Carol Deutsch - US grants

The proposed research is aimed at characterizing and investigating the cellular physiology of the human peripheral blood lymphocyte in order to determine the role of various regulatory mechanisms in cell activation. We shall investigate the molecular basis for regulation of intracellular pH, determine the effect of mitogens on this regulation, and examine the ability of mitogen-stimulated lymphocytes, at various stages of activation, to volume regulate in response to hypotonicity. In addition, ionic modulation of cell contents, cell membrane, cell volume, T-cell receptor expression and growth factor production, and stimulated proliferation will be evaluated in both normal and mitogen-treated lymphocytes. These studies will thus provide a greater understanding of the fundamental molecular mechanisms of lymphocyte homeostasis and blastogenesis.

The proposed research is aimed at determining cellular transmembrane pH gradients by non-invasive, continuous measurement using nuclear magnetic resonance. This, in part, entails the development of more sensitive, 19F NMR methodologies and indicators. Additional fluorinated amino acids and their esters will be synthesized and tested in order to find optimal indicators and indicator precursors for measuring organelle and cytosolic pH under physiologic conditions. As these methods are developed we will apply them to measurements of cytosolic and compartment pH in situ (human blood lymphocytes, isolated rat hepatocytes and cloned insulinoma cells), in order to study the relationship between cytosolic pH and compartment pH, metabolic and developmental processes.

The goal of this proposal is to determine the role of pH regulation and membrane conductance in fuel-stimulated insulin secretion from Beta-cells. These studies will be carried out on insulinoma cells from transplantable tumors and from clonal lines and from normal cultured Beta-cells from rat islets, which differ in their secretory response characteristics. Specifically, our goals will be (1) to elucidate the molecular mechanism(s) of regulation of intracellular pH, (2) to identify and characterize ionic membrane channels and (3) to test the hypothesis that stimulation of insulin released is mediated by alterations in intracellular pH and/or membrane conductance. These results will be closely correlated with secretion, bioenergetics and Ca++ homeostasis studies in these same cells through collaboration with members of the Diabetes Center.

The proposed research is aimed at characterizing and investigating the cellular physiology of the human peripheral blood lymphocyte in order to determine the role of various regulatory mechanisms in cell activation. We shall investigate the molecular basis for regulation of intracellular pH, determine the effect of mitogens on this regulation, and examine the ability of mitogen-stimulated lymphocytes, at various stages of activation, to volume regulate in response to hypotonicity. In addition, ionic modulation of cell contents, cell membrane, cell volume, T-cell receptor expression and growth factor production, and stimulated proliferation will be evaluated in both normal and mitogen-treated lymphocytes. These studies will thus provide a greater understanding of the fundamental molecular mechanisms of lymphocyte homeostasis and blastogenesis.

The proposed research is aimed at determining cellular pH by non-invasive, continuous measurement using 19F nuclear magnetic resonance. A variety of fluorinated molecules will be synthesized and tested in order to find optimal indicators and indicator precursors for measuring organelle and cytosolic pH under physiological conditions. As these methods are developed we will apply them to cytosolic and compartment pH in situ in isolated cells.

The proposed research is aimed at determining cellular pH by non-invasive, continuous measurement using 19F nuclear magnetic resonance. A variety of fluorinated molecules will be synthesized and tested in order to find optimal indicators and indicator precursors for measuring organelle and cytosolic pH under physiological conditions. As these methods are developed we will apply them to cytosolic and compartment pH in situ in isolated cells.

This research program proposes to study the biophysical properties and subunit interactions of voltage-gated K+ channels and their role in the physiology of T lymphocytes. This proposal is divided into three main projects. The first project determines the impact of K+ channel gating, inactivation kinetics and cumulative inactivation on membrane potential, regulatory volume decrease, and apoptosis. Wile-type and mutated K+ channels will be heterologously expressed in a mouse T cell line, CTLL-2, which lacks endogenous K+ channels. The second project determines turnover rates of functional K+ channel isoforms, and the physiological impact of irreversible ablation of K+ channels in primary human T lymphocytes. This will entail use of a new technique, chromophore-assisted laser inactivation. The third project evaluates specific models of assembly and suppression of T cell voltage-gated K+ channels, specifically, Kv1.3, in cell-free ad in vitro cellular expression systems. This will entail expression of wild-type and truncated K+ channel DNA sequences.

potassium channel; mathematical model; leukocyte activation /transformation; T lymphocyte; electrophysiology; protein isoforms; voltage gated channel; mutant; cooperative study; conformation; chemical kinetics; stoichiometry; electrical conductance; glycosylation; intermolecular interaction; protein structure; tissue /cell culture; cell free system; plasmids;

virus; proteins; lasers; AIDS; immunology; human tissue; communicable diseases; blood; lymphatic system; biomedical resource;

This proposal is designed to examine molecular mechanisms underlying the assembly and function of Kv1.3, a voltage-gated potassium channel (Kv) important in the physiology of T- lymphocytes. Kv1.3 is a typical member of Kv channels, which have diverse and critical roles in both excitable and non-excitable cells. The proposed experiments will employ a wide range of techniques, including biochemical and electrophysiological assays in heterologous expression systems (Xenopus oocytes, mammalian cells, and microsomal membranes). The first aim is to elucidate interaction surfaces, both within and between subunits, during the assembly of Kv changes into functional homotetramers. We hypothesize that conformational changes in oligomeric intermediates are generated along the assembly pathway, and that some are prerequisites for subsequent assembly steps. We will use association and cysteine accessibility assays and dominant negative suppression to explore channel topology and to test specific candidate sites of interaction. Substituted cysteine accessibility will be assayed in part by mass-tagging using a cysteine reagent conjugated with polyethylene glycol. The second aim is to study the functional role played by charged amino acids that are believed to play a role in both assembly and channel gating. We will explore voltage-dependent conformational changes of the S2, S3, and S4 transmembrane segments, using cysteine scanning methods in patch clamp recording. We will test the hypothesis that negatively charged residues in the S2 and S3 segments affect the voltage-dependent conformations of the S4 segment.

Our research program aims to understand how a voltage-gated potassium (Kv) channel is made. This is a complicated multi-step process that requires acquisition of local secondary, tertiary, and quaternary structures, either sequentially or as coupled events. How this happens is, for the most part, unknown. Yet the impact of these steps is profound, often with pathological consequences. We focus our attention on the human tetrameric Kv1.3 channel, particularly its early-stage folding, and the determinants that regulate these folding events in the exit tunnel of the ribosome and the ER membrane. Three Aims comprise this grant proposal. The ability of a protein to form helices is a fundamental prerequisite for protein folding and function in all proteomes. However, this process has not been defined in the confined and heterogeneous microenvironment of the ribosome exit tunnel where a protein is first made. Helicity must occur at the right time and place during translation. Failure to meet this requirement impairs peptide targeting, chaperone association, efficient bilayer insertion, and oligomerization. In Aim 1, we specifically ask when, where, how, and why these critical secondary structures arise in the Kv1.3 nascent peptide. We will determine the molecular mechanisms that delay or initiate helix formation and identify the underlying peptide-tunnel interactions that are responsible. To do this, we use biochemical approaches and cryo-EM single particle reconstruction of peptide-ribosome complexes. In Aim 2, we explore an exciting new field of fundamental importance to how proteins are made, namely, how a peptide's sequence generates piconewtons of force that fine tune Kv peptide's rate of elongation and folding efficiency. We use experimental approaches and molecular dynamics simulations to identify the type of peptide-tunnel interactions giving rise to force, the nature of the force, and its consequences as the peptide is elongated. Given that human Kv1.3 is expressed in neuronal and immune cells, and impaired expression produces chronic inflammatory disease and autoimmune disorders, it is compelling to ask whether human disease-linked variants of the KCNA3, the gene that encodes Kv1.3, introduce folding/assembly/trafficking defects. In Aim 3, we address this question using the recently developed ?genome- first? approach to determine the clinical consequences of specific KCNA3 rare variants and biophysical determinations of Kv1.3 folding and function to identify the molecular defects. Our overall vision of Kv folding includes complex coupled events between intrapeptide segments, the ribosome exit tunnel, and the ER membrane. We now expand this view by introducing two new concepts for further investigation: 1) repressor/activator activity acts as a molecular switch to govern the time and tunnel location of Kv helix initiation, and 2) cotranslational force generation modulates translation rates and folding. Both concepts represent paradigm shifts that reveal additional levels of regulation for Kv channel folding and may generalize to the biogenesis of other proteins.

DESCRIPTION (provided by applicant): Potassium channel activity in a cell is a critical determinant of normal cell function. This activity depends on an ensemble of channel properties including permeation and gating. Permeant ions themselves modulate these properties, thereby suggesting a potential means of autoregulation of channel activity, which could be important for homeostatic electrical activity. Our long term goal is to understand the autoregulatory mechanisms by which permeant ions modulate and synergize pore properties, gating (specifically slow inactivation), and movement of the voltage sensor. This proposal is divided into two aims. The first aim investigates the mechanisms of permeant ion modulation of gating and permeation in potassium channels. Several hypotheses will be considered by exploiting a series of Shaker mutants having different kinetics of slow inactivation. Specifically, we will test how mutants that enhance the rate of slow inactivation have drastically reduced macroscopic conductances to Cs + ions, whether this is due to altered permeabilities/selectivity, lower values of single channel conductance, altered ion occupancy of the pore, and/or decreased probabilities of opening. Among other electrophysiological approaches, we will use nonstationary noise analysis of currents expressed in HEK cells. The second aim will test whether ions in the selectivity filter modulate the movement of voltage sensors. These studies will entail measurements of kinetics and voltage dependence of gating currents in HEK cells.

DESCRIPTION (provided by applicant): Potassium channels are tetrameric membrane proteins that provide a highly selective conduit for potassium ions to diffuse across the hydrophobic barrier of cell membranes. As such, their formation and biophysical properties are critical for processes like neuronal excitability, secretion of hormones, and muscle contraction. The formation of ion channels includes biogenesis of monomeric channel subunits, assembly of subunits into the tetrameric channel, and trafficking of the channel to the appropriate cellular membrane where it performs its functional role. Although the structure and function of mature potassium channels have been studied extensively, little is known about the early folding events in channel biogenesis. Defects in translation and folding have downstream consequences for assembly, trafficking, and function of potassium channels, and underlie pathology. The long-term goal of our research program is to elucidate basic principles of translation and protein folding in the biogenesis of voltage-gated potassium (Kv) channels, including folding events in the ribosome-nascent peptide complex. This proposal is divided into four interrelated Projects, each having several Aims. Project I is devoted to understanding how the T1 domain, critical for assembly and targeting of Kv channels, moves through the tunnel and is persuaded to fold during biogenesis. No models of this progression exist. The expected outcomes of our studies will fill this gap. Project II maps the events and location of voltage- sensor (VS) formation. Thes findings bear on all voltage-gated channels and mechanisms for VS folding defects. Project III defines prerequisites for pore formation and the defects in pore architecture that underlie the impaired trafficking responsible for channel diseases, e.g., Long QT2 Syndrome. Project IV will generate new paradigms for allosteric communication in the ribosome-nascent peptide complex, challenge existing paradigms that refute the important role of peptide-tunnel dynamics. Our results will advance a new technology for determining rates of peptide movement in the tunnel during peptide elongation. The results of our studies bear not only on Kv channel formation and cellular levels of Kv protein, but also on broader issues in biogenesis and folding of proteins, and will provide a paradigm for rational design of therapeutics for Kv folding defects. Many of the methods and strategies used in these proposed studies (e.g., pegylation, intramolecular crosslinking assays of a nascent peptide attached to a ribosomal tunnel) were introduced and developed in my laboratory to assay secondary, tertiary, and quaternary folding events. The proposed experiments will use a range of techniques including novel biochemical micro-assays of Kv biogenic intermediates, electrophysiology of Xenopus oocytes, and computational analysis. PUBLIC HEALTH RELEVANCE: Formation of voltage-gated potassium channels includes synthesis of the channel protein, assembly of these proteins into multimeric channels, and delivery of these channels to the plasma membrane. Defects in any of these steps will result in altered expression of the channels at the cell surface, with pathological and often lethal consequences. Our studies of ion channel folding bear on basic principles of protein folding and pave the way for understanding the molecular basis of protein misfolding, a fundamental cause of many diseases.