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The human nongastric [H.sup.+][K.sup.+]-ATPase, ATP1AL1, shown to reabsorb [K.sup.+] in exchange for [H.sup.+] or [Na.sup.+], is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal [H.sup.+]-[K.sup.+]-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal [H.sup.+]-[K.sup.+]-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the [H.sup.+]-[K.sup.+]-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric [H.sup.+]-[K.sup.+]-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric [H.sup.+]-[K.sup.+]-ATPase plasma membrane expression. nongastric hydrogen-potassium-adenosine triphosphatase; Madin-Darby canine kidney cells

Cell migration of transformed renal epithelial cells (MDCK-F) depends - in addition to cytoskeletal mechanisms - on the polarized activity of a [Ca.sup.2+]-sensitive [K.sup.+] channel in the rear part of the cells. However, because of the lack of specific markers for this channel we are not able to determine whether a polarized distribution of the channel protein underlies its functional polarization. To determine whether the migrating MDCK-F cells have retained the ability to target [K.sup.+] channels to distinct membrane areas we stably transfected the cells with the voltage-dependent [K.sup.+] channel Kv1.4. Stable expression and insertion into the plasma membrane could be shown by reverse transcription-PCR, genomic PCR, Western blot, and patch-clamp techniques, respectively. The distribution of Kv1.4 was assessed with indirect immunofluorescence by using conventional and confocal microscopy. These experiments revealed that Kv1.4 is expressed only in transfected cells where it elicits the typical voltage-dependent, rapidly inactivating [K.sup.+] current. The Kv1.4 protein is clustered at the leading edge of protruding lamellipodia of migrating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion channels into the plasma membrane in an asymmetric way, which reflects the polarization of migrating cells in the plane of movement. These findings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane areas.

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 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 catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of C[O.sub.2]) reduced their magnitude. The data show 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 activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.