Peter K. Lauf - US grants

The cation dimorphism of high K+ (HK) and low K+ (LK) sheep red cells is genetically associated with differences in a) Na+K+ pumps, b) ouabain-sensitive Cl- dependent K+ transport which is activated through reaction of N-ethylmaleimide (NEM) with its sulfhydryl (SH) groups or by cell swelling in hyposmotic media, and c) the presence of the M and L membrane antigens. Both HK and LK red cells possess a ouabain-insensitive Na+/Cl exchange flux whose role is unexplained. The process of maturation of the reticulocy to the prospective LK red cell may involve all of the above transport systems operating consecutively or simultaneously to exchange cellular K+ for Na+ (HK-LK transition). For the study of regulation and development of membrane transport and its pathophysiology and its genetic basis in disease, the HK/LK sheep red cell system offers a unique, genetically defined model. To understand the precise mechanism of the HK-LK transition a further analysis is proposed of the individual transport systems in mature HK and LK red cells as well as an in vitro follow-up study of the maturational changes of all transport activities. In particular, it is proposed: 1. A further characterization of the SH-dependent, volume sensitive K/Cl pathway in LK red cells with respect to a) its biochemical and antigenic nature as ascertained by covalent binding of 3H-NEM or 3H-bumetanide, an inhibitory loop diuretic, and attachment of radiolabeled anti-L1 specifically reducing SH-dependent K/Cl flux; b) the hypothesis of regulation of SH-dependent K/Cl flux by cytoplasmic factors (ATP) or cellular metabolism; and c) the physiologic basis of Cl-requirement and inhibition by loop diuretics. These analyses will shed new light on the mechanism of other coupled ion transporters recently described in renal and intestinal physiology. 2. An analysis of ouabain-insensitive Na+ pathways of HK and LK red cells, in particular of the Na+/Na+ counter transport as a potential candidate for Na+/H+ exchange that together with outward K/Cl flux may be important for the development of the LK cell. 3. An investigation of the specific site of action of cellular K+ to inhibit the Na+K+ pump in LK cells, and an evaluation of the role of protons. 4. A further characterization of the M/L surface antigens with the aid of new electrophoretic techniques (electro blotting). 5. To test experimentally the hypothesis that all of the above transporters studied simultaneously or consecutively down-regulate cellular K+ levels from that in HK reticulocytes to those in mature LK red cells, and to understand the mode of inactivation of SH-dependent K/Cl transport in HK and of K+ pump fluxes in LK erythrocytes.

reticulocytes; ion transport; erythrocyte membrane; surface antigens; biological polymorphism; immunohematology; cytogenetics; passive transport; membrane permeability; scintillation counter; crosslink; stoichiometry; density gradient ultracentrifugation; affinity chromatography;

The cation dimorphism of high K+ (HK) and low K+ (LK) sheep red cells is genetically associated with differences in a) Na+K+ pumps, b) ouabain-sensitive Cl- dependent K+ transport which is activated through reaction of N-ethylmaleimide (NEM) with its sulfhydryl (SH) groups or by cell swelling in hyposmotic media, and c) the presence of the M and L membrane antigens. Both HK and LK red cells possess a ouabain-insensitive Na+/Cl exchange flux whose role is unexplained. The process of maturation of the reticulocy to the prospective LK red cell may involve all of the above transport systems operating consecutively or simultaneously to exchange cellular K+ for Na+ (HK-LK transition). For the study of regulation and development of membrane transport and its pathophysiology and its genetic basis in disease, the HK/LK sheep red cell system offers a unique, genetically defined model. To understand the precise mechanism of the HK-LK transition a further analysis is proposed of the individual transport systems in mature HK and LK red cells as well as an in vitro follow-up study of the maturational changes of all transport activities. In particular, it is proposed: 1. A further characterization of the SH-dependent, volume sensitive K/Cl pathway in LK red cells with respect to a) its biochemical and antigenic nature as ascertained by covalent binding of 3H-NEM or 3H-bumetanide, an inhibitory loop diuretic, and attachment of radiolabeled anti-L1 specifically reducing SH-dependent K/Cl flux; b) the hypothesis of regulation of SH-dependent K/Cl flux by cytoplasmic factors (ATP) or cellular metabolism; and c) the physiologic basis of Cl-requirement and inhibition by loop diuretics. These analyses will shed new light on the mechanism of other coupled ion transporters recently described in renal and intestinal physiology. 2. An analysis of ouabain-insensitive Na+ pathways of HK and LK red cells, in particular of the Na+/Na+ counter transport as a potential candidate for Na+/H+ exchange that together with outward K/Cl flux may be important for the development of the LK cell. 3. An investigation of the specific site of action of cellular K+ to inhibit the Na+K+ pump in LK cells, and an evaluation of the role of protons. 4. A further characterization of the M/L surface antigens with the aid of new electrophoretic techniques (electro blotting). 5. To test experimentally the hypothesis that all of the above transporters studied simultaneously or consecutively down-regulate cellular K+ levels from that in HK reticulocytes to those in mature LK red cells, and to understand the mode of inactivation of SH-dependent K/Cl transport in HK and of K+ pump fluxes in LK erythrocytes.

This project proposes a study of the kinetic, thermodynamic and regulatory properties of K-Cl cotransport of sheep red blood cells (RBCs), during the reticulocyte/mature RBC transition, and in ghosts. K- Cl cotransport, a ouabain-resistant (OR) and strictly Cl-dependent potassium (k) transporter, when activated by cell swelling or thiol group modification, may constitute a major fraction of the membrane's passive K permeability. Outwardly poised, K-Cl cotransport has been implicated in maturational RBC volume reduction of sheep and other RBCs, and pathologically in RBC dehydration of patients with certain hemoglobinopathies. In sheep, K-Cl cotransport occurs in all reticulocytes and in mature RBCs of the low K (LK) type but disappears in high K (HK) cells, The mechanism by which K-Cl cotransport is maintained moderately active in LK, however, inactivated in HK cells, is unknown. To understand the process of K-Cl cotransport involution in this model the following hypothesis will be tested: 1. The kinetic and thermodynamic characteristics of K-Cl cotransport, recently elucidated by us for the swelling activated system, are also common to hemoglobin-free ghosts, and to both reticulocyte precursor and mature RBCs. 2. Regulation involves membrane components of the transporter, as well as cytoplasmic factors. These general hypotheses will be tested as follows. The kinetic and thermodynamic properties of K-Cl cotransport will be compared by OR zero-trans K influxes and effluxes in reticulocytes and mature red cells of both LK and HK genotypes, and in resealed ghosts with those of the swelling induced K-Cl pathway. The regulation of K-Cl cotransport will be studied in LK cells with varied intracellular Mg, anions, and Ph, interventions known to cause activation or inactivation of the transporter. The presence of membrane-bound thiols crucial for K- Cl cotransport function will be tested by radio-labelling of selectively protected thiols and other groups, and electrophoretic verification of labelled membrane components, as well as by quantitative correlation of tracer incorporation with simultaneously measured K-Cl cotransprot activity. Understanding the process of transport changes in this model will elucidate why for example in human RBCs homozygous for hemoglobin S, K-Cl cotransport remains active and thus contributes to irreversible sickling.

[unreadable] DESCRIPTION (provided by applicant): The proposed project entails the use of new technological approaches to elucidate the biochemical nature of the M/L membrane blood group antigens. These sheep red blood cell (SRBC) proteins are functionally associated with the Na/K pump and K-CI cotransporter (COT). In SRBCs with low potassium (Ki) and high sodium (Nai) levels, a dominant genetic trait, Ki inhibits the Na/K pump causing the low Ki (LK) high Nai steady state levels. LK SRBCs possess two functionally separable L antigens, Lp and Li. AIIo-immune L antisera stimulate the Na/K pump several-fold due to the Lp- and inhibit K-CI COT by more than 60% due to the Li-antigen/antibody reactions. Thus, Lp is an inhibitor of the Na/K pump and Li an activator of K-CI COT. High Ki (HK) SRBC with a normal Na/K pump and low K-CI COT has M antigens not functionally associated with either transporter. The hypothesis is that Lp and Li, modulate Na/K pump and K-CI COT via signal transduction pathways, where one rate limiting step may be controlled by the LK gene. To understand the biochemistry of these antigens fully, the following strategies will be taken: [unreadable] 1. Preparation of high purity, highly functional Lp, Li and M antibodies from polyclonal L and M antisera by affinity chromatography on LK and HK SRBC membrane ghosts covalently immobilized on CNBr-activated Sepharose. 2. Biosensor-based interaction analysis to establish kinetic binding events between M and L antigen positive HK and LK SRBC ghosts, respectively, and their respectively immobilized affinity-purified L and M antibodies. Optimization of conditions relevant to the stabilization of immune complexes, and conversely determination of conditions useful for destabilizing immune complexes. 3. Use of affinity-purified L and M antibodies for i) antigen pull-down experiments on alkali-stripped, detergent-soluble, intrinsic membrane proteins, and ii) M and L epitope excision experiments on intact SRBC ghosts. 4. Mass fingerprinting and sequencing of L and M tryptic peptides followed by protein identification through database and Blast searches. 5. Functional proteomics by heterologous expression systems and verification of the identity of the L antigens by reversal of anti-Lp mediated Na/K pump stimulation and the anti-Li inhibition of K-CI COT by Rb influx measurements, and detection of the M antigen by inhibition of anti-M mediated immune hemolysis. Given the ubiquitous presence of the Na/K pump and K-CI COT in all mammalian cells, the molecular identification of the L and M antigens will explain the molecular basis of the HK/LK dimorphism and the regulation of these ion transporters and will potentially reveal a molecular basis for diseases where changes in ion transport do not correlate with either functional mutations or alterations in protein expression levels. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application requests partial support to host an International Symposium on Cell Volume & Signal Transduction at Wright State University September 20-24, 2003. This is third in a symposia series on the topic and originated in Smolenice, Slovakia (1997) and continued in Berlin, Germany (2000). As the first of its kind in the U.S., it builds on the previous 2 symposia's success. It draws together approximately 80 scientists from Europe and the United States in the fields of membrane transport and signal transduction in normal and pathologically altered cell volume regulation, in particular, the areas of cardiovascular, epithelial and neuronal cell physiology. Participants encompass both gender scientists whose eminence is well established in addition to those with emerging promise. The following topics' multidisciplinary nature guarantees cross-fertilization between the major research fields, a major goal of this meeting: 1. Hematological and vascular biology and pathology with special emphasis on erythrocytes, vascular smooth muscle and endothelial cells. 2. Renal and gastrointestinal biology and pathology, particularly of epithelial cells of the nephron, the gastrointestinal system and the liver. 3. Neurobiology, i.e., neuronal and glial cells. 4. Other systems. To achieve these goals, the conference will accommodate approximately 30 speakers (6-9 lectures/day) and 50 poster presenters in integrated sessions within the Wright State University campus. Opportunity will be provided for discussions during time intervals between talks and in afternoon poster sessions and evening lecture/social gatherings. Campus conference sites offer space for lectures, posters and meals, combined with gatherings/meetings at nearby offcampus historic sites, e.g., Dayton Engineers Club, the nationally-acclaimed Wright-Patterson Air Force Museum and Dayton Art Institute. The cost-effective setting is ideally situated with participant housing in hotels adjacent to campus and 3 airports (Dayton, Columbus and Cincinnati) nearby. The proximity of several major universities to WSU campus provides opportunity for greater attendance by scientists in the region. The meeting assumes a prominent position in the Ohio Bicentennial and Wright Brothers Invention of Flight Centennial celebrations. This meeting's success on U.S. soil will foster continuation of gathering of high-caliber scientists at subsequent international meetings