Title: Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation
Abstract: Article18 May 2006free access Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation Youssef Ben Ammar Youssef Ben Ammar Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Soichi Takeda Soichi Takeda Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Laboratory of Structural Biochemistry, RIKEN Harima Institute at SPring-8, Kouto, Mikazuki-cho, Sayo, Hyogo, Japan Search for more papers by this author Takashi Hisamitsu Takashi Hisamitsu Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Hidezo Mori Hidezo Mori Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Shigeo Wakabayashi Corresponding Author Shigeo Wakabayashi Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Youssef Ben Ammar Youssef Ben Ammar Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Soichi Takeda Soichi Takeda Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Laboratory of Structural Biochemistry, RIKEN Harima Institute at SPring-8, Kouto, Mikazuki-cho, Sayo, Hyogo, Japan Search for more papers by this author Takashi Hisamitsu Takashi Hisamitsu Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Hidezo Mori Hidezo Mori Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Shigeo Wakabayashi Corresponding Author Shigeo Wakabayashi Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan Search for more papers by this author Author Information Youssef Ben Ammar1,‡, Soichi Takeda2,3,‡, Takashi Hisamitsu1, Hidezo Mori2 and Shigeo Wakabayashi 1 1Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan 2Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan 3Laboratory of Structural Biochemistry, RIKEN Harima Institute at SPring-8, Kouto, Mikazuki-cho, Sayo, Hyogo, Japan ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565-8565, Japan. Tel.: +81 6 6833 5012; Fax: +81 6 6835 5314; E-mail: [email protected] The EMBO Journal (2006)25:2315-2325https://doi.org/10.1038/sj.emboj.7601145 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The plasma membrane Na+/H+ exchangers (NHE) require calcineurin B homologous protein (CHP) as an obligatory binding partner for ion transport. Here, we report the first crystal structure of CHP (CHP2 isoform) in complex with its binding domain in NHE1. We show that the cytoplasmic α-helix of NHE1 is inserted into the hydrophobic cleft formed by N- and C-lobes of CHP2 and that the size and shape of this crevice together with hydrogen bond formation at multiple positions assure a high degree of specificity for interaction with NHE members. Structure-based mutagenesis revealed the importance of hydrophobic interactions between CHP/NHE1 for the function of NHE1. Furthermore, the crystal structure shows the existence of a protruding CHP-unique region, and deletion of this region in CHP2 inhibited the NHE1 activity by inducing the acidic shift of intracellular pH dependence, while preserving interaction with NHE1. These findings suggest that CHP serves as an obligatory subunit that is required both for supporting the basic activity and regulating the pH-sensing of NHE1 via interactions between distinct parts of these proteins. Introduction Maintenance of intracellular pH (pHi), Na+ concentration, and cell volume is crucial for all living cells to survive and to ensure a variety of cellular functions, such as cell metabolic processes, muscle contraction, secretion, and higher-order brain activity. The Na+/H+ exchanger (NHE) is an important transporter regulating such ionic homeostasis and catalyzing the electroneutral countertransport of Na+ and H+ through the plasma membrane and other intracellular organellar membranes in various animal species (Wakabayashi et al, 1997; Counillon and Pouyssegur, 2000; Putney et al, 2002; Orlowski and Grinstein, 2004; Zachos et al, 2005). Of the nine different NHE isoforms identified to date (NHE1–NHE9), the ubiquitous isoform NHE1 has been studied most extensively. NHE1 is known to be activated rapidly in response to various extracellular stimuli, such as hormones, growth factors, and mechanical stressors (Wakabayashi et al, 1997; Orlowski and Grinstein, 2004). Such activation of NHE1 is often linked to various diseases. For example, elevated NHE1 activity is thought to be a risk factor causing heart failure and hypertrophy, as evidenced by the remarkable protective effects of specific inhibitors (Karmazyn, 2001; Engelhardt et al, 2002). Regulation of NHE1 is thought to occur through interaction of multiple signaling molecules with the carboxyl (C)-terminal cytoplasmic domain of NHE1 and subsequent conformational change of the amino (N)-terminal transmembrane domain responsible for catalyzing NHE (see Figure 1A for membrane topology). Importantly, this regulation of NHE1 is attributable to a change in the affinity for intracellular H+. A previous biochemical experiment using membrane vesicles suggested that the exchangers possess a cytoplasmic ‘H+-modifier’ or ‘pH-sensor’ site(s), distinct from the H+-transport site (Aronson et al, 1982; Wakabayashi et al, 2003a). The exchangers were thus considered to be activated through conformational changes caused by protonation of this regulatory site. Our recent study, using cells expressing NHE isoforms (NHE1–3), further supported this concept by measuring the reverse reaction of exchange (Na+ efflux) (Wakabayashi et al, 2003a). However, another recent study reported an allosteric model with no additional H+-modifier site to explain the sigmoidal cytosolic H+ dependence (Lacroix et al, 2004). Figure 1.Sequence alignments of CHP and its binding domain in NHE isoforms. (A) Membrane topology model of NHE1. NHE1 consists of the N-terminal transporter domain with 12 transmembrane domains (TM) and a long C-terminal regulatory domain. Six extracellular (EL) and five intracellular loops (IL) are mapped. CHP binds to the juxtamembrane cytoplasmic domain. Arg440 was identified as an important residue for NHE1 regulation. (B) Amino-acid sequences for human NHE1–NHE5 (Genbank Accession Numbers: NM_003047, NM_003048, NM_004174, NM_177084, and NM_004594, respectively) are aligned together with sequence of CNB-binding region in CNA (Q08209). Hydrophobic residues involved in interaction with CHP or CNB are highlighted in green. Residues in NHE1 forming hydrogen bonds with CHP2 are shown in red. (C) Amino-acid sequences of CHP1/2 and CNB (Q99653, Q9D869, and P63098). Secondary structural elements are based on the structure of CHP2/NHE1-peptide complex. Loops of four EF-hands are shown by green boxes, but EF1 and EF2 are ancestral sites that do not coordinate Ca2+. Hydrophobic residues involved in interaction with NHE1 or CNA are highlighted in blue and red, respectively. Central CHP-unique region is boxed. Residues in CHP2 forming hydrogen bonds with NHE1 are shown in red. Download figure Download PowerPoint Of the signaling molecules that interact with the exchanger, calcineurin B (CNB) homologous protein (CHP) is particularly important. CHP was initially identified as a protein (p22) involved in vesicular transport (Barroso et al, 1996) and that interacts with NHE (Lin and Barber, 1996). CHP is a Ca2+-binding protein with EF-hand motifs and is myristoylated at the N-terminus (Gly2) (Barroso et al, 1996; Lin and Barber, 1996). To date, three CHP isoforms with different tissue expression patterns have been identified in mammalian tissues. While CHP1 is expressed ubiquitously in virtually all tissues, the expression of CHP2 is restricted to cancer cells (Pang et al, 2002) and the small intestine (Inoue et al, 2003), and that of CHP3 (also called tescalcin) is restricted to the heart, brain, stomach, and testis (Mailander et al, 2001; Perera et al, 2001; Gutierrez-Ford et al, 2003). Previously, we reported that CHP (at least CHP1 and CHP2) is an essential cofactor supporting the physiological activity of the plasmalemmal NHE by interacting with the juxtamembrane cytoplasmic domain of NHE1 to NHE3 (Pang et al, 2001, 2002). CHP binding-defective mutant exchangers (NHE1-3) exhibited low exchange activity (Pang et al, 2001) (5–10% of the wild-type level) and greatly shifted the pHi-dependence to the acidic side (Pang et al, 2004), suggesting that the juxtamembrane region with bound CHP would therefore function as a key structure maintaining the physiologically active conformation of NHE1. Recently, the crystal structure of CHP1 was solved as a single polypeptide without the target molecule (Naoe et al, 2005). However, this structure did not provide information about the interaction(s) with NHE. To obtain insights into the molecular mechanisms of the interaction(s) with NHE and regulation of NHE by CHP, we determined the crystal structure of CHP (CHP2 isoform) complexed with its binding region in NHE1. Herein, we report that the α-helical CHP-binding region of NHE1 is inserted into the hydrophobic cleft formed by residues in the N- and C-lobes of CHP2 through strong hydrophobic interactions as well as hydrogen bond formation. Structure-based mutational analysis revealed that CHP may activate the exchanger by associating its hydrophobic cleft and also regulate the pH-sensing of NHE1 via its central CHP-unique region connecting the two lobes of CHP. Results Overall structure of CHP2 in complex with the cytoplasmic binding region of NHE1 We crystallized the His6-tagged human CHP2 (1–196) complexed with its binding domain (aa 503–545) in NHE1 (referred to as CHP2/NHE1-peptide) in the presence of yttrium, initially solved the structure by MAD phasing, and refined the structure model to 2.7 Å (Table I). Figure 2 shows the overall structure of the complex CHP2/NHE1-peptide. The structural model contains 181 amino acids from CHP2 (Ile11–Glu99 and Lys105–Lys196), and 25 amino acids from NHE1 fragment (Arg516–His540). The polypeptide chain of CHP2 with dimensions of 55 × 47 × 28 Å is folded into two globular domains (N- and C-terminal lobes) composed of 12 α-helices (α1–α12) and four short β-strands (β1–β4). Two Y3+ ions were found to associate with EF3 and EF4 in the C-lobe (Figure 2), consistent with biochemical data showing the high-affinity binding of Y3+ (∼1 nM) to CHP2 (Supplementary Figure 1). The overall structure of CHP2 is close to the Ca2+-bound form of NHE-free CHP1, which was solved recently (Naoe et al, 2005; Supplementary Figure 2). Indeed, the distances between metal ions and coordinating oxygen atoms are almost the same for Y3+ in CHP2 (2.38±0.2 Å) and Ca2+ in CHP1 (2.40±0.1 Å). In addition, the Y3+-bound form of CHP2 preserved the ability to interact with the full-length NHE1 (Supplementary Figure 1). Therefore, we consider our solved structure compatible with the Ca2+-bound form of CHP2. The overall structure of CHP2 is also similar in folding topology to the structures of other EF-hand-containing proteins, including CNB (Kissinger et al, 1995), K+-channel interacting protein (KChIP1) (Zhou et al, 2004; Supplementary Figure 2), AtCBL2 (Nagae et al, 2003), NCS-1 (Bourne et al, 2001), neurocalcin (Vijay-Kumar and Kumar, 1999), and CIB (Gentry et al, 2005). A common structural feature of these proteins is that they all have four EF-hands in pairs such that EF1 and EF2 form the N-lobe and EF3 and EF4 form the C-lobe. However, some local differences were observed between CHP2 and CNB or between CHP2 and KChIP1. For example, the orientation of EF-hand α-helices is different among these molecules because of the swiveling motion between the N- and C-lobes of each molecule, although each pair of EF hands is relatively fixed via hydrogen bonds. This may explain the incomplete superposition between overall structures of CHP2 and CNB (root mean square deviation, r.m.s.d. 1.7 Å), in contrast to the relatively good superposition between the N-lobes or between the C-lobes themselves (r.m.s.d. 1.3 or 1.0 Å, respectively). Although CHP2 and CNB have about 36% identity in their amino-acid sequences (Figure 1C), CHP2 coordinates two Ca2+ ions in EF3 and EF4, unlike CNB capable of coordinating four Ca2+ ions in its four EF-hands (Kissinger et al, 1995). Lack of Ca2+-coordination in EF1 and EF2 would be derived from significant deviation from the canonical EF-hand sequence and resulting atypical structure, as described previously in detail (Naoe et al, 2005). Figure 2.Stereo view of the CHP2/NHE1-peptide complex showing the overall structure. N- and C-lobes of CHP2 are colored red and blue, respectively. The NHE1 peptide is colored green. Pink spheres represent the two yttrium ions coordinated by EF3 and EF4. Download figure Download PowerPoint Table 1. Data collection, phasing and refinement statistics High resolution data MAD Data collection Space group P43 P43 Cell dimensions a, b, c (Å) 49.96, 49.96, 103.2 49.96, 49.96, 103.2 α, β, γ (deg) 90, 90, 90 90, 90, 90 Peak Inflection Remote Wavelength 1.0000 0.7270 0.7266 1.0000 Resolution (Å) 50–2.7 50–3.0 (3.11–3.00) 50–3.0 (3.11–3.00) 50–3.0 (3.11–3.00) Rmergea 4.8 (25.1) 0.084 (0.311) 0.073 (0.297) 0.072 (0.330) I/σIb 17.3 (4.5) 14.9 (4.1) 15.9 (5.0) 15.8 (3.6) Completeness (%)b 97.3 (83.1) 98.5 (87.5) 98.5(87.5) 97.8 (80.9) Redundancyb 4.0 (3.1) 7.2 (5.6) 7.3 (5.7) 7.1 (5.3) Refinement Resolution (Å) 50–2.7 No. reflections 6746 Rwork/Rfree 0.218/0.287 No. atoms Protein 1674 Ligand/ion 2 B-factors Protein 72.5 Ligand/ion 70.5 R.m.s. deviations Bond lengths (Å) 0.005 Bond angles (deg) 1.00 Ramachandran plot (%) Favorable 87.2 Allowed 12.8 Generously allowed 0 Disallowed 0 a , where Ii(hkl) is the ith intensity measurement of reflection hkl, and 〈I(hkl)〉 is its average. b Highest resolution shell is shown in parentheses. The most remarkable difference between CHP2 and other calcium binding proteins is that the N- and C-lobes of CHP2 are linked by a long flexible region, which protrudes by a length of about 14 Å for the determined region (Figure 3A). This domain, referred to as the CHP-unique region, was not found in other Ca2+-binding proteins. Five residues, Thr100 to Lys104, were not assigned in the structure due to the absence of their electron density map. Particularly, the region from aa 93–108 did not make any contact with other regions of CHP2. In addition, we found that the side chain oxygen atoms of Asp95 in this region coordinate Y3+ in the neighboring molecule in the crystal and thus participate in stabilization of this region. This observation explains why CHP2/NHE1-peptide was crystallized only in the presence of Y3+. These findings suggest that the CHP-unique region is very flexible in aqueous solution. This is consistent with the finding that this region was not seen in the structure of NHE-free CHP1 (Naoe et al, 2005), which was solved in different crystal packing. Figure 3.Target specificity of hydrophobic cleft. (A–C) Surface features of CHP2, CNB, and KChP1 are presented together with α-helices of target peptides, respectively. In CHP2, front (upper) and side (lower) views are shown. The N- and C-lobes are colored light blue and light green, while the CHP-unique region is colored red. (D, E) Surface view of NHE1 (left) and CNA (right) peptides, respectively. The upper panels indicate the side facing outside the cleft and the lower panels represent the side facing the cleft. Hydrophobic residues are colored yellow. Download figure Download PowerPoint Interaction between CHP2 and its binding domain in NHE1 Structure determination of the complex revealed the orientation of CHP2 toward the cytoplasmic region of NHE1. The N- and C-terminal halves of the cytoplasmic α-helix of NHE1 are inserted into the hydrophobic cleft in such a way that they face the C- and N-lobes of CHP2, respectively (Figure 3A). This cleft constitutes the region that traps the cytoplasmic α-helix of NHE1 and maintains the stability of the complex by means of hydrophobic interactions. The contact area between CHP2- and NHE1 peptide is 1466 Å2, which accounts for 13.0 and 59.8% of the total surface areas, respectively, implying that a large area is used for interaction between the two molecules. The NHE1 peptide (Arg516–His540) solved in this structure contains different hydrophobic residues, all of which face toward the hydrophobic cleft of CHP2 (Figure 3D). Ile518, Ile522, Phe526, Leu527, and Leu530 are accommodated into the cavity formed by hydrophobic residues provided mainly from the C-lobe of CHP2, whereas Leu531, Ile534, and Ile537 are inserted into the cavity formed mainly by the N-lobe of CHP2 (Figure 4A and B). All hydrophobic side chains of the NHE1 peptide, with the exception of Ile518, make van der Waals contacts with side chain atoms from many hydrophobic residues of CHP2. For example, side chain atoms of Ile534 make van der Waals contacts with those of Leu57, Ile66, Phe70, Val86, Leu87 Phe90, Tyr118, and Ile192 of CHP2 (Figure 4C), while side chain atoms of Leu527 make interactions with those of Ala119, Leu181, Val186, Met184, and Met190 (Figure 4A). In addition to hydrophobic interactions, hydrogen bonds strengthen the association between CHP2 and NHE1 peptide. In the N-lobe of CHP2, the guanidinium group of Arg30 makes hydrogen bonds with the main chain carbonyl oxygen of Gly539, while the guanidinium group of Arg34 makes hydrogen bonds with the main chain carbonyl oxygens of Asp536 and Ile537 (Figure 4B). These hydrogen bonds are very important for the C-terminal end of the NHE1 helix to fix to the edge of the hydrophobic cleft of CHP2. The conserved Gly539 residue of NHE1 would allow the following cytoplasmic stretch to leave the cleft by inducing clear bending of the NHE1 peptide (Figures 3A and 4B). Several other hydrogen bonds are also formed. The hydrogen bond between the imidazole group (Nδ) of His523 in NHE1 and the side chain oxygen of Tyr123 in CHP2 is particularly interesting because it is the only polar interaction in the hydrophobic surface of the cleft (Supplementary Figure 3). These polar interactions are important to determine the specificity and orientation of CHP2 toward the cytoplasmic α-helix of NHE1. Figure 4.Closeup view showing the interaction between CHP2 and NHE1-peptide. (A) The NHE1-peptide backbone is shown in magenta, while hydrophobic side chains are shown in yellow. The hydrophobic pocket surrounding Leu527 is marked in red. His523 of NHE1 and Tyr123 of CHP2 are marked in blue. (B) Closeup view showing interaction between the N-lobe of CHP2 and the C-terminus of NHE1-peptide. (C) Ile534 is accommodated into the hydrophobic pocket produced by residues mainly in the N-lobe of CHP2. Download figure Download PowerPoint Although CNB and KChIP1 have similar hydrophobic clefts (Figure 3B and C), their size and shape are clearly different from those of CHP2. Interestingly, target peptides show sequence features that just fit to their clefts. While NHE1 peptide shows a uniform distribution of hydrophobic residues all facing toward the cleft of CHP2 (Figure 3D), CNA contains more hydrophobic residues that face toward both the CNB crevice and the outside (Figure 3E). In addition, CNA peptide contains four aromatic residues (Phe346, Phe350, Trp352, and Phe356) in the N-terminus, which are accommodated in corresponding hydrophobic cavities in the C-lobe of CNB, whereas NHE1-peptide contains only one aromatic residue (Phe526) located in the central part of CHP2. These observations suggest that highly selective fitting of each peptide to its corresponding crevices may be caused by differences in the distribution of hydrophobic residues of the target peptides and in the shape of the cleft formed by α-helices of EF-hands. Effect of mutations on interaction between NHE1 and CHP2 in cells The present structure indicated that the correct CHP-binding domain of NHE1 consists of the region from aa 516–540, extended by 10 residues toward the C-terminus of the region (aa 510–530), as predicted previously (Pang et al, 2001). This prompted us to mutate residues in this extended region of NHE1 interacting with the N-lobe of CHP2. We mutated the Ile534 and Ile537 residues of NHE1 into charged residues (Lys or Asp) and stably expressed these mutants in exchanger-deficient PS120 cells. These mutants were expressed in the plasma membrane (as indicated by the existence of surface-expressed mature NHE1 in Supplementary Figure 4). We examined the interactions of these mutants with CHP2 by assessing whether stably transfected green fluorescent protein (GFP)-tagged CHP2 was localized to the plasma membrane. As shown in Figure 5A, most of the CHP2 was localized to the plasma membrane of cells expressing the wild-type NHE1 but not in nontransfected PS120 cells (not shown; see Pang et al, 2001), indicating a strong interaction. Of note, in most cells expressing the wild-type NHE1 almost no GFP-fluorescence was detected in the intracellular space (Figure 5A), despite the accumulation of immature NHE1 in the intracellular membranes under forced expression (data not shown), suggesting that CHP2 interacts predominantly with mature NHE1 in the plasma membrane. This was further confirmed by the finding that almost no immature NHE1 co-immunoprecipitated with CHP2 (Supplementary Figures 1 and 4). In contrast to the wild-type NHE1, the plasma membrane localization of CHP2 was almost completely abolished when transfected into cells expressing the NHE1 mutants, I534K, I534D and I537K (Figure 5, and also D for summarized data), indicating that these mutations drastically reduced the affinity for interaction with CHP2. Figure 5.Effects of mutations on the interaction between NHE1 and CHP2 in cells. (A) Low magnification confocal images of cells coexpressing GFP-tagged CHP2 and the wild-type (left) or I537K (right) mutant exchangers. Inset shows the intensity profile of GFP fluorescence along the dotted line in a marked cell. In most cells expressing the wild-type NHE1 but not I537K, strong fluorescent signals were detected at the cell edge. (B) Subcellular localization of CHP2 expressed in cells. GFP-tagged CHP2 was coexpressed in cells stably expressing the wild-type or mutant NHE1 variants and GFP-fluorescence was observed by confocal microscopy. (C) Subcellular localization of mutant CHP2. GFP-tagged CHP2 mutants were expressed in cells stably expressing the wild-type NHE1. Eleven residues from Glu94 to Lys104 of CHP2 were deleted in Δ94–104, while the 10 N-terminal residues from Met1 to Val10 were deleted in ΔN10. For one control experiment, GFP-tagged human CNB was expressed in NHE1-transfectants. (D) Summary data for membrane localization of GFP-tagged CHP2. Intensity profile analysis was performed on confocal images as shown in (A). The number of cells with strong fluorescence signal at the cell edge (at least three times more than the average of fluorescence in the internal cell region) was counted. Data are expressed as the mean±s.d. from 6–8 images (total cell number analyzed, 99–341). Download figure Download PowerPoint Furthermore, as expected, the plasma membrane localization of CHP2 was also markedly reduced in cells expressing the wild-type NHE1 when Leu87 or Tyr118 of CHP2 was changed to Lys (Figure 5C and D), indicating that these mutants are not able to replace completely endogenous CHP1 bound to NHE1. As the expression of GFP-tagged CHP2 was approximately 10-fold higher than that of endogenous CHP1 (data not shown), these results suggest that these mutations would reduce the affinity for interaction with NHE1 at least one-order of magnitude. Similar analysis was performed with mutation of the Arg residues (Arg30 and Arg34) in the N-terminal α-helix of CHP2. The plasma membrane localization of CHP2 was abolished when these two residues were simultaneously changed to Glu (Figure 5C and D), while it was still partially preserved when either Arg residue alone was changed to Glu. Similar results were also obtained for the NHE1/CHP2 interaction in a co-immunoprecipitation experiment (Supplementary Figure 4). Thus, the hydrophobic and polar interactions involving these residues would be important in determining the strong and highly specific association between NHE1 and CHP2, Furthermore, in addition to the C-lobe, the N-lobe of CHP2 plays a crucial role in the tight interaction with NHE1, suggesting that our crystal structure reflects the interaction between native NHE1 and CHP2 molecules expressed in cells. On the other hand, structure determination indicated the existence of a unique protruding region in CHP2. In contrast to mutations of residues involving formation of the cleft, a mutant with deletion of this CHP-unique region (Δ94–104) was mostly localized to the plasma membrane of cells expressing wild-type NHE1 (Figure 5C), indicating that deletion did not disrupt the interaction of CHP2/NHE1. Hence, as also predicted from structure, the CHP-unique region was not involved in the interaction between CHP2/NHE1. The 10 N-terminal residues of CHP2 were also found not to be involved in the interaction, as indicated by the plasma membrane localization of ΔN10 (Figure 5C). Functional consequence of mutations We measured the NHE activity in cells expressing mutant exchangers. Figure 6A demonstrates the pHi-dependence of EIPA-sensitive 22Na+-uptake activity. While the wild-type NHE1 exhibited high 22Na+-uptake activity, with pK ∼6.5 for pHi (Hill coefficient, ∼1.7), the activity of mutant exchangers, I534D, I534K and Ile537, which lack CHP-binding was very low (see Supplementary Table I for kinetic parameters). The inhibitory effect of the mutations was characterized by changes in two parameters: (i) a drastic reduction of maximal 22Na+-uptake activity (Vmax) at acidic pHi, and (ii) a large acidic shift of the pHi-dependence of uptake (>0.6 pH units) (see Figure 6A, inset for normalized activity). We then compared the pHi-dependence of 22Na+-uptake in cells co-expressing NHE1 with GFP-tagged CHP2 or its deletion mutant Δ94–104, which preserves interaction with CHP. Interestingly, expression of Δ94–104 significantly shifted the pHi-dependence of uptake towards acidic pHi (∼0.2 pH unit) without any change in Vmax, while it preserved the cooperative pHi dependence, with a Hill coefficient ∼1.5 (Figure 6B, see also inset for the sigmoidal internal H+ dependence), suggesting that the CHP-unique region is involved in regulation of pH-sensing of NHE1. In general, the sigmoidal H+-dependence has been interpreted as exhibiting involvement of at least two H+-binding sites: H+-transport and H+-regulatory sites in NHE (Wakabayashi et al, 1997). To examine the effect of deletion of a CHP-specific region on the H+-regulatory site, we measured the reverse reaction of NHE1, that is, 22Na+ efflux coupled to H+-influx. If we assume that NHE1 catalyzes a counter-transport reaction only involving the transport site, intracellular acidification should result in inhibition of 22Na+ efflux due to cytosolic Na+/H+ competition. In contrast to this thermodynamically expected inhibition, modest acidification from 7.5 to 7.2 dramatically stimulated the rate of EIPA-inhibitable 22Na+ efflux in cells co-expressing the wild-type NHE1 and CHP2 (Figure 6D), suggesting that protonation of H+-regulatory sites activated NHE1 (Figure 6C), as reported in detail, previously (Aronson et al, 1982; Wakabayashi et al, 2003a). Consistent with the acidic shift of pHi-dependence of 22Na+ uptake, we observed that deletion of a CHP-unique region significantly reduced the rate of 22Na+ efflux (Figure 6D). EIPA-sensitive fraction of 22Na+ efflux at pHi 7.2 during the initial 3 min was reduced to ∼30% upon expression of Δ94–104, suggesting that the affinity of the H+-regulatory site for H+ decreased upon deletion of this region. Figure 6.Effect of mutations on the exchange activity. (A), pHi-dependence of EIPA-sensitive 22Na+-uptake in cells expressing wild-type NHE1 (•) or CHP binding-defective mutants, I534D (○), I534K (▴) and I537K (▵). pHi was clamped at various values with K+/nigericin. Data were fitted to Hill equations with the kinetic parameters shown in Supplementary Table I and plotted after normalization by the maximal activity at pHi=5.4 (inset). (B) pHi-dependence of EIPA-sensitive 22Na+-uptake in cells co-expressing wild-type NHE1 and GFP-tagged CHP2 (•) or deletion mutant Δ94–104 of CHP2 (○). Data were fitted to Hill equations with the kinetic parameter