IMAGING EXCISED APICAL PLASMA-MEMBRANE PATCHES OF MDCK CELLS IN PHYSIOLOGICAL CONDITIONS WITH ATOMIC-FORCE MICROSCOPY

We combined the patch-clamp technique with atomic force microscopy (AF M) to visualize plasma membrane proteins protruding from the extracell ular surface of cultured kidney cells (MDCK cells). To achieve molecul ar resolution, patches were mechanically isolated from whole MDCK cell s by applying the patch-clamp technique. The excised inside-out patche s were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i.e. in fluid). Thus. the reso lution could be increased considerably (lateral and vertical resolutio ns 5 and 0.1 nm, respectively) as compared to experiments on intact ce lls, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pro nase. These proteins had a density of about 90 per mu m(2), with heigh ts between 1 and 9 nm, and lateral dimensions of 20-60 nm. Their frequ ency distribution showed a peak value of 3 nm for the protein height. A simplified assumption - modelling plasma membrane proteins as spheri cal structures protruding from the lipid bilayer allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM c an be used to study the molecular structures of membranes which were i solated with the patch-clamp technique. individual membrane proteins a nd protein clusters, and their arrangement and distribution in a nativ e plasma membrane can be visualized under physiological conditions, wh ich is a first step for their identification.