Experimental Methods of Studying Mechanosensitive Channels and Possible Errors in Data Interpretation

Abstract

In this review we discuss most widely used experimental methods of the membrane stretch which are used for investigation of mechanosensitive channels (MSCs) by patch-clamp. We have tried to discuss possible mistakes in interpreting the data received by various methods of MSCs investigation. In the conditions of single channel recording we briefly analyse positive and negative pressure as mechanical stimulation and demonstrate that MSC respond only to membrane tension. After gigaseal forming suction there appears resting patch for the reason of the patch adhesion to the glass and this creates a resting tension. It is shown that some channels can be active at zero pressure because the seal adhesion energy produces tension. Such a situation can be considered as pre-stretch. Related to this we discuss research showing that stretch-inactivated channels (SICs) do not imply the existence of a new type of channel, but inactivation of channel activity in response to suction can be explained by the activity of pre-stressing of stretch-activated channels (SACs). We also criticize the presence of pressure activated channels (PACs). According to the Laplace’s equation, positive or negative pressures should make equal contributions to the stress. In the conditions of whole cell recording we discuss the known methods of a cell direct mechanical stretching. That is homogeneous stretching of single cells with the use of two patch pipettes, three types of axial stretch - by two glass capillaries, by glass stylus and by two thin carbon fibres. We briefly discuss the merits and imperfections of cell swelling. We analyse the possibilities of paramagnetic microbead method that allows the application of controlled forces to the membrane at which those mechanical forces are transmitted by integrins. We discuss the possibilities of cell compression. Obviously the stresses are very complicated in compression and no one knows how to analyze the data in a mechanistic manner. We discuss the study of bacterial mechanosensitive channels. We discuss the limitation of the research using protein purification and functional reconstitution in planar lipid bilayers or in vesicles. Also, rarely used methods are presented. In this review we discuss most widely used experimental methods of the membrane stretch, which are used for investigation of mechanosensitive channels (MSCs) by means of patch-clamp method. We address possible mistakes in interpreting the data, obtained by means of various methods of MSCs investigation. Under conditions of single channel recording we briefly analyse positive and negative pressure in terms of mechanical stimulation and demonstrate that MSC respond only to membrane tension. Resting tension of the membrane is created after suction, which is applied for the purpose of gigaseal formation. It is shown that some channels can be active at zero pressure because the seal adhesion energy produces tension. Such situation can be considered as pre-stretch. In this respect we discuss reports, showing that stretch-inactivated channels (SICs) do not imply the existence of a new type of channels, when inactivation of channel activity in response to suction can be explained by the activity of pre-stressing of stretch-activated channels (SACs). We discuss the controversy about the presence of pressure activated channels (PACs). According to the Laplace’s equation, positive or negative pressures should make equal contributions to the stress. We also discuss reported methods of direct mechanical stretching of cells during whole cell recording. Discussion covers method of homogeneous stretching of a single cell by means of two patch pipettes and three types of axial stretch - by two glass capillaries, by glass stylus and by two thin carbon fibres. We briefly discuss the merits and imperfections of cell swelling. We analyse the possibilities of paramagnetic microbead method that allows the application of controlled forces to the membrane, at the level of which those mechanical forces are transmitted by integrins. We discuss possible methods of cell compression. Obviously distribution of forces is very complicated during compression and no one knows how to analyze the data in a mechanistic manner. We discuss the study of bacterial mechanosensitive channels. We discuss the limitation of the research using protein purification and functional reconstitution in planar lipid bilayers and in vesicles. Also, rarely used methods are presented