Hector Rasgado-Flores - US grants

muscle cells; hormone regulation /control mechanism; cell morphology; solute; osmosis; calcium; sarcolemma; radiotracer; Cirripedia;

Intracellular Mg2+ (Mgi) is a cofactor for hundreds of enzymes and modulates membrane receptors, ionic channels, and transporters. Hormones induce massive efflux of Mgi from cells but the underlying mechanism of plasmalemmal transport of Mg2+ is unknown. Squid exons, because of their large size, are ideally suited to study Mg2+ transport: internal perfusion and voltage-clamping permits measurement and control of all the relevant driving forces for Mg2+ transport. In these cells, the concentration of intracellular free Mg2+ ([Mg2+]i) is about 600 times lower than expected if Mg2+ ions were at electrochemical equilibrium across the plasmalemma. Active extrusion of Mg2+ maintains [Mg2+]i under steady-state conditions. An ATP-dependent Na/Mg exchanger has been proposed as the sole mechanism responsible for Mg2+ extrusion. This hypothesis explains numerous experimental observations but leaves others unexplained: electrophysiological evidence indicates that Na/Mg exchange is voltage insensitive (consistent with a 2 Na+:1 Mg2+ exchange). However, thermodynamic considerations suggest that neither a stoichiometry of 1, 2, or 3 Na+:1 Mg2+ can predict the measured [Mg2+]; in excitable cells. Numerous observations in various cell types suggest that in addition to Na+, K+ and C1- may also be involved in the regulation of [Mg2+]i. Likewise, we have found preliminary evidence that, in squid axons, the electrochemical gradients of K+ and C1- may be coupled to Mg2+ transport: i) intracellular K+ and C1- are required for Mg2+-dependent Na+ fluxes; and ii) extracellular Mg2+ promotes simultaneous equimolar efflux of Na+-K+ and of Na+-C1-. However, an stoichiometry of 1Na+ + 1K+ = 1C1-:1 Mg2+ does not predict the measured steady-state [Mg2+]i and is inconsistent with an electroneutral exchanger. On the other hand, an stoichiometry of 2Na+ + 2K+ + 2C1-:1 Mg2+ explains the body of available information about Mg2+ transport and accurately predicts the steady-state [Mg2+]i in squid exons. Our aim is to assess if a 2Na+ + 2K+ + 2C1-:1 Mg2+ exchanger is the mechanism responsible for transporting Mg2+ across the plasmalemma and for maintaining [Mg2+]i under steady-state conditions in squid axons. Two main studies will be performed: i) determine the ionic-dependence and stoichiometry of the expected ionic fluxes (using radiolabelled ions); and ii) assess the effect of changes in the electrochemical gradients of Na+,K+ and C1- on the steady-state free and total [Mg]i (measured with flame photometry and fluorescent dyes). Intact, injected and internally dialyzed and voltage-clamped squid axons will be used.

? DESCRIPTION (provided by applicant): Living organisms necessitate the uptake of energy and of building blocks, and the elimination of waste. Therefore, the barrier that separates them from the environment cannot be completely tight to the movement of water, ions, and small organic molecules. As a consequence, cells from their beginning created basic mechanisms to maintain and regulate their water content and volume. When disrupted, these basic functions can have severe consequences for an organism, and diseases of salt and water transport are involved in both acute and chronic conditions that impact over 50% of the population, and have been documented to cost the health care system billions of dollars annually. The goal of the proposed symposium Cell Biology of Volume Regulation and Fluid Homeostasis, an 11th International Symposium on Cell Volume Regulation is to cover both the basic mechanisms of cell volume regulation and their implications in several major diseases including hypertension, brain disorders and lung and kidney diseases. Specifically, we propose 10 scientific sessions: The first part of the conference Cell Biology of Volume Regulation and Fluid Homeostasis, is divided into 5 sessions: I.1 Molecular Mechanisms of Cell Volume Regulation: Transporters and Ion Channels; I.2 Salt-sensitive Mechanisms in Regulation of Apoptosis and Autophagy; I.3 Cell Volume Regulation in Cell Proliferation and Migration; I.4 Lipid Regulation of Osmotic- and Mechano-sensitive Ion Transport Mechanisms; and I.5 Hydration and water transport through the membrane. The second part of the conference Diseases of Volume Regulation and Fluid Homeostasis, is divided into 4 sessions: II.1 Role of Salt Transport in Hypertension; II.2 Fluid-Electrolyte Contributions to Disease Progression in Polycystic Kidney Disease; II.3 Osmoregulation and Hydration in Cystic Fibrosis; and II.4 Osmoregulation in Neurological and Brain Disorders. A 10th scientific session will be dedicated to Young Investigators. In addition, we propose a new educational outreach initiative that will include high school and community college students. This will be achieved via a Lunch and Learn with a Professor sessions that will focus on salt and water in the diet and on small groups discussions about science and scientists personal experiences. It is the first time in the last decade that an International Symposium on Cell Volume Regulation is organized in the United States, which we believe is critically important to facilitate the interaction of the American scientists to the international community and foster international collaborations in this area. Narrative: The major goal of this international scientific meeting on Cell Volume Regulation and Fluid Homeostasis is to cover both the basic mechanisms of cell volume regulation and the roles of these mechanisms in several major diseases including hypertension, brain disorders and lung and kidney diseases. In addition, the meeting will include an educational outreach program that will include high school students and community college students to foster the interest of the students to science.