Abstract
The Na+/K+/2Cl− cotransporter isoform 1 (NKCC1) mediates electroneutral transport of Na+, K+ and 2Cl− into the cell, and plays crucial roles in homeostasis of cellular Cl− content and cell volume regulation. Turnover of many protein is mediated by covalent attachment of the small protein ubiquitin. Polyubiquitinated proteins are degraded by 26S proteasome. Monoubiquitinated plasma membrane proteins undergo endocytosis and degraded in lysosome, although polyubiquitination is involved in degradation of plasma membrane proteins in some case. We previously demonstrated that NKCC1 is essential for the NGF-induced neurite outgrowth in rat pheochromocytoma PC12 and PC12D cells. The aim of this study is to declare 1) which pathway, or both pathways, is responsible for the degradation of NKCC1 protein, and 2) whether the rate of neurite elongation has correlation with the protein level of NKCC1. Using western blotting and microscopy technique, we found that: 1) the protein level of NKCC1 and the neurite elongation rate in PC12D cells were much higher than those in PC12 cells, 2) the degradation of NKCC1 protein in PC12D cells was much slower than that in PC12 cells, and 3) the degradation rate of NKCC1 in PC12 cells was diminished by treatment with MG132 or clasto-lactacystin β-lactone (a proteasomal inhibitor), but not with chloroquine (a lysosomal inhibitor). These results suggest that the rate of degradation and turnover of NKCC1 protein is mainly mediated by the proteasomal system, and that the high expression level of NKCC1 in PC12D cells is due to a slow degradation rate compared with PC12 cells. [J Physiol Sci. 2008;58 Suppl:S205]
