a myriad of small molecules across the plasma membrane. In humans, there are six SGLT subtypes labeled 1-6 that are expressed widely in the small intestine, kidney, lung, muscle, and brain. Due to their role in sugar reabsorption, SGLTs are currently exploited as drug targets for the treatment of type 2 diabetes, especially hSGLT2, which is responsible for 98% of glucose reabsorption in the kidneys. Current inhibitors are chemical derivatives of the naturally occurring small molecule phlorizin, which is expressed in the bark of fruit trees, such as apple and pear. The structural basis of binding is not known, in part, because high-resolution structures of mammalian SGLTs do not exist. However, the inward-facing structure of the bacterial homologue from vibrio parahaemolyticus (vSGLT) has been solved both in apo and in complex with galactose, and our collaborators recently solved the outward-facing structure of a closely related homologue (unpublished). Here we combine homology modeling, virtual screening techniques and molecular dynamics simulations to achieve two goals: 1) model the outward-facing state of SGLTs, and 2) predict the binding mode of phlorizin and its derivatives hSGLT1 and 2. As a result, the spectroscopic data (double electron-electron resonance) probing outward facing state of SGLTs validate our homology model and mutagenesis studies testing binding to hSGLT1 and 2 are in agreement with our predicted binding modes.

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

The current report provides a detailed analysis of the changes in the first two components of the auditory evoked potential (AEP) that accompany associative learning. AEPs were recorded from the primary auditory cortex before and after training sessions. Experimental subjects underwent one (n=5) or two (n=7) days of conditioning in which a tone, serving as a conditioned stimulus (CS), was paired with mild foot shock. Control subjects received one (n=5) or two (n=7) days of exposure to the same stimuli delivered randomly. Only animals receiving paired CS-US training developed a conditioned tachycardia response to the tone. Our analyses demonstrated that both early components of the AEP recorded from the granular layer of the cortex undergo CS-specific associative changes: (1) the first, negative component (occurring ∼21ms following tone onset) was significantly augmented after one and two days of training while maintaining its latency, and (2) the second, positive component (occurring ∼50ms following tone onset) was augmented after two days of training, and showed a significant reduction in latency after one and two days of training. We view these changes as evidence of increased cortical synchronization, thereby lending new insight into the temporal dynamics of neural network activity related to auditory learning.

Sodium dependent glucose transporters (SGLTs) are members of the large solute carrier (SLC) family of proteins that exploit the sodium ion concentration gradient to transport glucose across the plasma membrane. In humans, there are six subtypes (SGLT1-6) expressed widely in the small intestine, kidney, lung, muscle and brain. Due to their role in sugar reabsorption, SGLTs are currently exploited as a drug target for the treatment of type 2 diabetes, especially hSGLT2, which is responsible for 98% of glucose reabsorption in the kidneys. Current inhibitors are chemical derivatives of the naturally occurring small molecule phlorizin, which is expressed in the bark of fruit trees, such as apple and pear. The structural basis of binding is not known, in part because high-resolution structures of mammalian SGLTs do not exist. However, the structure of the bacterial homologue from vibrio parahaemolyticus (vSGLT) has been solved in complex with galactose opening up the possibility for structure based drug design. vSGLT is in an inward-facing conformation poised to deliver the sugar to the cytoplasmic space. Here, we combine homology modeling, pharmacophore searching, virtual screening techniques and molecular dynamics simulations to achieve two goals: 1) determine how known inhibitors bind to hSGLT2 to better understand the mode of action of these drugs, and 2) identify binders to vSGLT that can be used to trap the transporter in different states for use in biophysical experiments.

Key points Glucose transporters are central players in glucose homeostasis. There are two major classes of glucose transporters in the body, the passive facilitative glucose transporters (GLUTs) and the secondary active sodium‐coupled glucose transporters (SGLTs). In the present study, we report the use of a non‐invasive imaging technique, positron emission tomography, in mice aiming to evaluate the role of GLUTs and SGLTs in controlling glucose distribution and utilization. We show that GLUTs are most significant for glucose uptake into the brain and liver, whereas SGLTs are important in glucose recovery in the kidney. This work provides further support for the use of SGLT imaging in the investigation of the role of SGLT transporters in human physiology and diseases such as diabetes and cancer. AbstractThe importance of sodium‐coupled glucose transporters (SGLTs) and facilitative glucose transporters (GLUTs) in glucose homeostasis was studied in mice using fluorine‐18 labelled glucose molecular imaging probes and non‐invasive positron emission tomography (PET) imaging. The probes were: α‐methyl‐4‐[F‐18]‐fluoro‐4‐deoxy‐d‐glucopyranoside (Me‐4FDG), a substrate for SGLTs; 4‐deoxy‐4‐[F‐18]‐fluoro‐d‐glucose (4‐FDG), a substrate for SGLTs and GLUTs; and 2‐deoxy‐2‐[F‐18]‐fluoro‐d–glucose (2‐FDG), a substrate for GLUTs. These radiolabelled imaging probes were injected i.v. into wild‐type, Sglt1–/–, Sglt2–/– and Glut2–/– mice and their dynamic whole‐body distribution was determined using microPET. The distribution of 2‐FDG was similar to that reported earlier (i.e. it accumulated in the brain, heart, liver and kidney, and was excreted into the urinary bladder). There was little change in the distribution of 2‐FDG in Glut2–/– mice, apart from a reduction in the rate of uptake into liver. The major differences between Me‐4FDG and 2‐FDG were that Me‐4FDG did not enter the brain and was not excreted into the urinary bladder. There was urinary excretion of Me‐4FDG in Sglt1–/– and Sglt2–/– mice. However, Me‐4FDG was not reabsorbed in the kidney in Glut2–/– mice. There were no differences in Me‐4FDG uptake into the heart of wild‐type, Sglt1–/– and Sglt2–/– mice. We conclude that GLUT2 is important in glucose liver transport and reabsorption of glucose in the kidney along with SGLT2 and SGLT1. Complete reabsorption of Me‐4FDG from the glomerular filtrate in wild‐type mice and the absence of reabsorption in the kidney in Glut2–/– mice confirm the importance of GLUT2 in glucose absorption across the proximal tubule.