IMAGING ROMK1 INWARDLY RECTIFYING ATP-SENSITIVE K-FORCE MICROSCOPY( CHANNEL PROTEIN USING ATOMIC)

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been show n by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a v ariety of biological macromolecules. Recently, a fusion protein of glu tathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to pr oduce a polyclonal antibody for immunolocalization of ROMK1. We have u sed atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica, ROMK1-GST appears in imag es as a particle composed of two units of similar size. Analyses of im ages indicate that the two units have volumes of approximate to 118 nm (3), which is close to tile theoretical volume of a globular protein o f approximate to 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with th e ROMK1-GST fusion protein's existence as a heterodimer. In experiment s on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solutio n produced a change in height of the aggregates. This change (which wa s reversible) suggests that ATP induces a structural change in the ROM K1 protein. The data show that atomic force microscopy is a useful too l for examination of purified protein molecules under near-physiologic al conditions, and furthermore, that structural alterations in the pro teins may be continuously investigated.