Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Abstract

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na + /Ca 2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the 1 st five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using GST pulldown assay, we demonstrated that the intracellular loop, but not the N-or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na + /Ca 2+ exchangers consisting of N-or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. While expression of Nterminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N-and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Coimmunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase

“…1,2-Dioleoyl-sn-glycero-3-phosphocholine was from Avanti Polar Lipids (Alabaster, AL). Vitamin K 1 (20) (10 mg/mL) was from Abbott Laboratories (Chicago, IL). Vitamin K 1 (25) was from GLsynthesis Inc. (Worcester, MA).…”

“…When limited structural information about a protein is available, functional splitting and reassembly of multispanning membrane proteins can be employed to study their structure and function (17)(18)(19)(20)(21)(22). It is a useful approach to study the function of protein domains, the role of individual transmembrane helices, and the helical packing within the membrane.…”

Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain

“…HEK-293 cells were transfected with control pAdTrack-CMV vector alone (3 g), vector (2 g) ϩ pAdTrack-CMV-NCX1 (WT or deletion mutant, 1 g), or vector ϩ pAdTrack-CMV-PLM ϩ pAdTrack-CMV-NCX1 (WT or deletion mutant, 1 g each) as described in detail previously (1,38,39). Empty vector was used to control for the amount of DNA (3 g for each plate) used in transfection.…”

“…NMR (10) and infrared spectroscopy (2) showed that the TM domain of PLM reconstituted in liposomes is an ␣-helix with a maximum tilt of 15-17°. Specifically, NMR spectroscopic studies of highly purified PLM in model micelles indicate that the molecule consists of four ␣-helices: H1 (residues [12][13][14][15][16][17] is in the extracellular NH 2 terminus, H2 (residues [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] is the main TM helix followed by the short H3 (residues 39 -45), and H4 (residues 60 -68) in the COOH terminus is connected to H3 by a flexible linker (36). PKA phosphorylates Ser 68 , whereas PKC phosphorylates Ser 63 and Ser 68 , of PLM (37).…”

“…Specifically, the exchange inhibitory peptide (XIP) region (residues 219 -238) (18) associated with regulation of NCX1 activity by Na ϩ , Ca 2ϩ , and phosphatidylinositol 4,5-bisphosphate (13,21), the proximal linker domain (residues 259 -370), the two consecutive Ca 2ϩ -binding domains 1 (residues 371-508) (17) and 2 (residues 501-650) (14), and the interaction site for endogenous XIP (residues 562-679) (20) reside within the intracellular loop. Using glutathione S-transferase (GST) pull-down assays and split NCX1 exchangers consisting of NH 2 -or COOH-terminal domains with varying lengths of intracellular loop (26), we previously demonstrated that PLM interacts and associates with a region encompassing residues 218 -358 of the intracellular loop of NCX1 (38).…”