To investigate the biosynthetic mechanisms involved in the expression of the renal epithelial inward rectifying K(+) channel, ROMK1 (Kir1.1a), a six amino acid epitope (AU1) was introduced onto the extreme N-terminus for efficient immunoprecipitation. As expressed in Xenopus oocytes, the AU1 epitope did not modify the functional properties of the ROMK1 channel. To analyze kinetics of ROMK1 synthesis in renal epithelial cells, the AU1-ROMK1 construct was stably transfected in MDCK cells and pulse chase experiments were conducted. When the cells are grown at 37 degrees C, the ROMK1 protein was unstable, being rapidly degraded with a t(1/2) < 1 hour. Furthermore, whole cell patch clamp experiments failed to detect functional ROMK1 channels at the plasma membrane in cells grown at 37 degrees C. In contrast, the degradation process was minimized when the cells were grown at 26 degrees C (t(1/2) > 4 hours), allowing ROMK1 channels to be functionally expressed on the plasma membrane. In summary, in a mammalian epithelial expression system maintained at a physiological temperature, wild-type ROMK1 is bio-synthetically labile and incapable of efficient traffic to the plasmalemma. These observations are reminiscent of temperature sensitive biosynthetic defects in mutant plasma membrane proteins, suggesting that wild-type ROMK1 may require other factors, like the association of a surrogate subunit, for appropriate biosynthetic processing.
Copyright 1999 Academic Press.