MIGRATING TRANSFORMED MDCK CELLS ARE ABLE TO STRUCTURALLY POLARIZE A VOLTAGE-ACTIVATED K+ CHANNEL

Cell migration of transformed renal epithelial cells (MDCK-F) depends- in addition to cytoskeletal mechanisms-on the polarized activity of a Ca2+-sensitive K+ channel in the rear part of the cells. However, beca use of the lack of specific markers for this channel we are not able t o determine whether a polarized distribution of the channel protein un derlies its functional polarization, To determine whether the migratin g MDCK-F cells have retained the ability to target K+ channels to dist inct membrane areas we stably transfected the cells with the voltage-d ependent K+ channel Kv1.4. Stable expression and insertion into the pl asma membrane could be shown by reverse transcription-PCR, genomic PCR , Western blot, and patch-clamp techniques, respectively. The distribu tion of Kv1.4 was assessed with indirect immunofluorescence by using c onventional and confocal microscopy. These experiments revealed that K v1.4 is expressed only in transfected cells where it elicits the typic al voltage-dependent, rapidly inactivating K+ current, The Kv1.4 prote in is clustered at the leading edge of protruding lamellipodia of migr ating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion ch annels into the plasma membrane in an asymmetric way, which reflects t he polarization of migrating cells in the plane of movement, These fin dings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane are as.