Regulation of function and protein expression of noradrenaline transporter by protein kinases

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Regulation of noradrenaline transporter (NAT) function by calmodulin-related protein kinases was studied in PC12 cells, which endogenously express NA transporters. 1. We determined a full sequence of rat NAT cDNA and found five consensus sites for phosphorylation by Ca^<2+>/calmodulin-dependent (CaM) kinase II.A synthetic peptide whose sequence was contained in the intracellular COOH-terminal domain of rat NAT was phosphorylated by purified brain CaM kinase II.2. Extensive NAT-like immunoreactivity or fluorescence of transfected GFP-NAT fusion protein in plasma membrane of PC12 cells disappeared after incubation with KN-93 or in the absence of Ca^<2+>. 3. In clonal cell lines of PC12 cells which overexpressed CaM kinase II α subunit, [^3H]NA uptake as well as expression of NAT mRNA and protein was markedly increased compared with wild type cells. Content of phosphorylated CREB, an active form of a transcription factor was also increased in CaM kinase II α-overexpressed cell lines. The increased uptake and mRNA expression were significantly suppressed by chronic treatment of KN-93. These results suggest that NA uptake might be facilitated by Ca^<2+>/calmodulin-dependent protein kinases through direct phosphorylation of COOH-terminal domain of NAT and/or through stimulated translocation of NAT to plasma membrane, and that activation of CaM kinase II might chronically enhance expression of NAT.