1.A.54 The Presenilin ER Ca2+ Leak Channel (Presenilin) Family

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder that affects ~2% of the population in industrialized countries. Mutations in presenilins 1 and 2 (PS1 and PS2) account for ~40% of familial AD cases (Tandon and Fraser, 2002). Familial AD mutations and genetic deletions of presenilins have been associated with calcium (Ca2+) signaling abnormalities. Presenilins regulate capacitative calcium entry independently of gamma-secretase activity. Tu et al. (2006) have demonstrated that wild-type presenilins, but not PS1-M146V and PS2-N141I familial AD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout mouse embryonic fibroblasts, they found that presenilins account for 80% of passive Ca2+ leak from the endoplasmic reticulum. Deficient Ca2+ signaling in double knockout fibroblasts can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca2+ leak function of presenilins is independent of their γ-secretase activity. The data of Tu et al. (2006) suggest a Ca2+ signaling function for presenilins and provide support for the 'Ca2+ hypothesis of AD.' The presenilin 1 calcium leak conductance pore involves TMSs 7 and 9, but not 6, as well as a hydrophilic catalytic cavity (Nelson et al., 2011).  Presenilins play a role in calcium-mediated lysosomal fusion (Bezprozvanny 2012; Coen et al. 2012).  Individual presenilin TMSs play roles in the cleavage of C99 and the generation of Abeta peptides (Schmidt et al. 2023).

Familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) (M146L) and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+ signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing, an important feature of AD pathology. These observations provide molecular insights into the 'Ca2+ dysregulation' hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention (Cheung et al., 2008).

Most cases of AD are idiopathic and are characterized by late onset (>60 years of age). A small fraction of AD cases (familial AD) are characterized by an earlier onset and genetic inheritance. Presenilins are 50 kDa proteins that contain nine transmembrane domains (Laudon et al., 2005) and reside in the endoplasmic reticulum (ER) membrane. They maintain a 9 TMS topology throughout the secretory pathway (Spasic et al., 2006). The complex of presenilins with nicastrin (NCT), APH-1 and PEN-2 subunits functions as γ-secretase, which cleaves the amyloid precursor protein (APP) and releases the amyloid β-peptide (Aβ), the principal constituent of the amyloid plaques in the brains of AD patients. Consistent with the role of presenilins as catalytic subunits of γ-secretase, familial AD mutations in presenilins affect APP processing. These familial AD mutations also result in deranged calcium (Ca2+) signaling (reviewed in Smith et al., 2005). Tu et al. (2006) established that presenilins function as passive ER Ca2+ leak channels and that the familial AD mutations of presenilins affect their ability to conduct Ca2+ ions. These results provide new insight into the normal physiological function of presenilins and strengthen the emerging connection between deranged neuronal Ca2+ signaling and AD.

Presenilin-1 (γ-secretase) is a multisubunit aspartate protease requiring the coordinated action of presenilins (PSs) (Wolfe and Kopan, 2004), nicastrin (NCT), PEN-2 and APH-1 and is crucial for the intramembrane proteolysis of type I membrane proteins such as the amyloid precursor protein (APP) and Notch. The catalytic component, PS1, is a polytopic membrane protein that undergoes endoproteolysis resulting in stable PS1 NH2- and COOH-terminal fragments (PS1-NTF and -CTF). PS1 has 9 TMSs (Spasic et al., 2006).

Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid, belonging to a family of evolutionarily conserved integral membrane proteases that participate in signaling. The yeast orthologue is involved in mitochondrial fusion. PARL associates with presenilins 1 and 2 and cleaves type 1 transmembrane proteins. Parl-/- mice undergo progressive atrophy leading to death, due to increased apoptosis. Parl-/- cells are not protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1 (Cipolat et al., 2006).

The hydrophilic 'catalytic pore' structure of γ-secretase is formed by TMSs 6, 7 and 9 of presenilin 1 (PS1), the catalytic subunit of γ-secretase in the membrane. The first hydrophobic region, putative TMS1 of PS1, is located in proximity to the catalytic GxGD and PAL motifs within the C-terminal fragment of PS1, facing directly the catalytic pore (Takagi et al., 2010).

Presenilins show a region of similarity (residues 217-305 in PS-2) with the 'Bacteroides development protein' (TC #9.A.18.1.2) BacA (residues 53-149 in this 420 aa protein) of Rhizobium meliloti (e-value of 0.001). Most homologues are from eukaryotes (both plants and animals), but a distant homologue is found in the halobacterial archaeon, Haloquadratum walsbyi (TC #1.A.54.2.1). Many other archaea encode homologues in their genomes, but they do not appear to be present in bacteria.

Kuo et al. 2015 examined two archaeal GxGD proteases (PSH and FlaK), with known three-dimensional structures. Both are in the same GxGD family as presenilin, a protein mutated in Alzheimer's Disease. They demonstrated that PSH and FlaK form cation channels in lipid bilayers. A mutation that affected the enzymatic activity of FlaK rendered the channel catalytically inactive and altered the ion selectivity, indicating that the ion channel and the catalytic activities are linked. Thus, PSH and FlaK, are true 'chanzymes' with interdependent ion channel and protease activity conferred by a single structural domain embedded in the membrane, supporting the proposal that higher-order proteases, including presenilin, have channel function.

Intramembrane proteolysis involves the cleavage of substrate proteins within their hydrophobic TMSs. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 (see TC subfamily 1.A.54.3). As presenilin homologues, they employ a similar catalytic mechanism as the well-studied gamma-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. Mentrup et al. 2020 summarized how phenotypes  are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events provide specific regulatory switches. Then many pathways are influenced including signal transduction, membrane trafficking and protein glycosylation.

The transport reactions catalyzed by presenilins is:

Ca2+ and other cations (out) → Ca2+ and other cations (in)



Annaert, W. and B. De Strooper. (2002). A cell biological perspective on Alzheimer's disease. Annu. Rev. Cell Dev. Biol. 18: 25-51.

Bezprozvanny, I. (2012). Presenilins: a novel link between intracellular calcium signaling and lysosomal function? J. Cell Biol. 198: 7-10.

Cheung, K.H., D. Shineman, M. Müller, C. Cárdenas, L. Mei, J. Yang, T. Tomita, T. Iwatsubo, V.M. Lee, and J.K. Foskett. (2008). Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating. Neuron. 58: 871-883.

Cipolat, S., T. Rudka, D. Hartmann, V. Costa, L. Serneels, K. Craessaerts, K. Metzger, C. Frezza, W. Annaert, L. D'Adamio, C. Derks, T. Dejaegere, L. Pellegrini, R. D'Hooge, L. Scorrano, and B. De Strooper. (2006). Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126: 163-175.

Coen, K., R.S. Flannagan, S. Baron, L.R. Carraro-Lacroix, D. Wang, W. Vermeire, C. Michiels, S. Munck, V. Baert, S. Sugita, F. Wuytack, P.R. Hiesinger, S. Grinstein, and W. Annaert. (2012). Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells. J. Cell Biol. 198: 23-35.

Dehury, B., N. Tang, and K.P. Kepp. (2019). Insights into membrane-bound presenilin 2 from all-atom molecular dynamics simulations. J Biomol Struct Dyn 1-27. [Epub: Ahead of Print]

Eckert, G.P. and W.E. Müller. (2009). Presenilin 1 modifies lipid raft composition of neuronal membranes. Biochem. Biophys. Res. Commun. 382: 673-677.

Hu, J., Y. Xue, S. Lee, and Y. Ha. (2011). The crystal structure of GXGD membrane protease FlaK. Nature 475: 528-531.

Jules, F., E. Sauvageau, K. Dumaresq-Doiron, J. Mazzaferri, M. Haug-Kröper, R. Fluhrer, S. Costantino, and S. Lefrancois. (2017). CLN5 is cleaved by members of the SPP/SPPL family to produce a mature soluble protein. Exp Cell Res 357: 40-50.

Kim, J. and R. Schekman. (2004). The ins and outs of presenilin 1 membrane topology. Proc. Natl. Acad. Sci. USA 101: 905-906.

Kuo, I.Y., J. Hu, Y. Ha, and B.E. Ehrlich. (2015). Presenilin-like GxGD membrane proteases have dual roles as proteolytic enzymes and ion channels. J. Biol. Chem. 290: 6419-6427.

Laudon, H., E.M. Hansson, K. Melen, A. Bergman, M.R. Farmery, B. Winblad, U. Lendahl, G. von Heijne, and J. Naslund. (2005). A nine-transmembrane domain topology for presenilin 1. J. Biol. Chem. 280: 35352-35360.

Li, X., S. Dang, C. Yan, X. Gong, J. Wang, and Y. Shi. (2013). Structure of a presenilin family intramembrane aspartate protease. Nature 493: 56-61.

Mentrup, T., F. Cabrera-Cabrera, R. Fluhrer, and B. Schröder. (2020). Physiological functions of SPP/SPPL intramembrane proteases. Cell Mol Life Sci 77: 2959-2979.

Moliaka, Y.K., A. Grigorenko, D. Madera, and E.I. Rogaev. (2004). Impas 1 possesses endoproteolytic activity against multipass membrane protein substrate cleaving the presenilin 1 holoprotein. FEBS Lett. 557: 185-192.

Naing, S.H., S. Kalyoncu, D.M. Smalley, H. Kim, X. Tao, J.B. George, A.P. Jonke, R.C. Oliver, V.S. Urban, M.P. Torres, and R.L. Lieberman. (2018). Both positional and chemical variables control proteolytic cleavage of a presenilin ortholog. J. Biol. Chem. 293: 4653-4663.

Nelson, O., C. Supnet, A. Tolia, K. Horré, B. De Strooper, and I. Bezprozvanny. (2011). Mutagenesis mapping of the presenilin 1 calcium leak conductance pore. J. Biol. Chem. 286: 22339-22347.

Romero-Molina, C., F. Garretti, S.J. Andrews, E. Marcora, and A.M. Goate. (2022). Microglial efferocytosis: Diving into the Alzheimer''s disease gene pool. Neuron. 110: 3513-3533.

Sato, C., Y. Morohashi, T. Tomita, and T. Iwatsubo. (2006). Structure of the catalytic pore of γ-secretase probed by the accessibility of substituted cysteines. J. Neurosci. 26: 12081-12088.

Schmidt, F.C., K. Fitz, L.P. Feilen, M. Okochi, H. Steiner, and D. Langosch. (2023). Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin. J. Biol. Chem. 299: 104626. [Epub: Ahead of Print]

Smeijers, A.F., K. Pieterse, A.J. Markvoort, and P.A. Hilbers. (2006). Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion. J Phys Chem B 110: 13614-13623.

Smith, I.F., K.N. Green, and F.M. LaFerla. ((2005)). Calcium dysregulation in Alzheimer's disease: recent advances gained from genetically modified animals. Cell Calcium 38: 427-437.

Spasic, D., A. Tolia, K. Dillen, V. Baert, B. De Strooper, S. Vrijens, and W. Annaert. (2006). Presenilin-1 maintains a nine-transmembrane topology throughout the secretory pathway. J. Biol. Chem. 281: 26569-26577.

Takagi, S., A. Tominaga, C. Sato, T. Tomita, and T. Iwatsubo. (2010). Participation of transmembrane domain 1 of presenilin 1 in the catalytic pore structure of the γ-secretase. J. Neurosci. 30: 15943-15950.

Tandon, A. and P. Fraser. (2002). The presenilins. Genome Biol. 3: (E-pub).

Tu, H., O. Nelson, A. Bezprozvanny, Z. Wang, S.F. Lee, Y.H. Hao, L. Serneels, B. De Strooper, G. Yu, and I. Bezprozvanny. (2006). Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell 126: 981-993.

Wolfe, M.S. and R. Kopan. (2004). Intramembrane proteolysis: theme and variations. Science 305: 1119-1123.


TC#NameOrganismal TypeExample

Presenilin-1 (PS-1; PS1; PSEN1; PSNL1; STM-1; E5-1; AD; AD3) of 467 aas and 9 or 10 TMSs in a 6 or 7 + 3 TMS arrangement. Ca2+ leak channel (part of the γ-secretase complex; expression alters the lipid raft composition in neuronal membranes (Eckert and Müller, 2009)). The first 5 TMSs of presenilin-1 are homologous to the 5 TMS CD47 antigenic protein, a constituent of the osteoclast fusion complex (1.N.1.1.1), and CD47 is therefore a presenilin homologue.  The active site of gamma-secretase resides in an aqueous catalytic pore within the lipid bilayer and is tapered around the catalytic aspartates (Sato et al. 2006). TMS 6 and TMS 7 contribute to the hydrophilic pore. Residues at the luminal portion of TMS 6 are predicted to form a subsite for substrate or inhibitor binding on the α-helix facing the hydrophilic milieu, whereas those around the GxGD catalytic motif within TMS 7 are water accessible (Sato et al. 2006). Mutations in PSEN1 or PSEN2 (TC# 1.A.5.1.2) can lead to Altzheimer's disease (Romero-Molina et al. 2022).


Presenilin-1 of Homo sapiens (P49768)


Presenilin-2 (PS-2; STM-2; E5-2; AD3 LP; AD5 PSN-2) Ca2+ leak channel of 448 aas and 9 TMSs. Presenilins 1 and 2 (PS1 & PS2) are main genetic risk factors of familial Alzheimer's disease (AD) that produce the beta-amyloid (Abeta) peptides. They also function in calcium signaling (Dehury et al. 2019). Mutations in both cause AD. The 9-TMS channel structure is substantially controlled by major dynamics in the hydrophilic loop bridging TMS6 and TMS7, which functions as a "plug" in the PS2 membrane channel. TMS2, TMS6, TMS7 and TMS9 flexibility controls the size of this channel. Most pathogenic PS2 mutations reduce stability relative to random mutations (Dehury et al. 2019).


Presenilin-2 of Homo sapiens (448 aas; P49810)


TC#NameOrganismal TypeExample

Archaeal presenilin homologue (DUF1119; COG3389; PSN). Members of the peptidase A22B superfamily (found in many archaea, but not bacteria, shows some sequence similarity to members of the LIV-E family, e.g., 2.A.78.2.1))


PSN of Haloquadratum walsbyi (339 aas; 9 TMSs; CAJ51633)


Presenilin homologue (DUF1119) of 301 aas and 9 TMSs with known 3-d structure. The amino-terminal domain, consisting of TM1-6, forms a horseshoe-shaped structure, surrounding TM7-9 of the carboxy-terminal domain. The two catalytic aspartate residues are located on the cytoplasmic side of TMS 6 and TMS 7, spatially close to each other and approximately 8 Å into the lipid membrane surface. Water molecules gain constant access to the catalytic aspartates through a large cavity between the amino- and carboxy-terminal domains. (Li et al. 2013).  Both protease and ion channel activities have been demostrated, and these two activities share the same active site (Kuo et al. 2015). Cleavage is controlled by both positional and chemical factors (Naing et al. 2018).


Presenilin homologue of Methanoculleus marisnigri


TC#NameOrganismal TypeExample

Signal peptide peptidase-2A (SPP2A; 523 aas; 8TMSs) There is no evidence for a transport function for this protease. The functions of these SPP and SPPL proteases have been reviewed (Mentrup et al. 2020).


SPP2A of Mus musculus (Q9JJF9)


Signal peptide peptidase like 2A, SPPL2A


SPPL2A of Homo sapiens


Signal peptide peptidase, SppL3 of 385 aas and 9 TMSs. Cleaves the single TMS in the neuronal ceroid lipofuscinoses (NCLs), a group of proteins causing recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death (Jules et al. 2017). CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. It is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3 into a mature soluble protein consisting of residues 93-407 and an N-terminal fragment is then further cleaved by SPPL3 and SPPL2b and degraded in the proteasome (Jules et al. 2017).


Spp of Homo sapiens


Signal peptide peptidase, Spp


Spp of Drosophila melanogaster


Impas 1 (IMP1, HM13, PSL3, APP, MARP086) possesses endoproteolytic activity against multipass membrane protein substrates, cleaving the presenilin 1 holoprotein (Moliaka et al. 2004).

Impas 1 of Homo sapiens


TC#NameOrganismal TypeExample

The pre-flagelin peptidase of 230 aas and 6 TMSs, FlaK, with known 3-d structure (3.6Å resolution) (Hu et al. 2011).  This protein is a member of the presenilin/GxGD membrane protein family; it plays a dual role as protease and ion-conducting channel and is therefore called a "channzyme" (Kuo et al. 2015).


FlaK of Methanococcus maripaludis


Leader peptidase of 342 aas


Leader peptidase of Natrinema pellirubrum


Type IV leader peptidase of 289 aas and 7 TMSs.


peptidase of Methanobrevibacter smithii


Peptidase of 375 aas


Peptidase of Thermococcus sibiricus


Peptidase of 260 aas


Peptidase of Methanosphaerula palustris


TC#NameOrganismal TypeExample

Prepilliin peptidase A24 of 167 aas and 6 TMSs.


Peptidase of Desulfotomaculum hydrothermale


Peptidase A24 prepilin type IV of 158 aas


Peptidase of Aminobacterium colombiense


Peptidase of 286 aas


Peptidase of Acinetobacter pittii


Leader peptidase, PppA or YghH of 269 aas and 8 TMSs. May be able to flip phospholipids from one lipid monolayer to another as a scramblase (Smeijers et al. 2006).

PppA of E. coli


TC#NameOrganismal TypeExample

Uncharacterized protein of 229 aas and 6 TMSs.


UP of Thermoplasma volcanium