Evidence for the role of cell stiffness in modulation of volume-regulated anion channels F. J. Byfield, 1,2 B. D. Hoffman, 3 V. G. Romanenko, 1,4 Y. Fang, 1,2 J. C. Crocker 1,3 and I. Levitan 1,2 1 Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA 3 Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA 4 Department of Physiology, University of Rochester, Rochester, NY, USA Received 17 October 2005, accepted 25 January 2006 Correspondence: I. Levitan, Department of Medicine, University of Illinois Chicago, 840 S. Wood Str, Chicago, IL 60612, USA. E-mail: levitan@uic.edu Abstract Aim: To investigate the link between cell stiffness and volume-regulated anion current (VRAC) in aortic endothelium. Method: Bovine aortic endothelial cells (BAECs) were exposed to methyl- b-cyclodextrin (MbCD) to deplete cellular cholesterol and the changes in cellular stiffness were measured by micropipette aspiration. VRAC density was measured electrophysiologically in the same cell populations. Further- more, to probe the effects of cholesterol depletion on the mechanics of ‘deep’ cytoskeleton, we employ a novel technique to analyse correlated motion of intracellular particles. Results: We show that cholesterol depletion results in cellular stiffening and an upregulation of VRAC density. Replenishing cellular sterol pool with epicholesterol, a chiral analogue of cholesterol, abrogates both of these ef- fects. This indicates that cholesterol sensitivity of both cell mechanics and VRAC are due to changes in the physical properties of the membrane rather than due to specific sterol–protein interactions. We also show that choles- terol depletion increases the stiffness of the ‘deep cytoskeleton’ and that disruption of actin filaments abolishes both cell stiffening and upregulation of VRAC due to cholesterol depletion. Furthermore, comparing BAECs to human aortic endothelial cells (HAECs), we show that BAECs that are inherently stiffer also develop larger VRACs. Conclusions: Taken together, our observations suggest an increase in the cytoskeleton stiffness has a facilitatory effect on VRAC development. We suggest that stiffening of the cytoskeleton increases tension in the membrane– cytoskeleton layer and that in turn facilitates VRAC. Keywords anion channels, cell mechanics, cholesterol, volume regulation. Activation of volume-regulated anion channels (VRAC) is a key step in maintaining cell volume homeostasis and in prevention of pathological cell swelling in a variety of cell types (reviewed by Nilius & Droogmans 2001). Our studies focus on investigating molecular mechanisms that regulate VRAC in vascular endothelial cells, where cell swelling was shown to have a major detrimental effect by (i) narrowing of capillary luminal space which increases capillary hydraulic resistance and decreases blood flow (Mazzoni et al. 1989, 1995, Menger et al. 1997) and (ii) disruption of endothelial junctional integrity leading to the disruption of endothelial per- meability barrier (Gilmont et al. 1998). The latter results in the diffusion of oxidized lipids into the vascular wall, which is believed to constitute the initial stage of atherosclerosis development. Importantly, Acta Physiol 2006, 187, 285–294 Ó 2006 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2006.01555.x 285