Membrane residence time and degradation of KCa3.1 in polarized Fischer rat epithelial (FRT) cells

Abstract

In this study, we examined the membrane localization, residence time and degradative pathway of KCa3.1, for the first time in a polarized epithelium. To determine the membrane localization of KCa3.1‐BLAP, FRT cells were grown on filters and incubated with either apically‐applied streptavidin or basolaterally‐applied streptavidin. Untransfected cells were used as a negative control. We demonstrated, that KCa3.1‐BLAP was trafficked exclusively to the basolateral membrane of the cells (n=5, both IF and IB). Next, we examined the residence time of KCa3.1 in which FRT cells were incubated with basolaterally‐applied streptavidin and returned back to 37C for 0, 1, 3, 5, 8 and 12 hours. After 2 hours, membrane KCa3.1‐BLAP channels were localized into intracellular vesicles (by IF, n=5) and by 3–4 hours the presence of intracellular KCa3.1‐BLAP was reduced by half (by IB, n=5). Finally, to determine the degradative pathway of KCa3.1‐BLAP, we examined the effects of the lysosomal inhibitors, leupeptin (100 μM) and pepstatin (1μg/ml), on the intracellular fate of KCa3.1‐ BLAP. The presence of leupeptin and pepstatin reduced the degradation of KCa3.1‐BLAP suggesting the channel is degraded via a lysosomal‐dependent pathway (IB, n=5). Our results clearly demonstrate that KCa3.1 is trafficked exclusively to the basolateral membrane of polarized FRT cells, the channels have a half‐life of ~4 hours and that the channels are degraded in the lysosome. This work was supported by the Department of Physiology, Univ. of Otago (KLH) and NIH (DCD).