title: Residues 248-252 and 300-304 of the cardiac Na+/Ca2+ exchanger are involved in its regulation by phospholemman.
abstract: Using split cardiac Na(+)/Ca(2+) exchangers (NCX1), we previously demonstrated that phospholemman (PLM) regulates NCX1 by interacting with the proximal linker domain (residues 218-358) of the intracellular loop of NCX1. With the use of overlapping loop deletion mutants, interaction sites are localized to two regions spanning residues 238-270 and residues 300-328 of NCX1. In this study, we used alanine (Ala) linker scanning to pinpoint the residues in the proximal linker domain involved in regulation of NCX1 by PLM. Transfection of human embryonic kidney (HEK)293 cells with wild-type (WT) NCX1 or its Ala mutants but not empty vector resulted in NCX1 current (I(NaCa)). Coexpression of PLM with WT NCX1 inhibited I(NaCa). Mutating residues 248-252 (PASKT) or 300-304 (QKHPD) in WT NCX1 to Ala resulted in loss of inhibition of I(NaCa) by PLM. By contrast, inhibition of I(NaCa) by PLM was preserved when residues 238-242, 243-247, 253-257, 258-262, 263-267, 305-309, 310-314, 315-319, 320-324, or 325-329 were mutated to Ala. While mutating residue 301 to alanine completely abolished PLM inhibition, mutation of any single residue 250-252, 300, or 302-304 resulted in partial reduction in inhibition. Mutating residues 248-252 to Ala resulted in significantly weaker association with PLM. The NCX1-G503P mutant that lacks Ca(2+)-dependent activation retained its sensitivity to PLM. We conclude that residues 248-252 and 300-304 in the proximal linker domain of NCX1 were involved in its inhibition by PLM.
authors: Division of Nephrology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors: Xue-Qian
authors: XQ
authors: Zhang
authors: JuFang
authors: J
authors: Wang
authors: Jianliang
authors: J
authors: Song
authors: Angi M
authors: AM
authors: Ji
authors: Tung O
authors: TO
authors: Chan
authors: Joseph Y
authors: JY
authors: Cheung
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman; D000409:Alanine