Phospholemman inhibition of the cardiac Na<sup>+</sup>/Ca<sup>2+</sup> exchanger: Role of phosphorylation.
Abstract
We have previously demonstrated that phospholemman (PLM), a 15 kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na/Ca exchanger (NCX1). In addition, p rotein kinase A phosphorylates serine while protein kinase C phosphorylates both serine and serine of PLM. Using HEK293 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 to alanine (S63A) preserved PLM’s sensitivity to forskolin in terms of suppression of INaCa, whereas mutating serine to alanine (S68A) abolished PLM’s inhibitory effect on INaCa. Mutating serine to glutamic acid (phosphomimetic) resulted in additional suppression of I NaCa as compared to wild-type PLM. These results suggest that PLM phosphorylated at serine inhibited INaCa. The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knockout (KO) mice. When compared to wild-type myocytes, INaCa was significant larger in PLM-KO myocytes. In addition, 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, inhibits Na/Ca exchange in the heart.
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