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.

Fig. 1.

Topology model for Na⁺/Ca²⁺ exchanger (NCX1). The mature NCX1 molecule is modeled to consist of 9 transmembrane (TM) segments with 2 reentrant loops (between TM2 and TM3 and between TM7 and TM8) as part of the conserved α-repeat motifs, which are important in ion transport activity (16, 23, 24). The NH₂-terminus is extracellular and COOH-terminus is intracellular. Between TM5 and TM6 is a large intracellular loop (residues 218–764), which contains the regulatory domains of the exchanger. Specifically, the proximal linker domain (residues 218–358), which interacts with phospholemman (39, 46), the exchange inhibitory peptide (XIP) region (residues 219–238) (19), the 2 calcium binding domains (CBD) 1 (residues 371–500) and 2 (residues 505–689) connected in tandem by a short linker (residues 501–504) (11, 15, 27), and the interaction site for endogenous XIP (residues 562–679) (20) all reside within the intracellular loop. The 2 specific segments [residues 248–252 (PASKT) and residues 300–304 (QKHPD)] in the proximal linker domain that mediates inhibition of NCX1 by phospholemman are shown.

Fig. 2.

Mutating residues 248–252 to alanine (Ala) results in loss of inhibition of Na⁺/Ca²⁺ current (I_NaCa) by phospholemman (PLM) in transfected human embryonic kidney (HEK)293 cells. I_NaCa measured in HEK293 cells transfected with NCX1 Ala mutants (5 consecutive Ala substitutions at a time) spanning the region encompassing residues 238–267 with (diamonds; n = 6, 7, 8, 6, 2, and 6 for 238–242A, 243–247A, 248–252A, 253–257A, 258–262A, and 263–267A mutants, respectively) or without PLM (triangles; n = 4, 6, 8, 6, 6, and 8 for 238–242A, 243–247A, 248–252A, 253–257A, 258–262A, and 263–267A mutants, respectively) are shown. HEK293 cells transfected with pAdTrack-CMV vector have very low endogenous currents under our measurement conditions (open circles, top left panel; n = 8). Symbols represent means ± SE. Error bars are not shown if they fall inside the boundaries of symbols. Except for 248–252A mutant (group, P < 0.54; voltage, P < 0.0001; and group × voltage interaction effects, P < 0.52), two-way ANOVA indicated significant (P < 0.0001 for group, voltage, and group × voltage interaction effects) inhibition of I_NaCa by PLM for all other NCX1 Ala mutants are shown.

Fig. 3.

Mutating residues 300–304 to Ala results in loss of inhibition of I_NaCa by PLM in transfected HEK293 cells. I_NaCa measured in HEK293 cells transfected with NCX1 Ala mutants (5 consecutive Ala substitutions at a time) spanning the region encompassing residues 300–329, with (diamonds; n = 4, 4, 5, 6, 9, and 6 for 300–304A, 305–309A, 310–314A, 315–319A, 320–324A, and 325–329A mutants, respectively) or without PLM (triangles; n = 9, 14, 7, 6, 7, and 9 for 300–304A, 305–309A, 310–314A, 315–319A, 320–324A, and 325–329A mutants, respectively) are shown. Symbols represent means ± SE. Error bars are not shown if they fall inside the boundaries of symbols. Except for 300–304A mutant (group, P < 0.16; voltage, P < 0.0001; and group × voltage interaction effects, P < 0.07), two-way ANOVA indicated significant (P < 0.0001 for group, voltage, and group × voltage interaction effects) inhibition of I_NaCa by PLM for all other NCX1 Ala mutants are shown.

Fig. 4.

Effects of single residue mutation on inhibition of I_NaCa by PLM. I_NaCa measured in HEK293 cells transfected with single NCX1 Ala mutants, with (diamonds; n = 5, 6, 8, 10, 3, 10, 6, 7, and 7 for 248A, 250A, 251A, 252A, 300A, 301A, 302A, 303A, and 304A mutants, respectively) and without PLM (triangles; n = 4, 11, 9, 8, 5, 4, 7, 5, and 10 for 248A, 250A, 251A, 252A, 300A, 301A, 302A, 303A, and 304A mutants, respectively) are shown. Residue 249 of wild-type rat NCX1 is Ala. I_NaCa in cells transfected with NCX1-G503P mutant, which lacks Ca²⁺-dependent activation (21), with (diamonds; n = 4) and without PLM (triangles; n = 6) is also shown. Symbols represent means ± SE. Error bars are not shown if they fall inside the boundaries of symbols. Except for 301A mutant (group, P < 0.91; voltage, P < 0.0001; and group × voltage interaction effects, P < 0.56), two-way ANOVA indicated significant (group, P < 0.037; voltage, P < 0.0001; and group × voltage interaction effects, P < 0.0001) inhibition of I_NaCa by PLM for all other single NCX1 Ala mutants are shown. For NCX1-G503P mutant, two-way ANOVA indicated significant (P < 0.0001 for group, voltage and group × voltage interaction effects) inhibition of I_NaCa by PLM.

Fig. 5.

Reciprocal copurification of PLM with wild-type (WT) NCX1, 248–252A and 300–304A mutants in transfected HEK293 crude membrane extracts. Top: immunoblots of WT NCX1, 248–252A, and 300–304A immunoprecipitates from 400 μg of solubilized membrane preparations using 5 μg of anti-NCX1 antibody (R3F1), or 5 μg of anti-PLM antibody (C2), or control rabbit serum (IgG) and probed with R3F1. Crude membranes (input) were prepared as previously described (1, 39). Molecular markers are indicated on the left.

Fig. 6.

Physical association of PLM with residues 218–270 was weakened by mutating residues 248–252 to Ala. Purified GST or GST-NCX1 fusion proteins linked to GSH-sepharose beads were incubated with His-tagged PLM (1 μg), and GST pull-down assay performed as described in methods. Top: GST and GST-NCX1 fusion proteins detected by Coomassie blue staining. MW, molecular weight marker. 218–270 Mut, NCX1 fragment spanning residues 218–270 in which residues 248–252 were mutated to Ala; 300–373 Mut, NCX1 fragment spanning residues 300–373 in which residues 300–304 were mutated to Ala. Signal intensities (normalized to signal intensity of GST) of 218–270, 218–270 Mut, 300–373 and 300–373 Mut are 0.31, 0.47, 0.45, and 0.70, respectively. Bottom: His-tagged PLM detected by C2 antibody. Signal intensities (normalized to signal intensity of input His-PLM) of His-PLM pulled down by 218–270, 218–270 Mut, 300–373 and 300–373 Mut are 0.49, 0.21, 0.52, and 0.22, respectively. For 218–270 Mut, the His-PLM signal corrected for the difference in GST-fusion products is (0.21 × 0.31)/0.47 = 0.14. Therefore, compared with 218–270, 218–270 Mut is 0.14/0.49 or 28.5% as efficient in terms of pulling down His-PLM. For 300–373 Mut, the efficiency in pulling down His-PLM is 26.9% compared with 300–373. Composite results from three separate GST pulldown experiments are given in results.