Expression and Biophysical Characterization of Bacterial Mechano-Sensitive Ion Channel of Large Conductance into Mammalian Cells

Abstract

Mechanobiology is an emerging field of science focused on the role of physical parameters in determining cell morphology and physiology. Mechano-sensistive ion channels represent one of the more important cellular elements sensing and transducing mechanical forces in chemical signaling activating biological pathways.The mechanosensitive channel of large conductance (MscL) was the first mechano-sensitive ion channel to be isolated, cloned and sequenced, and therefore is the more characterized. In Escherichia Coli, MscL acts as an emergency release valve when the tension in the membrane lipid bilayer is getting near to the lytic limit. In the past, the MscL has been used as a comparative model for newly discovered mechano-sensitive channels, and recently, it has been recognized as a tool for potential application in nanotechnology. For example, the MscL have been successfully expressed in mammalian cells to achieve controlled delivery of bioactive molecules through application of mechanical forces.In the present work, we report the expression of the MscL channel into mammalian cells, in order to develop an experimental model of cells highly sensitive to mechanical forces. In order to fully characterize the biophysical features of this transmembrane protein, we developed an optical tweezers setup integrated with a patch-clamp recording system to apply calibrated forces on single cells and simultaneously record the electrophysiological response of the genetically modified cells. We strongly believe that heterologous cellular expression of the MscL may become a useful tool to achieve targeted modulation of cellular activity, as well as to understand the role of mechanical properties of the materials currently used for the development of cellurized scaffolds.