Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Neurobiology; Pathophysiology; Gene Expression; Signal Transduction
Neuronal firing patterns are highly diverse because neurons regulate a wide variety of different behaviors and physiological functions including cognition and memory. Whether a neuron exhibits regular spiking, burst firing, adaptation or high frequency firing will largely be determined by which specific ion channel genes a neuron chooses to express. I am interested in a class of potassium channels that are sensitive to intracellular sodium. There are two members in this family, known as Slack and Slick, and both channel subunits are expressed in many different types of neurons. I am particularly interested in how these channels contribute to the firing patterns of pain-sensing neurons and neurons of the cerebral cortex. Understanding when, where and how these channels are working should provide important information on sensory and cortical processing and will provide insights on nociception, psychiatric disorders such as schizophrenia and bipolar disorder and neurological diseases such as epilepsy.
Gastroenterology; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular and Cellular Biology
Research in my laboratory concerns neurotransmitter and hormone-mediated anion secretion by gastrointestinal secretory tissues like intestinal crypts and liver ducts. I am determining the mechanisms that regulate the basolateral membrane K+ channel, KCNQ1, in anion secretion because these channels play a critical role in secretion by maintaining membrane potential as a driving force for anion exit across the apical cell membrane. Characterization of KCNQ1 K+ channels will help us to understand and remedy defects in anion secretion, especially in diseases like cystic fibrosis. I use electrophysiological techniques, including Ussing chamber, patch-clamp, and Fura-2 fluorescence techniques. I am also studying the mechanisms by which K+ channel antagonists (e.g., Zn2+) block KCNQ1 channels so that anti-secretory, anti-diarrheal drugs can be developed. I have past experience determining the mechanisms by which neurotransmitters regulate K+ channels via inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release transduction pathways. I am also collaborating with Dr. John Crane to define the mechanisms by which Zn2+ inhibits the effects of Enteropathogenic E. coli (EPEC) on epithelial cell death and EPEC-stimulated phosphorylation and activation of the CFTR Cl- channel. There is considerable controversy concerning the role and basis of GI disorders associated with autism. In collaboration with Drs. Randall Rasmusson and Glenna Bett, I am investigating the mechanistic link between autism susceptibility and abnormal GI function. I propose that disorders of cellular Ca2+ homeostasis play a key role in the GI disorders of autism. Using mouse models derived from Cav1.2 Ca2+ channel defect that produces the human disorder, Timothy Syndrome, I am characterizing muscle tension and electrophysiological properties of the Ca2+ channel in intestinal smooth muscle. This information will lead to new approaches to identify therapeutic targets and treatments for autistic spectrum GI disorders and symptoms.
Bioinformatics; Genomics and proteomics; Immunology; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Neurobiology; Gene Expression
The current focus of my lab is on iron metabolism in animals and humans. From the practical viewpoint, iron is an important nutrient, but its ability to act in the ferrous and ferric state also makes it toxic. Thus, iron deficiency is the most frequent disorder in the world and hereditary hemochromatosis (HH) is the most common Mendelian disorder in the United States. Our research is related to erythroid differentiation on the fundamental level and to genetic and acquired diseases on the applied level, with four long-term themes: 1.) analysis of the molecular basis of differential gene expression among tissues and during development, with hemoglobin synthesis and red blood cell (RBC) development as models; 2.) application of molecular and genetic advances to inherited diseases; 3.) iron metabolism; 4.) study of gene variation in populations and divergence of gene loci during evolution. New vistas have opened recently for the anemia of chronic diseases, leading us to re-exam how microbes and their human hosts fight for iron. We approach these issues by working on rodent models like the Belgrade rat, plus a series of genetically engineered mice. The rat has a hypochromic, microcytic anemia inherited as an autosomal recessive. The defect is in an iron transporter called DMT1 (or slc11a2, previously called Nramp2 or DCT1) that is responsible for iron uptake by enterocytes and is also responsible for iron exiting endosomes in the transferrin cycle. The rats appear to have a severe iron deficiency, and although dietary iron and iron injection increase the number of RBCs, they do not restore the RBCs nor the rat itself to a normal phenotype. Recent discoveries show that DMT1 is ubiquitous and responsible for transport of other metals such as Mn and Ni. It occurs in the kidney, brain and lung at even higher levels than in the GI tract or in erythroid cells. It also has multiple isoforms, and we have cloned them and developed cell lines that express high levels of particular isoforms. We have specific antibodies to the isoforms and assays for each of the mRNAs too. Future projects in my lab will continue to address whether DMT1 is dysregulated in HH. We will also tackle how DMT1 functions in neurons, pneumocytes and other tissues, look at isoforms of DMT1 under circumstances where we suspect that they must have different functions from one another, and examine DMT1’s relevance to iron metabolism and human disease. Because we cloned the gene and identified the mutation, a number of molecular and cellular approaches can now be used. As evidence indicates that metal ion homeostasis fails in Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, research on DMT1 has opened new vistas for these disorders.
Inherited Metabolic Disorders; Membrane Transport (Ion Transport); Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure; Transgenic organisms; Vision science
Most physiological processes and numerous disease states influence or are influenced by pH. Even relatively small deviations in whole body pH can have devastating consequences for our health. Our bodies are subject to a constant challenge from dietary and metabolic acids, thus it is critical for the body to have mechanisms that tightly regulate pH. Blood plasma pH is maintained at a value close to 7.4, predominantly thanks to the buffering action of 24 mM bicarbonate (HCO3-). HCO3- neutralizes acid, generating carbon dioxide and water (HCO3- + H+ to CO2 + H2O), preventing lethal acidosis. I study the SLC4 family of membrane proteins that move acid/base equivalents across cell membranes. Notable members include  the Na/2HCO3 cotransporter NBCe1-A that reclaims HCO3- from filtered blood plasma in kidney tubules (preventing loss of vital plasma HCO3- to the urine),  NBCe1-B that promotes fluid removal from the corneal stroma (preventing corneal edema and vision loss),  the Cl-HCO3 exchanger AE1 that promotes O2-CO2 exchange in red blood cells, and  SLC4A11 that conducts H+ and promotes corneal clarity. Dysfunction of SLC4 family members is associated with renal tubular acidosis, blindness, cancer, deafness, epilepsy, and hypertension.
Neurodegenerative disorders; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Neurobiology; Neuropharmacology; Protein Function and Structure; Signal Transduction
We investigate the activation mechanisms of fast neurotransmitter receptors. We seek to define the activation pathway, modulatory mechanisms and structure-function relationships of the N-methyl-D-aspartate (NMDA) receptor to better understand the roles played by this protein in the brain. NMDA receptors are the most abundant glutamate-stimulated, Ca2+-conducting ion channels in brain and spinal cord. They are the predominant molecular devices for controlling synaptic development and plasticity and govern memory and learning processes. Understanding the mechanisms that control their activity may lead to more effective strategies to treat neuropathies including stroke, neurodegenerative conditions, chronic pain and addiction as well as mental disorders such as schizophrenia and epilepsy.
Pulmonary & Critical Care Medicine; Immunology; Membrane Transport (Ion Transport)
My clinical responsibilities include working as one of five Intensive Care Unit physicians at the Buffalo VA Medical Center (Buffalo VAMC). I also have a pulmonary medicine outpatient clinic at the UBMD multispecialty practice in Williamsville. My outpatient clinic accepts all patients, with a focus on interstitial lung disease (associated with collagen vascular disorders such as systemic lupus erythematousus [SLE]), scleroderma, rheumatoid arthritis, Sarcoidosis, the idiopathic pulmonary pneumonitis syndromes (IPF/UIP) and advanced chronic obstructive pulmonary disease (COPD). We are one of the founding sites of the Greater New York Sarcoidosis Consortium and work closely with our neurology colleagues in managing patients with respiratory complications of neuromuscular disorders such as ALS, Duchenne‘s Muscular Dystrophy and others. I focus my research on understanding the impaired immune response to infection that occurs in patients with COPD. This impaired immune response leads to more frequent disease exacerbations and more rapid disease progression. My lab has optimized a noninvasive macrophage model (monocyte-derived macrophage) to study how to restore the immune function of alveolar macrophages in patients with COPD. Using this model, we study cell surface receptor expression, cytokine responses and intracellular signaling using flow cytometry, bead arrays and molecular biology techniques. My research team collaborates with Dr. Sanjay Sethi and other researchers in the areas of pulmonary, sleep medicine, critical care, infectious disease and microbiology. Undergraduates, medical students, residents and fellows are welcome in my lab. I am committed to teaching the next generation of physicians. I teach medical students in years two through four; in addition, I teach the residents and the pulmonary and critical care medicine fellowship trainees who rotate with me on the inpatient and outpatient services. I also teach the internal medicine and anesthesia residents and provide grand rounds lectures for the Buffalo VAMC, Buffalo General Medical Center and Erie County Medical Center. I serve on the Resident Clinical Competency Committee and as a formal resident advisor, shepherding my mentees through residency and helping them launch their professional careers. My door is always open to any trainee at any level.
Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Molecular and Cellular Biology; Protein Folding; Protein Function and Structure; Signal Transduction
Work in my lab seeks to elucidate the transduction mechanisms of ion channels involved in thermal sensation and pain, such as the heat-activated vanilloid receptors (TRPV1-4) and the cold-activated TRPM8 – the so-called thermal TRP channels. Expressed in peripheral afferent nerve endings, these channels function as an array of thermometers for sensing ambient temperature from noxious cold to noxious hot. While all proteins are thermally sensitive, thermal TRP channels are gated by temperature and possess unprecedentedly high temperature dependence. But the mechanisms of their temperature gating has remained mysterious, in contrast to our abundant knowledge on other types of ion channel gating (e.g. voltage or ligand-driven). Thermal TRP channels are also distinct for their polymodal responsiveness. TRPV1, for example, is responsive to heat, voltage, pH, capsaicin (i.e. the hot ingredient of chili peppers) among many other irritant compounds. The channels are thus informative for deciphering how biological proteins achieve multitasking. Thermal TRP channels also have receptor-like roles in mediating intracellular signaling. The calcium influx through the channels has potentially a broad spectrum of functional consequences, one of which is the desensitization of the channels themselves, a phenomenon that is believed to underlie peripheral analgesics. Our research is centered on problems like these, and we approach them by a combination of techniques such as recombinant mutagenesis, patch-clamp recording, fluorescence measurements, quantitative modeling, etc, which together allow us to draw insights into functions of the channels at mechanistic levels. Complementing our experimental studies, we are also interested in development of methodology to ever extend experimental resolutions. For example, to time-resolve temperature-dependent activation of thermal TRP channels, we have developed a laser diode-based temperature clamp apparatus, which achieves for the first time a submillisecond resolution (>105 oC/s) while capable of clamping temperature constant. For the past decade we have also been developing sophisticated algorithms for statistical analysis of single-molecule measurements such as single-channel patch-clamp recordings, which can help unravel the richness of data pertaining to molecular mechanisms at high resolutions. Together, these approaches provide us with unique abilities for in-depth studies of structure-mechanisms of ion channels.
Cardiopulmonary physiology; Cytoskeleton and cell motility; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Molecular Basis of Disease; Signal Transduction
My research interests center on mechanical and electrical biophysics, from molecules to organs, and the development of new tools. And, in recent years I worked in transitional science; bringing basic science to the clinic and to industry. My basic research interests are on cell mechanics and the mechanisms by which mechanical forces are transduced into messages such as voltage and chemicals such as ATP and Ca2+. I discovered mechanosensitive ion channels in 1983. My methodology has included patch clamp, high resolution bright field light microscopy, low light fluorescence microscopy, high speed digital imaging, TIRF, digital image analysis, high voltage EM with tomography, Atomic Force Microscopy, molecular biology, natural product and recombinant protein biochemistry, NMR and microfabrication and microfluidics. We discovered the only known specific inhibitor of mechanosensitive ion channels and uncovered its remarkable mode action by using a combination of electrophysiology and chiral chemistry. We have demonstrated potential clinical applications of the peptide for cardiac arrhythmias, oncology, muscular dystrophy, and incontinence. We have developed many scientific tools. Recently we developed a sensor chip to measure cell volume in real time, and that is now entering production with Reichert Instruments of Buffalo. We also have an Small Business Innovation Research contract to develop a microfluidic, bipolar, temperature jump chip with ALA Scientific and developed a microfabricated Atomic Force Microscopy probe that is an order of magnitude faster and more stable than any commercial probes. We have made probe operable with two independent degrees of freedom on a standard Atomic Force Microscopy. This permits us to remove all drift and coherent noise by using one axis to measure the substrate position and the other the sample position. These probes are being produced by a new company in Buffalo, kBtwist. We have used the Atomic Force Microscope combined with electrophysiology to study the dynamics of single voltage dependent ion channels. This technique provides a resolution of >0.01nm in a kHz bandwidth. I have developed other hardware including the first automated microelectrode puller, a micron sized thermometer and heater and a high speed pressure servo. Some of these devices have been patented by the University of Buffalo and some are in current production. To analyze the reaction kinetics of single molecules, we developed and made publicly available (www.qub.buffalo.edu) a complete software package for Windows that does data acquisition and Markov likelihood analysis. The development was funded by the National Science Foundation, National Institutes of Health and Keck over the last fifteen years, and has been applied to ion channels, molecular motors and the even the sleep patterns of mice. We have taught at UB hands-on course to use the software, and the course was attended by an international group of academic scientists and students, government and industry.
Behavioral pharmacology; Cardiac pharmacology; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Neurobiology; Neuropharmacology; Signal Transduction; Transgenic organisms
With over 400 genes coding for them in humans, ion channels play a significant role in most physiological functions. Drug-induced channel dysfunction often leads to a variety of disorders and results in significant incidence of serious injury and death. We investigate molecular mechanisms underlying neurodegenerative disorders and cardiac arrhythmias induced by ion channel dysfunction arising from genetic factors and/or drug interactions. The tools used for these investigations include genetic, electrophysiologic, pharmacologic, molecular and cell culturing methods. Preparations used for experiments include Drosophila as a genetic model system, and human cell lines expressing human ion channels that play an important role in critical-to-life functions including cardiac rhythm, respiration and the central nervous system.
Cell growth, differentiation and development; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Signal Transduction; Inherited Metabolic Disorders; RNA
Regulation of Kidney Epithelial Cell Growth, Transport and Differentiation Our laboratory is investigating the molecular mechanisms by which hormones, growth factors and extracellular matrix proteins regulate kidney tubule epithelial cell growth and functional differentiation in vitro. An established canine kidney epithelial cell line, MDCK, and isolated "mutants" are currently being utilized to examine the actions of growth regulatory on the expression of several proteins including the Na+, K+-ATPase and laminin, a glycoprotein in the extracellular matrix. The effects of novel growth regulatory factors on the expression of proteins involved in gluconeogenesis, membrane transport, renal disease and growth control in primary renal cell cultures are being examined. Primary kidney epithelial cells differentiate into nephrons in a reconstituted extracellular matrix proteins is the subject of study.
Cytoskeleton and cell motility; Genomics and proteomics; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Pathophysiology; Regulation of metabolism
1. Mechanism and regulation of gastric acid secretion: Regulation of gastric acid secretion is the major treatment of many GI diseases including GERD, gastric, duodenal and esophageal ulcers. The spending in treating these conditions is substantial. The gastric parietal cell, lining the lumen of the stomach, is responsible for the secretion of isotonic HCl (0.15M) into stomach. One ATP is consumed for every proton secreted into the stomach lumen and a lot of proton pump (H,K-ATPase, the alpha and beta subunits of this enzyme were discovered in 1967(1) and 1990(2)) is required for this job. To accommodate these many proton pumps, the apical plasma membrane, in the resting state, is expanded in the form of numerous invaginations which express relatively short microvilli, and a large compartment of cytoplasmic membranes, commonly called tubulovesicles, fully loaded with proton pumps. Upon stimulation by hismatine initiated PKA signaling, these tubulovesicles traffic to and fuse with apical membrane, forming densely packed microvilli comparable to those found on the brush border membrane of small intestine. This intracellular trafficking and fusion events bring proton pumps to their post for active acid secretion. In time, these proton pumps are brought back into the cytoplasm (by way of endocytosis) for a reliable mechanism to turn off acid secretion. Although the membrane recycling theory was raised a long time ago(3), there are still many major gaps in the understanding of the mechanism for the regulation of acid secretion, which are the research interests of our laboratory. Techniques employed include isolation and primary culture of gastric parietal cells, measurement of acid secretion, fractionation of different membranes by differential and gradient centrifugation. 2. Using gastric parietal cell model to study general cell biological questions: how membrane trafficking is regulated by small G-proteins, how filamentous actin supports the dynamic change of microvilli on apical membrane. The parietal cell has a remarkably large volume of intracellular membrane trafficking adapted to the elegant mechanism for the regulation of acid secretion. This means that this cell is abundant in those protein machineries required for membrane trafficking and fusion, exocytosis and endocytosis. For instance, no other cell type expresses the amount of syntaxin3 found in parietal cell. Therefore, parietal cell is the top choice for elucidating many of the core questions in cell biology. Techniques used to attack these questions include immunoabsorption, differential ultra-centrifugation, IMAC, 2D-electrophoresis, LC-MSMS, and confocal microscopy. 3. Pathogenesis of Nonalcoholic Steatohepatitis (NASH) - NASH research is funded by the Peter and Tommy Fund. NASH is a disease of the liver that is associated with obesity and adult onset, or type II, diabetes. NASH is not a benign disease. Many people with NASH have a shorter life expectancy than those who no not have NASH. NASH is associated with cirrhosis and is the third most common reason for liver transplantation in adults. No one knows what causes NASH, but it is known that in obese people there is increased fat in the liver. In addition to fat, cells that cause inflammation are found in the liver in patients with NASH. It is thought that these inflammatory cells may cause liver damage that results in fibrosis, cirrhosis and ultimately liver failure. The purpose of this research is to understand the relationship between obesity and the molecular factors that control inflammation so the interaction of the two can be better understood and treatments developed. NASH and alcoholic steatohepatitis share many histological features. Both NASH and alcoholic steatohepatitis patients exhibit macrovesicular and microvesicular fat in hepatocytes. The number and size of Mallory bodies, and the pattern of pericellular fibrosis are also indistinguishable between two disease groups. Previous studies suggested that intestinal bacteria produced more alcohol in obese mice than lean animals. Therefore, we hypothesized and provided the first molecular evidence that alcohol metabolism contributes to the pathogenesis of NASH (Baker et al, 2010). Fatty liver is a prerequisite for the development of NASH. The homeostasis of hepatic lipid depends on the dynamic balance of multiple metabolic pathways. Previous studies focusing on individual pathway or enzyme drew conflicting conclusions on the molecular mechanism for the accumulation of lipid in hepatocytes. With a high through-put technique, we compared all the major pathways in parallel. We are expecting to publish the exciting results in the near future. Oxidative stress is believed to be a major factor mediating the transition from simple steatosis to NASH. The prevention or mitigation of oxidative stress in patients with simple steatosis could prevent NASH. Our current research examines two facets of this problem: 1) what are the molecular mechanisms causing oxidative stress; 2) what are the molecular mechanisms that our body take to fight oxidative stress. Many novel findings have been observed in the lab and we are in the process of confirming these observations. 4. Pathogenesis of Inflammatory Bowel Diseases (IBD): The etiology of IBD is unknown, but a body of evidence from clinical and experimental observation indicates a role for intestinal microflora in the pathogenesis of this disease. An increasing number of both clinical and laboratory-derived observations support the importance of luminal components in driving the inflammatory response in Crohn‘s disease. Members of the Toll-like receptor family are key regulators of both innate and adaptive immune responses. These receptors bind molecular structures that are expressed by microbes but are not expressed by the human host. Activation of these receptors initiates an inflammatory cascade that attempts to clear the offending pathogen and set in motion a specific adaptive immune response. Defects in sensing of pathogens or mediation of the inflammatory cascade may contribute to the pathophysiology of disease and injure the host by activating a deleterious immune response, such as in inflammatory bowel disease. The focus of this research is to identify specific toll-like receptor mutations that may be associated with the development of inflammatory bowel disease.