Phospholemman Inhibition of the Cardiac Na+/Ca2+ Exchanger ROLE OF PHOSPHORYLATION

Abstract We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (INaCa) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in INaCa, (ii) attenuation of the increase in INaCa by PMA, and (iii) additional reduction in INaCa in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of INaCa, whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on INaCa. Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of INaCa as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited INaCa. The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, INaCa was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in INaCa was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on INaCa in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.