1985 — 2005 |
Knauf, Philip A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ion Transport Mechanisms in Blood Cells @ University of Rochester
The transmembrane anion exchange protein band 3 (B3) and band 3-related proteins (B3RP) are widely distributed in cells ranging from red blood cells (RBC), where B3 catalyzes the C1/HCO3, exchange that improves blood CO2 transport, to HL60 promyelocytic leukemic cells and brain neurons, where B3RP's probably play central roles in cell pH and volume regulation. B3 and B3RP's are also important in epithelia, particularly those of the kidney and digestive system that are involved in secretion of acid or base. B3 operates by a ping-pong mechanism, in which the transport site has two conformations: E1, with the transport site facing inward, and E0 with it facing outward. When a suitable anion such as C1- is bound to form ECli or EClo, the transport site can reorient to face the opposite side of the membrane, thus transporting the anion. To understand the structural changes that B3 undergoes during substrate binding and transport site reorientation, it is necessary to know what fraction of B3 is in each conformation, and to be able to alter the conformational distribution experimentally. 35Cl NMR techniques will be used to define for the first time the conformational distribution of B3 in C1- media. Inhibitory probes, including a newly discovered compound, diBA, that inhibits at 10/9 M, will be used to sense the conformational changes and to fix the protein in defined conformations for structural analysis. AE2, a B3RP that is expressed in HL60 cells and many other cell types, is structurally very similar to B3, but differs greatly from B3 in inhibitor sensitivity (e.g, 1000-fold less for diBA) and apparently even in its basic kinetic mechanism, which seems not to be a simple ping-pong mechanism based on transport measurements in HL60 cells. A recently- developed system for expressing human AE2 in insect cells, together with HL60 cells, will be used to compare more closely the kinetic mechanism of AE2 with that of B3. Site-directed mutants and AE2/B3 hybrid proteins will help to determine what portions of Be/AE2 are involved in inhibitor binding and in the transporting conformational change. Analysis of the structure and mechanism of these proteins is necessary for a better understanding of basic cellular processes, such as volume and pH regulation, as well as their modifications in disease. The very high-affinity probes investigated here, or their analogues, could also prove to be useful drugs for selectivity modifying these processes, thereby altering function in blood cells or kidney, stomach, and other epithelia.
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1998 — 2002 |
Knauf, Philip A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cell Ion Regulation in Relation to Microcirculatory Function @ University of Rochester
There is good evidence that many of the interactions between blood cells and endothelial cells lining microvessels. which play an important role in such pathological processes as ischemia-reperfusion injury, involve signaling mechanisms in which the regulation of cellular ion fluxes and cell volume play a major role. This project focuses primarily on basic mechanisms of regulation of anion content and volume, in both neutrophils and endothelial cells, with an emphasis on using the unique opportunities of this program project to relate this cellular regulatory processes to functional studies of their effects on the behavior of cells in microcirculatory model systems. The first aim is directed toward understanding the mechanism of and effects of the initial decrease and later increase of neutrophil C1 content ("C1 pulse"), which is a common feature of activation by various agonists and where there is abundant pharmacological evidence for functional importance. A combination of isotope marker, chemical probe, and electrophysiological techniques will be used to determine the mechanisms operative in each phase of this process. This knowledge will be used in collaborative studies with other projects to assess the functional importance of the C1 pulse in affecting cell mechanical properties, granule release, display of surface adhesion molecules, and migration through endothelial cell layers. The second aim involves analysis of the way in which volume regulatory processes in endothelial cells may contribute to pathological events in hemorrhagic shock and ischemia, and on how these proteins could affect interaction with circulating neutrophils. Research will focus on the mechanism of endothelial cell swelling under ischemic and acidic conditions, and the reason for failure of volume-regulatory mechanisms, as well as alterations of neutrophil interactions with endothelial cells under theses conditions. Also, the effects of shear stress on endothelial cell volume and on ion regulation will be examined, under conditions where endothelial cells are grown on surfaces with various curvatures. These studies provide a basis for understanding changes in endothelial cell function under conditions of circulatory stress in terms of fundamental alterations of cell regulatory mechanisms.
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