Interdisciplinary Programmes

Membrane Biophysics

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We study the mechanisms of transport of small solutes and water molecules across cell membranes. Our primary interest lies in the physiology of epithelial cells, the cells that line surfaces of the body and are the key transporting cells of organs like the intestine, kidneys and eyes. In many of these epithelial cell systems, it is still not clear as to what the driving forces for water absorption and secretion are. The technical challenge of working with small living structures in vitro requires experimental models dedicated to each cell system and the parameters being measured. Where information is sought at a single membrane level we use artificial planar bilayer membranes with model channels like the antibiotic gramicidin inserted in them. Our current experiments involve:

Insect physiology: The Drosophila midgut has recently been shown to have progenitor cells that determine the architecture of the midgut of the fly with a plan strikingly similar to the development of the mammalian gut. However, very little is known of the physiology of the Drosophila midgut. As the gut is segmented, we perfuse individual segments in vitro, and test epithelial transport models with a defined number of cells and negligible barriers. Complete ultrastructural stereology has been done of all relevant membrane areal and volume densities. With excellent control of transepithelial voltage, pressure and solute composition gradients, we have identified a V-H+-ATPase present predominantly or exclusively on the basolateral membranes that is a powerful transporter for acid-base regulation. We are now analyzing the larval and adult epithelium to understand the role of the carbonic anhydrase catalysed intracellular H+ transport pool, and the localisation and other transporters involved in transepithelial H+ transport.

Ocular physiology: The mammalian retinal pigment epithelium is critically required by the neural retina for its nutrition and physiology, and many diseases of the back of the eye like age-related macular degeneration and retinal detachment are thought to have their origins in pigment epithelium and Bruch's membrane dysfunction. One of the main functions of this epithelium is solute-coupled water absorption across the back of the eye. Determination of the small osmotic and hydrostatic gradients that drive fluid transfer across epithelial cell layers by further refinement of the capacitance probe technique will allow us to model solute-coupled water transport in general, and also understand the relative importance of the epithelium and Bruch’s membrane water permeabilities. In addition to refining models of intraepithelial solute-solvent coupling, we have shown that the osmotic forces driving water transport in epithelia are much larger than previously believed, and that unstirred-layer corrections are not as large as generally supposed, and certainly tractable.

Solute-solvent interactions in interfaces and inside membrane channels reconstituted into bimolecular lipid membranes: With these techniques, it has been possible to determine the number of water molecules (5 to 7) coupled to a Group IA cation in its transit through an ion channel and also the binding constants for ions inside the channel. The ion-selective microelectrode technique for measuring streaming potentials has the advantage that it makes no presuppositions about the molecular mechanism of ion-water coupling inside carriers like Valinomycin and ion channels. Most of the work has been done on very narrow channels like the antibiotic gramicidin, through which ions and water can permeate selectively, but cannot go past each other. Present work is centered on the effects of lipid bilayer thickness and composition on the conduction process.

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