STRUCTURAL ACTIVITY OF A CLONED POTASSIUM CHANNEL (ROMK1) MONITORED WITH THE ATOMIC-FORCE MICROSCOPE - THE MOLECULAR-SANDWICH TECHNIQUE

The atomic force microscope (AFM) was used to continuously follow heig ht changes of individual protein molecules exposed to physiological st imuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial pota ssium channel known to be highly pH sensitive) and lowered onto atomic ally flat mica surface until the protein was sandwiched between AFM ti p and mica. Because the AFM tip was an integral part of a highly flexi ble cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of t he cantilever that was monitored by means of a laser be am. With this system it was possible! to resolve vertical height changes in the ROMK 1 protein of greater than or equal to 0.2 nm (approximately 5% of the molecule's height) with a time resolution of greater than or equal to 1 msec. When bathed in electrolyte solution that contained the catalyt ic subunit of protein kinase A and 0.1 mM ATP (conditions that activat e the native ion channel), eve found stochastically occurring height f luctuations in the ROMK1 molecule. These changes in height were pa-dep endent, being greatest at pH 7.6, and lowering the DH (either by titra tion or by the application of CO2) reduced their magnitude. The data s how that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM ''molecular-sandwich'' technique, called MOST, measures structural ac tivity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the mol ecular level.