THE ATOMIC-FORCE MICROSCOPE DETECTS ATP-SENSITIVE PROTEIN CLUSTERS INTHE PLASMA-MEMBRANE OF TRANSFORMED MDCK CELLS

Plasma membrane proteins are supposed to form clusters that allow 'fun ctional cross-talk' between individual molecules within nanometre dist ance. However, such hypothetical protein clusters have not yet been sh own directly in native plasma membranes. Therefore, we developed a tec hnique to get access to the inner face of the plasma membrane of cultu red transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding f rom the cytoplasmic membrane surface. We used the K+ channel blocker i beriotoxin (IBTX), a positively charged toxin molecule, that binds wit h high affinity to plasma membrane potassium channels and to atomicall y flat mica. Thus, apical plasma membranes could be 'glued' with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out me mbrane patches was imaged with AFM in electrolyte solution mimicking t he cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9 +/- 0.02 nm. Multiple p roteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. Large protrusions were most lik ely protein clusters. Addition of the proteolytic enzyme pronase to th e bath solution led to the disappearance of the proteins within minute s. The metabolic substrate ATP induced a shape-change of the protein c lusters and smaller subunits became visible. ADP or the non-hydrolysab le ATP analogue, ATP-gamma-S, could not exert similar effects. It is c oncluded that plasma membrane proteins (and/or membrane associated pro teins) form 'functional clusters' in their native environment. The 'ph ysiological' arrangement of the protein molecules within a cluster req uires ATP. (C) 1997 Academic Press.