Evidence showing an intermolecular interaction between KChIP proteins and Taiwan cobra cardiotoxins

Direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated in the present study. It was found that KChIPs bound with CTX3, in which KChIP and CTX3 formed a 1:1 complex as evidenced by the results of chemical cross-linking. Pull-down assay revealed that the intact EF-hands 3 and 4 of KChIP1 were critical for CTX3-binding. Likewise, removal of EF-hands 3 and 4 distorted the ability of KChIP1 to bind with Kv4.2 N-terminal fragment (KvN) as well as fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS). In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca(2+)-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca(2+)-bound KChIP1, but not Ca(2+)-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K(+) channel, the potentiality of CTX for this biomedical application could be considered.     

Effects of Metal-Binding Properties of Human Kv Channel-Interacting Proteins on their Molecular Structure and Binding with Kv4.2 Channel

The goal of the present study is to explore whether Ca²⁺ and Mg²⁺-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca²⁺-binding sites, respectively, and only one type of Mg²⁺-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca²⁺, but the binding affinity for Mg²⁺ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg²⁺ and Ca²⁺. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg²⁺ and Ca²⁺. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg²⁺- and Ca²⁺-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.     

Interactions of KChIP4a and its mutants with Ca or Kv4.3 N-terminus by affinity capillary electrophoresis

The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca²⁺ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (K_b). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 10⁶ L mol^–1 and (5.26 ± 0.71) × 10⁶ L mol^–1, respectively. In addition, in the presence of calcium (10 μmol L^–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 10⁷ L mol^–1. In addition, the binding constant of KChIP4a with Ca²⁺ was (7.1 ± 1.5) × 10⁷ L mol^–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca²⁺, and the integrity of the molecular structure of KChIP4a was important for Ca²⁺ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.     

Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

Functional implication with the metal-binding properties of KChIP1

To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca(2+)-binding sites, high-affinity and low-affinity Ca(2+)-binding sites. However, only low-affinity-binding site for Mg(2+), Sr(2+), and Ba(2+) was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca(2+)-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1.     

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.     

Structural insights into the functional interaction of KChIP1 with Shal-type K(+) channels

Four Kv channel-interacting proteins (KChIP1 through KChIP4) interact directly with the N-terminal domain of three Shal-type voltage-gated potassium channels (Kv4.1, Kv4.2, and Kv4.3) to modulate cell surface expression and function of Kv4 channels. Here we report a 2.0 Angstrom crystal structure of the core domain of KChIP1 (KChIP1*) in complex with the N-terminal fragment of Kv4.2 (Kv4.2N30). The complex reveals a clam-shaped dimeric assembly. Four EF-hands from each KChIP1 form each shell of the clam. The N-terminal end of Kv4.2 forming an alpha helix (alpha1) and the C-terminal alpha helix (H10) of KChIP1 are enclosed nearly coaxially by these shells. As a result, the H10 of KChIP1 and alpha1 of Kv4.2 mediate interactions between these two molecules, structurally reminiscent of the interactions between calmodulin and its target peptides. Site-specific mutagenesis combined with functional characterization shows that those interactions mediated by alpha1 and H10 are essential to the modulation of Kv4.2 by KChIPs.     

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca²⁺) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca²⁺, DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca²⁺. In the absence of Ca²⁺, DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca²⁺-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μm as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional ¹H,¹⁵N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to ¹⁵N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca²⁺-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca²⁺ also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH₂-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.     

Mutational analysis of Raf-1 cysteine rich domain: Requirement for a cluster of basic aminoacids for interaction with phosphatidylserine

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R¹⁴³, K¹⁴⁴, and K¹⁴⁸ appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.     

Modulation by Clamping: Kv4 and KChIP Interactions

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) α subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K⁺ current, I_SA, in neurons and transient outward current, I_TO, in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein–protein interaction for treatment of membrane excitability-related disorders. Special issue article in honor of Dr. Ji-Sheng Han.     

Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions

Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca²⁺-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca²⁺-dependent binding that became partially Ca²⁺ independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca²⁺-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5 mM Ca²⁺. We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.     

Studies of the slowly exchanging chloride in Photosystem II of higher plants

³⁶Cl^- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of ³⁶Cl^-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10^-4 s^-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on ³⁶Cl^-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pK_a of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br^- and NO₃ ^-. The slowly exchanging Cl^- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S₂ state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S₂ state EPR signals.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - MWCO molecular weight cut off - PPBQ phenyl-p-benzoquinone - PS II Photosystem II     

Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca²⁺ and Cl⁻, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca²⁺ and Cl⁻ retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.     

Expression and high glucose-mediated regulation of K channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K⁺ channels K_V4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K_V4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K⁺ current and an alteration in the inactivation of the macroscopic outward K⁺ currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K_V4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.     

Enhanced Trafficking of Tetrameric Kv4.3 Channels by KChIP1 Clamping

The cytoplamsic auxiliary KChIPs modulate surface expression and gating properties of Kv4 channels. Recent co-crystal structure of Kv4.3 N-terminus and KChIP1 reveals a clamping action of the complex in which a single KChIP1 molecule laterally binds two neighboring Kv4.3 N-termini at different locations, thus forming two contact interfaces involved in the protein–protein interaction. In the second interface, it functions to stabilize the tetrameric assembly, but the role it plays in channel trafficking remains elusive. In this study, we examined the effects of KChIP1 on Kv4 protein trafficking in COS-7 cells expressing EGFP-tagged Kv4.3 channels using confocal microscopy. Mutations either in KChIP1 (KChIP1 L39E-Y57A-K61A) or Kv4.3 (Kv4.3 E70A-F73E) that disrupt the protein–protein interaction within the second interface can reduce surface expression of Kv4 channel proteins. Kv4.3 C110A, the Zn²⁺ binding site mutation in T1 domain, that disrupts the tetrameric assembly of the channels can be rescued by WT KChIP1, but not the KChIP1 triple mutant. These results were further confirmed by whole cell current recordings in oocytes. Our findings show that key residues of second interface involved in stabilizing tetrameric assembly can regulate the channel trafficking, indicating an intrinsic link between tetrameric assembly and channel trafficking. The results also suggest that formation of octameric Kv4 and KChIP complex by KChIPs clamping takes place before their trafficking to final destination on the cell surface. Special issue article in honor of Dr. Ji-Sheng Han.     

Pro-apoptotic function of calsenilin/DREAM/KChIP3.

Apoptotic cell death and increased production of amyloid b peptide (Ab) are pathological features of Alzheimer's disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro-apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide-induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+-ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3-induced apoptosis was suppressed by caspase inhibitor Z-VAD and by Bcl-XL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPSW) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C-terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Ab42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis-associated Ab42 production of calsenilin/DREAM/KChIP3.