Monomeric α-synuclein activates the plasma membrane calcium pump
Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+-ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.
Plasma-membrane Ca2+-ATPase (PMCA) is shown here to be activated by α-synuclein (aSN) in the presence of negatively charged lipids. Colocalization of aSN and PMCA at synapses and aSN stimulated, PMCA dependent calcium extrusion indicate a role in neuronal calcium homeostasis that is supported by mathematical modeling.
The alpha-synuclein protein (aSN, 140 residues and 14.5 kDa molecular weight) is well recognized and highly studied for its pathological role in neurodegeneration. Oligomerization, fibrillization, and abnormal aggregation in neurons are linked to synucleinopathies, e.g., Lewy body dementia or Parkinson's disease (PD) (Burre et al, 2018; Oliveira et al, 2021). However, the normal function of native aSN remains an open question. Importantly, aSN is highly abundant in the presynaptic region of neurons, where concentrations reach 5–50 μM (Bodner et al, 2009; Theillet et al, 2016; Perni et al, 2017). It is likely involved in homeostasis of synaptic vesicle release (Sulzer & Edwards, 2019) although mechanisms remain unclear, but a chaperone function of SNARE proteins has been proposed (Burre et al, 2010). A tetrameric form was isolated from cells after in vivo cross-linking (Dettmer et al, 2013, 2015a); however, the intact aSN appears structurally disordered and monomeric in cells (Fauvet et al, 2012; Theillet et al, 2016). aSN is also a peripheral membrane protein adopting an α-helical structure of the positively charged N-terminal region 1–95 through interaction with acidic phospholipids (Dikiy & Eliezer, 2012). The membrane interaction of aSN was suggested to play a role in clustering of synaptic vesicles (Jo et al, 2000; Lautenschlager et al, 2018), regulation of the presynapse size (Vargas et al, 2017), and neurotransmitter release (Burre et al, 2010). A disease-related mutation A30P—strongly linked to the inherited form of PD—diminishes the lipid-binding properties (Jensen et al, 1998). The C-terminal region, comprising residues 96–14, appears as constitutively disordered, has a strong negative charge, and binds calcium ions with low affinity (Eliezer et al, 2001; Nielsen et al, 2001; Lautenschlager et al, 2018).
Regulation of intracellular calcium homeostasis is essential for the proper functioning of cells. Resting concentration of free Ca2+ in the cytosol of a healthy cell is around 100 nM, while the extracellular concentration is in the millimolar range. In a calcium signaling event, rapid calcium influxes via calcium channels increase intracellular calcium concentrations and must be followed by efficient recovery by specialized proteins. The key players are (i) the plasma membrane calcium ATPase (PMCA) and (ii) the sodium-calcium exchanger (NCX), both removing Ca2+ to the extracellular space, and (iii) the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) filling intracellular calcium stores. Furthermore, calcium-binding proteins, in particular calmodulin (CaM), function as a calcium buffer and calcium-dependent regulators of multiple proteins (Cali et al, 2018).
Increasing evidence reveals a link between calcium dysregulation and propagation of synucleinopathies. Voltage-gated CaV1 channels and CaV1.3 mRNA are upregulated as an early feature of PD in areas not associated with overt loss of neurons or Lewy body formation (Hurley et al, 2013, 2015). Fibrillar oligomers of aSN, which are formed with the disease development, can enhance the Ca2+-permeability of plasma membrane (Cali et al, 2014; Di Scala et al, 2016; Rcom-H'cheo-Gauthier et al, 2016) and have been found to activate SERCA (Betzer et al, 2018), thus contributing to disturbances in calcium homeostasis. The SERCA interaction was identified by aSN pull-down experiments, and PMCA was also identified from these experiments (Betzer et al, 2015).
Neurons have a large complexity and a highly polarized architecture; calcium signaling events in these cells, therefore, are highly localized. Dramatic, but extremely local calcium influxes are related to signals leading to vesicular exocytosis in the presynaptic termini. In the presynaptic bouton, the signaling events take place in nanodomains within 50 nm from Ca2+ channels (Augustine et al, 2003) and free calcium concentrations can rise locally by > 1,000-fold (Long et al, 2008). Terminating rather than dissipating a local signal causes a high demand for an efficient and flexible calcium removal system with fine-tuning to a required level of resting concentrations. PMCA plays a key role in this process.
PMCA is a transmembrane protein of 130–140 kDa and belongs to the P2B subfamily of P-type ATPases. Being a high affinity and low-capacity active transporter, it can fine-tune the resting free calcium ion concentration in cytosol (Carafoli, 1994; Strehler et al, 2007b). In humans and other mammals, four PMCA isoforms (PMCA1-4) are encoded by separate genes (Strehler et al, 2007a). PMCA1 is considered a “housekeeping” pump and together with PMCA4 ubiquitously expressed in all tissues. PMCA2 and 3 have specific expression patterns and are mostly found in excitable tissues and often referred to as neuron-specific isoforms (Domi et al, 2007; Strehler & Thayer, 2018). Through alternative RNA splicing at two different sites (“A” and “C”), the four isoforms can be produced in more than 20 different variants (Strehler & Zacharias, 2001). Splicing at site C affects the length of the C-terminal tail with CaM-binding sites (variants denoted a through d), and splicing at site A impacts the length of the first intracellular loop (variants denoted w, x, y, z), which leads from the A-domain to the transmembrane segment 3 (TM3) (Strehler, 2015). The different splice variants are in many cases tissue-specific and differ in the degree of activation and autoinhibition (Kessler et al, 1992; Caride et al, 1999).
PMCA is regulated through interactions with protein partners as well as phospholipids. A classical regulatory feedback mechanism happens via the C-terminal autoinhibitory domain of PMCA. In the resting cell, the domain interacts with the cytoplasmic domains and autoinhibits calcium transport. Upon an increase in intracellular calcium, calcium-bound CaM binds to the autoinhibitory domain resulting in a rise in PMCA activity. The degree and calcium threshold of activation may depend on how many CaM-binding sites—one or two—are present within the autoinhibitory domain (Tidow et al, 2012). Furthermore, PMCA regulation by acidic phospholipids has been mapped to two binding sites—one in the autoinhibitory domain, the other at the cytosolic loop between the A-domain and TM3. The mechanism of how acidic lipids regulate the pump is not understood in detail; however, they appear to be both modulators of activation by CaM as well as stand-alone activators (Niggli et al, 1981a; Zvaritch et al, 1990; Brodin et al, 1992; Pinto Fde & Adamo, 2002; Tidow et al, 2012; Lopreiato et al, 2014; Penniston et al, 2014). Hence, the (auto)regulatory regions also correspond to the sites of variations by alternative splicing.
Here, we show that aSN in its soluble, monomeric form acts as a very potent activator of human PMCAs. The effect relies on the presence of acidic phospholipids and is independent of the CaM-binding autoinhibitory domain. Our findings suggest that the activation mechanism is based on interactions of the N-terminal segment of aSN, negatively charged lipids, and a phospholipid binding site of PMCAs. We propose that aSN complements CaM in local compartments such as the presynapse and that aSN unlike CaM provides a pool of activated PMCA through a Ca2+-independent mechanisms that can respond immediately to calcium spikes. The findings provide a new perspective on the physiological role of native aSN in the presynapse and demonstrate how the lipid environment affects the critical calcium extruding activity of PMCA.
aSN co-localizes with PMCA and stimulates calcium extrusion
Co-immunoprecipitation experiments with brain homogenate from aSN-knockout mice supplemented with purified aSN monomer revealed aSN binding, and a similar experiment with aSN oligomer showed the same result indicating no discrimination on aSN conformation (Fig 1A, left). Co-immunoprecipitation and western blotting of endogenous aSN and PMCA from detergent extracts of total brain homogenates from wild-type C57BL/6 mice show that endogenous PMCA interacts with endogenous aSN (Fig 1A, right). PMCA appears with multiple bands, or a smear on western blots from the two co-IPs, likely due to the varying amounts of post-translational modifications of PMCA (Tolosa de Talamoni et al, 1993). Moreover, a pull-down assay performed with purified proteins showed aSN binding to PMCA immobilized on Calmodulin sepharose (Fig 1B).
The interaction between aSN and PMCA was investigated further in primary hippocampal neurons derived from newborn C57BL/6 mice and with neurons from aSN-KO mice as negative controls. Primary hippocampal neurons were fixed and analyzed after 14 days in culture by immunofluorescence labeling of synapses by synaptophysin and an aSN/PMCA proximity ligation assay (PLA). The PLA is used for in situ detection of protein interactions and is based on two primary antibodies from different species, here ASY-1, a polyclonal aSN antibody generated in rabbit, and 5F10, a monoclonal pan PMCA antibody generated in mouse. The secondary antibodies were labeled with oligonucleotides that enzymatically can be ligated when in proximity and then amplified, hence generating concatemeric sequences. These sequences can bind fluorescently labeled oligonucleotides yielding a red fluorescent signal when aSN and PMCA are located within approximately 40 nm. PMCA and aSN were found to be in this proximity of each other and located to the synapses (Fig 1C).
The functional effect of PMCA calcium transport was investigated in primary hippocampal neurons from aSN-KO neurons transiently transfected with either aSN and mCherry, or mCherry alone as negative control. SERCA was inhibited by thapsigargin (4 μM) before recording. Cellular calcium responses were monitored by the calcium-sensing dye Fura2-AM upon depolarization by 8 mM KCl in the extracellular medium. Neurons expressing aSN expelled calcium to a markedly higher degree than the mCherry expressing neurons (Fig 1D and E). Vanadate is a well-known and potent inhibitor of P-type ATPases (Cantley et al, 1978; Bond & Hudgins, 1980; Dupont & Bennett, 1982) and was used in this experiment as a PMCA inhibitor, since SERCA was inhibited by thapsigargin from the start. Vanadate decreased the calcium expulsion of aSN mCherry neurons to the same level as mCherry-only neurons treated with vanadate. This supports that increased efflux induced by aSN is driven by PMCA.
Calcium extrusion experiments similar to those in neurons were also attempted in three different cell lines, SH-SY5Y, PC12, and Chromaffin cells, to investigate further variations of background conditions. In the human neuroblastoma cell line SH-SY5Y, with doxycycline controllable wild-type aSN expression, the response to K+ dependent depolarization had great variability, as have been observed previously (Morton et al, 1992). For cells with a clear response to K+ dependent depolarization, the SH-SY5Y cells overexpressing aSN exhibited increased calcium expulsion compared to SH-SY5Y with no aSN expression (Appendix Fig S1A–C). In PC12 cells, the response to K+-dependent depolarization gave a good signal, but no difference between control cells and cells overexpressing aSN was observed (data not shown). This can have many causes, but we did not explore it further. For Chromaffin cells, we did not detect a significant response to K+-dependent depolarization. Hence, in particular primary hippocampal neurons, and to some extend also SH-SY5Y cells with inducible overexpression of wild-type aSN, show aSN-stimulated PMCA activity.
Monomeric aSN activates human PMCA in a lipid-dependent manner and independent of the autoinhibitory domain of PMCA
The measurements of calcium-dependent ATPase activity of PMCA were performed with two human PMCA—ubiquitous PMCA1 and neuron-specific PMCA2 isoforms (specifically the splice variants PMCA1d and PMCA2w/a). The experiments were performed with PMCAs relipidated either with porcine brain phosphatidylcholine (BPC, neutral lipids) or with bovine brain lipid extract Folch fraction I (Sigma-Aldrich, from here on referred to as brain extract lipids, BE), of which 60% are negatively charged phosphatidylinositol and phosphatidylserine lipids (Folch et al, 1957; Boura & Hurley, 2012). We observed monomeric aSN causing a strong increase in the activity of both PMCA isoforms, however only for PMCAs relipidated with BE, not BPC. Ca2+ titration revealed that the elevation of ATPase activity is also accompanied by a three- to five-fold increase in the apparent calcium affinity (Fig 2A, left, center; Table 1). Additionally, the activation is gradually abolished by increasing the content of neutral BPC in the relipidation mixture (Fig 2A, right). To rule out potential, unspecific effects of other ATPases or impurities in the PMCA preparations, we prepared an inactive variant of PMCA (PMCA1dD475N), in which the reactive aspartyl residue in the active site was replaced by asparagine. In terms of purity, these preparations were no different from the wild type, as confirmed by SDS–PAGE and size exclusion chromatography (Appendix Fig S2A–D), and they showed no ATPase activity, neither with nor without aSN (Fig 2A left, 2B).
|, microM||Brain lipid extract (BE) basal||Brain lipid extract (BE) + aSN||Brain PC basal||Brain PC + aSN|
|PMCA1d||0.56 ± 0.02||0.082 ± 0.005||0.38 ± 0.02||0.35 ± 0.02|
|PMCA1-ΔC||0.16 ± 0.01||0.06 ± 0.01||0.11 ± 0.01||0.10 ± 0.01|
|PMCA2w/a||0.38 ± 0.05||0.13 ± 0.01||0.62 ± 0.03||0.44 ± 0.02|
To further investigate the acidic phospholipid dependence, we performed aSN titration comparing effects of wild-type aSN and the A30P variant with reduced membrane-binding ability (Jensen et al, 1998). In BE, the activation of PMCA was significantly attenuated by the A30P mutation. As could be expected, the aSN variant was ineffective in the neutral environment of BPC (Fig 2B).
Next, we examined the potential interplay between aSN and the well-known protein activator of PMCA, namely Calmodulin (CaM). For both PMCA isoforms, the susceptibility for activation by CaM and aSN appears to be lipid-dependent, with the acidic lipids promoting the effect of aSN and neutral lipids promoting the effect of CaM (Fig 2C). To confirm that the action of aSN was independent of the autoinhibitory domain, we designed a truncated construct (PMCA1-ΔC), lacking 184 residues of the C-terminus including the autoinhibitory domain with the CaM-binding site(s) and a long, intrinsically disordered linker region (Fig 2D, top). Foreseeably, loss of the C-terminal tail resulted in a constitutively active protein, insensitive to CaM (Appendix Fig S3A) and with basal activity higher than that of the full-length wild type (Fig 2D, bottom left). However, the stimulation by aSN was preserved and appeared like for the full-length pump, indicating the aSN effect to be independent of the autoinhibitory mechanism associated with the C-terminal tail (Fig 2D, bottom right). The apparent Ca2+ affinity of PMCA1d was increased by the truncation itself, and aSN strengthened that effect (Table 1). Again, the activation was lipid-dependent, occurring in the PMCA1-ΔC variant reconstituted in BE and not in BPC (Appendix Fig S3A).
Additionally, we examined the oligomeric form of the full-length aSN, which was previously established to have a great activating effect on SERCA (Betzer et al, 2018). For PMCA, however, the effect of oligomers was smaller than that of the monomer, occurring only at higher concentrations and to a lower fold of activation (Appendix Fig S3B). The activation might simply be explained by dissociating monomer from the oligomer, which we have found to be present in oligomer preparations (Appendix Fig S4). Moreover, we measured the PMCA activity in presence of two other synuclein isoforms, beta- and gamma-synuclein. Both stimulated the pump, but to a lesser extent (Fig EV1A and B).
PMCA alternative splicing events correlate with aSN expression level
PMCA is extensively regulated by alternative splicing, which results in over 20 variants, differing in distribution, kinetic properties, and CaM sensitivity (Strehler & Zacharias, 2001; Krebs, 2015). We wondered if PMCA splice variants could vary also in how they respond to aSN. We used available transcriptomics data from the VastDB database, http://vastdb.crg.eu/ (Tapial et al, 2017), containing information on alternative splicing events. The level of alternative splicing events in different tissues are indicated by PSI values (“percent spliced in”), and we asked whether specific splicing events correlate with aSN expression levels. First, we analyzed the splice site C, which is located at the C-terminal region of PMCA and contains the autoinhibitory domain that interacts with CaM. Alternative splicing at this site gives variants that may differ in the degree of autoinhibition or rate of activation by CaM. The splice variant “a,” with a shortened autoinhibitory domain, is known to have higher basal activity and to be less sensitive to CaM stimulation (Caride et al, 2007). The available data regarded PMCA1, PMCA3, and PMCA4, and the analysis displayed in Fig 3A shows that in the case of PMCA1 and PMCA4, incorporation leading to the variant “a” positively correlates with the expression level of aSN with only a few outliers. Only PMCA3 did not correlate and the PSI value of the “a” variant was relatively high regardless of aSN expression interval. The results suggest that in tissues expressing aSN at high levels, PMCA is alternatively spliced in a way that favors less CaM-sensitive variants. The PMCA “a” variant is mainly expressed in brain tissues and indicates a positive correlation between PMCA and aSN. Besides brain tissues, aSN is also expressed in high amounts in melanocytes and bone marrow, which account for the two outliers in the plot.
The other splice site—termed A—is located at the loop between the A-domain and TM3 (from here on referred to as “A-TM3 loop”). This loop is much longer in PMCAs compared to other P-type ATPases. Among isoforms, PMCA1 is not being spliced in this region, while in PMCA2 alternative exon composition leads to four different variants named “z,” “x,” “y,” and “w,” with all of them except “y” having been detected in humans (Strehler et al, 2007b; Di Leva et al, 2008). Figure 3B displays the analysis of the PMCA2 splicing events, where three exons can be incorporated leading to variants “w” (full length containing sequences marked in orange, yellow, and blue) and “x” (containing the blue sequence). The “z” variant occurs when none of the three exons are incorporated (Fig 3C). We observed incorporation of the exons leading to variants with a longer A-TM3 loop insert in tissues with low aSN expression and barely any incorporation of these exons for tissue with high expression of aSN. This suggests that low aSN expressing tissues would preferentially transcribe the longest PMCA2w, and tissues richer in aSN would express more PMCA2x and z.
The extended, variable A-TM3 loop of PMCA is predicted to be structurally disordered; a trait which was confirmed using circular dichroism (CD) spectroscopy analysis of a recombinantly expressed PMCA2wA-TM3 loop (Fig 3D). Near TM3, the loop contains several positively charged residues that mediate the activation by acidic phospholipids (Brodin et al, 1992; Pinto Fde & Adamo, 2002; Brini et al, 2010). We speculated about the importance of the loop length in the interaction with disordered aSN and performed the free calcium titration experiment with PMCA2 variants w/a, x/a, and z/a. All PMCA2 constructs used in the experiment were transformed anew into yeast cells derived from one colony and were expressed, purified, and tested simultaneously. The activity assays show that all three variants are activated by aSN (Fig 3E). The most remarkable difference is that PMCA2w/a has the lowest maximum activity among variants when activated by aSN. It was also the only one with a substantial increase in the apparent calcium affinity induced by aSN, the other having high affinity also in the basal activity state. PMCA2z/a, having the shortest loop region, showed the highest basal activity. The results implicate the A-TM3 loop in aSN interactions.
Indirect interaction between aSN and PMCA occurs between the N-terminal region of aSN and the acidic phospholipid binding site of PMCA
To determine whether the activation of PMCA was triggered by a direct protein–protein interaction of aSN with the PMCAA-TM3 loop, we performed interaction studies using NMR spectroscopy. We incubated recombinant PMCA2wA-TM3 loop with 15N-labeled aSN, however observing no chemical shift perturbations (CSPs; Fig EV2A), in both the absence (Fig EV2B) and presence of Ca2+ (Fig EV2C). Further, we found no indications of a direct interaction between full-length PMCA2w/a and aSN, as no CSPs (Fig EV2D) or changes in NMR peak intensities (Fig EV2E) could be seen in the aSN spectra. This suggests the interaction between aSN and PMCA is dependent on a more complex environment and likely is occurring via a mechanism facilitated by the membrane.
To map the potential interaction site on the aSN sequence (1–140), we performed a comparison of shortened aSN variants, truncated N-terminally (Δ14, Δ29) to compromise membrane binding (Cholak et al, 2020) or C-terminally (1–95, 1–61) to compromise low-affinity calcium binding and to remove the NAC binding region (Skaanning et al, 2020) respectively (Fig 4A). Most notably, the Δ14 variant had a greatly weakened effect on PMCA and Δ29 had almost lost its ability to activate PMCA, displaying only minor effects above 5 μM concentration. The C-terminally truncated (1–95) variant generally maintained function although to a lesser extent, activating to a lower fold (Fig 4B, Appendix Fig S3C). The (1–61) variant of aSN did not reach saturation within the examined concentration range, which suggests greatly reduced apparent affinity to the PMCA association.
The disordered C-terminal tail of aSN has calcium-binding properties (Lautenschlager et al, 2018) with a relevant affinity of ~ 20–50 μM (Newcombe et al, 2021), and thus we asked if it would impact the apparent calcium affinity of PMCA. Here, we compared activation of PMCA by the full length and C-terminally truncated (1–95) aSN in the Ca2+ titration experiment. However, despite a lower-fold activation, the C-truncated construct did not differ in shifting PMCA's apparent Kd for Ca2+ (Appendix Fig S3C).
Within certain limitations, deletions of large fragments of the lengthy A-TM3 loop of PMCA, including its binding site for acidic, negatively charged lipids, can retain basal activity (Pinto Fde & Adamo, 2002). Guided by this, we designed constructs and expressed and purified deletion variants of PMCA2, where we removed large parts of the loop. Sequences of the targeted region of PMCA2w/a and three designed variants (Δ292–383, Δ298–383, Δ298–372) are displayed in Fig 4C. The activity assay in the presence of BE (Fig 4D) showed differences in the basal activity and response to aSN. We observed a complete loss of ATPase activity of the Δ292–383 variant. Keeping the six N-terminally located residues of the loop resulted in the Δ298–383 variant with a basal activity preserved (0.84 ± 0.02 nmol Pi/μg PMCA/min.), but not response to aSN titration. Preserving further the 11-residue-long binding site for negatively charged lipids at the C-terminal end of the loop close to TM3 (Δ298–372) boosted basal activity (3.4 ± 0.5 nmol Pi/μg PMCA/min) and reinstalled the activation by aSN to the level observed in wild type PMCA2w/a (which has however a lower basal activity of 2.3 ± 0.3 nmol Pi/μg PMCA/min). This points to the binding site for negatively charged lipids as crucial for the interaction with aSN, and we propose the aSN-PMCA interaction to be mediated by acidic phospholipids involving the N-terminal region (1–95) of aSN and the PMCA binding site for negatively charged lipids (Fig 4E).
The activation by aSN is specific to mammalian calcium pump
PMCA activation by aSN is observed both for the ubiquitous PMCA1 and the more tissue-specific PMCA2 and was also verified for the C-terminally truncated PMCA4x isoform (Appendix Fig S3D), which likely colocalizes with aSN in synaptic terminals of retinal neurons (Krizaj et al, 2002; Bodis-Wollner et al, 2014; Cali et al, 2017). The broad impact on PMCA isoforms raised the question whether the stimulatory effect of aSN is at all specific to PMCA.
Interestingly, we found that rabbit SERCA1a also seems to be activated by the aSN monomer in the same lipid-dependent manner as PMCA (Fig 5A). From the two experiments performed, the effect was only predominant in low (60 nM) free Ca2+ concentration, whereas at high 1.8 μM free Ca2+, SERCA was activated to a lower fold. This indicates that aSN may also modulate the apparent Ca2+ affinity of SERCA. With continuous ER extending into presynaptic compartments (Wu et al, 2017; Singh et al, 2021), this is also of relevance to aSN function, and most likely it is also related to the previously reported activation by aSN oligomers (Betzer et al, 2018).
Next, we turned to a plant homolog of PMCA, namely the autoinhibited calcium ATPase 8 of Arabidopsis thaliana (ACA8), belonging to the same P2B subfamily of P-type ATPase (Axelsen & Palmgren, 2001), but not coexisting with aSN, which is only found in vertebrates. Structurally, ACA8 and PMCA differ in the localization of the autoinhibitory domain, which is situated on the N-terminus of ACA8 and the C-terminus in PMCAs, and ACA8 is lacking the long, disordered loop for the A-TM3 loop with the putative lipid-binding site in PMCA (Brodin et al, 1992; Pinto Fde & Adamo, 2002; Brini et al, 2010). In the presence of BE, ACA8 is not activated by aSN, but rather inhibited, and the effect is reversed by CaM. This may suggest an interplay between aSN, CaM, and acidic lipids, that results in a different effect, albeit weak, for the plant pump (Fig 5B). In the presence of soy PC, the pump is autoinhibited and can be activated by CaM, but not aSN. Furthermore, the human Na,K-ATPase (α1 isoform) was not activated by aSN (Fig 5C). These results suggest the specificity of the aSN interaction for mammalian Ca2+-ATPases.
A mathematical model describes the effect of aSN activation of PMCA on Ca2+ levels
To conceptualize the findings on lipid-mediated aSN binding to PMCA and to assess the influence on the calcium concentration in the presynaptic terminal cytosol, we developed a mathematical model for the presynaptic calcium ion regulation. It was based on a quantitative model for presynaptic calcium regulation, which integrates the classical Hodgkin-Huxley model for action potential propagation in order to simulate calcium dynamics in response to membrane potential changes (Hodgkin & Huxley, 1952; Erler et al, 2004; Keener & Sneyd, 2009). The model presented by Erler et al considers Ca2+ fluxes through PMCA (ATP-driven Ca2+ pump), the sodium-calcium exchanger (NCX), voltage-gated calcium channels (VGCC), and passive fluxes through unknown transport pathways and leakage across the membrane (Fig 6A). We adapted the model such that the PMCA-mediated Ca2+ flux depends on the cytosolic aSN concentration, by fitting a kinetic equation for non-essential activation as described in the Supplements (Appendix Fig S8, Equation S1) (Baici, 2015).
Using this model, we investigated the effect of different levels of aSN on the steady state Ca2+ concentration (Fig 6B) and the increase in cytosolic calcium in response to one or several action potentials (Fig 6C and D). The simulations showed an increase in the steady-state calcium concentration from 0.1 μM to around 0.25 μM, with the increase being most prominent at aSN concentrations below 10 μM. During an action potential, not only the height but also the duration of the calcium wave were increased. The predominant increase of the calcium wave duration (or width) was caused by an increase of the calcium clearance time (post-peak width, Fig 6D).
To investigate the influence of aSN on the calcium concentration during an action potential spike train, we analyzed the mean calcium concentration during this process. In general, these simulations resulted in an oscillating increase of the calcium concentrations as observed in vitro (Chamberland et al, 2020) (Fig 6C). Figure 6D (lower right panel) shows the maximum of the mean calcium concentration during an action potential spike train (as visualized in Fig 6C). In response to decreased aSN concentrations, the average calcium concentration increased because of the larger calcium waves at aSN concentrations below 10 μM (Fig 6D). Hence, the presence of monomeric aSN not only stabilizes the steady-state concentration of Ca2+, but also affects the calcium dynamics in response to one or several action potentials.
Here we show a strong, activating effect of soluble, monomeric aSN on PMCAs and to a lower degree also SERCA, which conversely was found earlier to be activated by oligomeric aSN (Betzer et al, 2018). Our results suggest that a physiological role of aSN is PMCA stimulation for calcium clearance by increasing Vmax and/or apparent calcium affinity. Due to the very specific localization of aSN, this mechanism would be restricted to specialized cells or cellular compartments like presynaptic termini of neurons. This is supported by our proximity ligation study showing an intracellular colocalization of PMCA and aSN in nerve terminals, and by experiments in primary neurons, where we show that aSN increases calcium expulsion after depolarization. Furthermore, the aSN-stimulated PMCA activity can respond instantly to calcium influx, unlike the calcium-dependent mechanism of CaM activation that requires several binding steps before going into effect.
The charge of phospholipids in the PMCA environment has a great influence on its regulation, and acidic lipids alone can activate PMCA to some extent via specific interaction sites (Carafoli, 1994; Lopreiato et al, 2014; Strehler, 2015). In a similar way to the inhibition by tau protein (Berrocal et al, 2017), we found that the effect of aSN on PMCA is enabled by negatively charged and not neutral phospholipids in the membrane. The opposite lipid dependence is observed for CaM, consistent with earlier findings (Niggli et al, 1981a,b; Lopreiato et al, 2014). The composition of the local lipid environment could potentially switch PMCA modes from being responsive either to aSN or CaM.
With its strong effect and accumulation at presynaptic compartments, aSN would allow for a rapid, stimulated response to incoming calcium signals and integration with other calcium-sensitive presynaptic functions such as neurotransmitter release. This, together with aSN activating PMCA independently of the autoinhibitory PMCA C-terminal tail, suggests aSN complements CaM as the activator of PMCA in this specific neuronal compartment.
Alternative splicing of PMCA generates many variants among isoforms and has been for a long time proposed to be a mechanism of fine-tuning calcium handling in cells affecting expression, localization, and membrane trafficking to specific compartments (Strehler & Zacharias, 2001; Antalffy et al, 2011; Krebs, 2015). Splice site “A” variants of PMCA (w, x, y, z) differ in the size of the lengthy and disordered A-TM3 loop (Fig 3C). That region was not resolved in a cryo-EM structure of PMCA1 (Gong et al, 2018), and its impact on the enzymatic properties is not clear, but the proximity to the membrane interface and cytosolic Ca2+ entry suggests a modulatory role of the A-TM3 loop, and its involvement in PMCA-lipid interaction and membrane trafficking has been proposed (Adamo & Penniston, 1992; Antalffy et al, 2011; Enyedi & Strehler, 2011). Indeed, our mutational studies indicate a modulation of the Ca2+ transport activity. In PMCA, the deletion of the intrinsically disordered A-TM3 loop (deleting residues 298–383 in PMCA2w/a) retained basal enzymatic function, but the protein was unresponsive to aSN. However, the stimulation was preserved in a Δ298–372 variant, where 11 residues upstream of the TM3 are maintained. This fragment, highly specific in sequence to PMCA, is rich in positive charge and was previously shown to interact with acidic lipids (Adamo & Penniston, 1992; Pinto Fde & Adamo, 2002), further discussed below. Additionally, this variant showed increased basal activity, which may indicate a role of the A-TM3 loop in some level of autoinhibition that complements the autoinhibitory domain at the C-terminal end of PMCA.
The correlations found between aSN expression level and splicing events at the “C” site of PMCA isoforms 1 and 4 suggest that an “aSN/CaM balance” of PMCA activation can be managed by alternative splicing, leading to the synthesis of less CaM-sensitive variants in tissues destined for aSN activity. Variations obtained at the splice site “A” of PMCA2 suggest preference for variant “w” in low-aSN-expressing tissues and shorter variants “x” and “z” being expressed together with increasing aSN level. In the activity assay, the PMCA2 splice variants responded differently to aSN; PMCA2w/a was the only ones having their apparent calcium affinity increased by aSN, but was conversely stimulated to the lowest maximum activity and had relatively low apparent calcium affinity in a basal (non-activated) state. The longest A-TM3 loop of PMCA2w (Fig 2D) can be associated with the furthest flexibility in the regulation of both affinity and rate. For PMCA2x and -z, only the rate was stimulated. These two variants are typically expressed in aSN-abundant tissues (Fig 3), where higher capacity for calcium extrusion is required, such as in neurons.
The specific function of soluble, monomeric aSN is debated (Cheng et al, 2011; Sulzer & Edwards, 2019), but preferential association to membranes with acidic phospholipids is a recurring theme (Davidson et al, 1998; Jo et al, 2000). The ability to activate PMCA appears strongly related to its lipid-binding properties. The aSN A30P mutant form, associated with familial early-onset PD, is known for reduced ability to bind acidic phospholipids (Jo et al, 2002), and we find here that it correlates with weak PMCA activation. Similar dependence emerges from the characteristics of the N-terminus of aSN. Monomeric aSN interacts with acidic phospholipids via the ~ 95 residues long N-terminal part adopting amphipathic helical segments (Ulmer et al, 2005; Uversky & Eliezer, 2009). The aSN binding to lipid bilayers has been proposed to be maintained by avidity between the 1 and 14 region, strongly interacting and anchoring into the lipid bilayer, and the downstream amphipathic helix having weak surface binding capacity (Cholak et al, 2020). Indeed, here we observed a gradual loss of the PMCA activating properties by deletion of 1–14 or 1–29 residues, with the Δ(1–29) construct being almost incapable of activating PMCA (Fig 4B). Moreover, the importance of the N-terminus is also indicated by the observation that both beta- and gamma-synuclein can activate PMCA, as this is a highly conserved region among synuclein isoforms (Hayashi & Carver, 2022).
From a structural and mechanistic point of view, we propose that PMCA is activated by a dynamic vis-à-vis interaction between the N-terminus of the monomeric aSN and the PMCA binding site for negatively charged lipids, located at the C-terminal side of the disordered A-TM3 loop, and mediated by acidic lipids (Fig 4E). The bulk part of the loop can play a tuning role together with the disordered C-terminal tail of aSN. With medium-affinity Ca2+-binding properties (Kd ~ 20–50 μM) (Lautenschlager et al, 2018; Newcombe et al, 2021), this region of aSN could potentially increase the local concentration of Ca2+ at the cytosolic entry site of PMCA. It appears to play a role in stimulation of the PMCA rate but does not have a visible impact on apparent calcium affinity.
The lipid-binding site of PMCA is likely functionalized by the many positively charged residues in the 11-residue sequence defining it (RKKASMHKKEK, see also Appendix Fig S5). The equivalent sequence in the plant ortholog ACA8 (MASISEDNGEE, residue 352–362) is negatively charged, as is the N-terminal part of the A-TM3 loop in the Δ298–383 deletion construct of PMCA2 (TLLGAGGEEEE). Both ACA8 and PMCA2-Δ298–383 show significant basal activity in presence of acidic lipids but are not activated by aSN. The corresponding region in SERCA (RDQMAATEQDK, residues 236–246 of rabbit SERCA1a) shows a mixed positive and negative charge, which may explain a mild activation by aSN and negatively charged lipids. We note that aSN contains several K-rich motifs in its N-terminal region (1–61) that have resemblance to the acidic-lipid binding sequence of the disordered loop of PMCA; an observation that together with the absence of a direct interaction in simple systems (Fig EV2) and a neurotoxic effect of their mutation (Dettmer et al, 2015b) strengthens the suggestion of a lipid-mediated interaction as the mechanism of aSN activation of PMCA in neurons.
The function of aSN oligomers and fibrils has been thoroughly studied, and among a plethora of cytotoxic capacities, they have been linked to calcium dysregulation by over-activation of SERCA (Danzer et al, 2007; Rcom-H'cheo-Gauthier et al, 2016; Betzer et al, 2018). We show here that native, monomeric aSN exerts a strongly activating effect on PMCA, whereas oligomeric aSN only activates weakly, which perhaps can be ascribed to the small amount of released monomer (Appendix Fig S4).
The mathematical modeling of presynaptic calcium levels shows aSN activation of PMCA affects both the steady-state concentration of Ca2+ and the calcium dynamics (Fig 6). At low levels of aSN in the presynapse, the basal Ca2+ concentration is increased and Ca2+ homeostasis is disrupted during sustained neuronal activity. The accumulation of Ca2+ only happens at monomeric aSN concentration below 10 μM ensuring that small fluctuations in aSN concentrations in the presynapse do not impact calcium regulation.
This novel function of aSN is relevant to calcium homeostasis of neurons and specifically presynaptic compartments, where PMCA plays a key role in calcium homeostasis; however, potentially it affects also the extracellular environment, where the exchange of each calcium ion for two protons by PMCA leads to transient alkalinization of nanodomains at the synaptic cleft with consequences to postsynaptic NMDA receptor fluxes (Chen & Chesler, 2015; Feghhi et al, 2021). Furthermore, the activated proton import of PMCA may be important for neutralization of an acidified environment at the synaptic cleft with large release activity of acidified neurotransmitter vesicles.
The role of aSN in hematopoietic system has been discussed (Pei & Maitta, 2019). The interaction with PMCA can be potentially important to calcium homeostasis in erythrocytes, where aSN is also very abundant (Barbour et al, 2008). Erythrocytes lack ER and mitochondria, and their calcium homeostasis depends solely on PMCA, so aSN could contribute to the very efficient calcium clearance mechanism of erythrocytes, which maintains resting calcium on a very low, 30–60 nM level (Bogdanova et al, 2013).
In the presynapse, PMCA-dependent control of the local calcium-depletion zones could be enforced by aSN and acidic lipids. This may for instance have importance for synaptic vesicle release and recycling where PMCA was proposed to functionally separate simultaneous calcium signals (Krick et al, 2021). Oligomerization and aggregation/fibrillation of aSN on the other hand would impair this function and lead to impaired calcium handling. Our finding can then also have implications regarding aSN pathology. Lipid-related dysfunctions have been linked to PD (Fanning et al, 2020). Early manifestations of PD could result from calcium dyshomeostasis caused by aggregating aSN having loss of function on PMCA activation, or by lipid-affinity-affecting mutations like A30P, which indeed leads to early neuronal dysfunctions (Kruger et al, 2001), or changes in the distribution and content of acidic plasma membrane lipids. Furthermore, impaired control of extracellular pH can be affected and cause neurotoxic effects. These findings merit a focus on the coupled function of PMCA, SERCA, aSN, and calcium homeostasis in neurodegenerative disorders.
Materials and Methods
Brains from C57BL/6 mice (Janvier Labs) or aSN-KO (Sncatm1Rosl [C57BL/6, The Jackson Laboratory]) were homogenized in 7× w/v homogenization buffer (320 mM sucrose, 4 mM HEPES–NaOH, 2 mM EDTA, and complete protease inhibitor mix [Roche], pH 7.4) using a loose-fitting glass-Teflon homogenizer (10 up-and-down strokes, 700 rpm). Debris was removed from the homogenate by centrifugation for 10 min at 1,000 g in a Sorvall RC 5C plus centrifuge. The resulting supernatant was centrifuged for 1 h at 100,000 g. The supernatant was removed, and the pellet was resuspended in the original volume (7×) of RIPA (50 mM Tris pH 7.4, 159 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS) for 3 h, whereafter samples were spun at 20,000 g, for 30 min at 4°C. Protein concentration was measured using the bicinchoninic acid assay. aSN oligomer or monomer (2 μg/ml) was mixed with 0.5 mg/ml aSN-KO mouse brain homogenate diluted in PBS, 0.5% Triton X-100 and incubated overnight at 4°C.
aSN binding (ASY-1) or control (non-immune IgG) was performed as previously described (Betzer et al, 2015). The samples were incubated for 2 h with rotation. The Sepharose beads were isolated and washed twice with PBS, 0.5% Triton X-100, and Co-IP proteins were eluted by incubation in a non-reducing SDS loading buffer at room temperature. Proteins were resolved on 10–16% gradient SDS–PAGE under reducing conditions followed by immunoblotting for PMCA (primary antibody: 5F10 anti-pan-PMCA, Abcam, ab2825, secondary antibody: anti-mouse-HRP, Dako) and aSN (primary antibody: anti-Syn-1, BD Transduction Laboratory, 610787 secondary antibody: anti-mouse-HRP, Dako, P0260). The interaction between endogenous aSN and the endogenous PMCA was studied in the extracts from C57BL/6 mice as described above.
Primary hippocampal neuronal cultures and cytosolic Ca2+ measurements
Primary hippocampal neurons were cultured from newborn (P0) aSN-KO mice (Sncatm1Rosl [C57BL/6], The Jackson Laboratory). Hippocampi were dissected in ice-cold Hank's balanced salt solution, dissociated in 20 U/ml papain in Hibernate A medium (Gibco) supplemented with 1xB27 and 0.3 g/l L-glutamine for 20 min at 37°C, washed twice, and triturated in plating medium (MEM [Gibco] supplemented with 5 g/l glucose, 0.2 g/l NaHCO3, 0.1 g/l transferrin, 0.25 g/l insulin, 0.3 g/l L-glutamine, and 10% fetal bovine calf serum [heat-inactivated]). Hippocampal neurons were seeded on Matrigel® matrix (Corning®)-coated coverslips. After 24 h, the medium was changed to growth medium (MEM supplemented with 5 g/l glucose, 0.2 g/l NaHCO3, 0.1 g/l Transferrin, 0.075 g/l L-glutamine, 1× B-27 supplement, 2 μM cytosine arabinoside and 5% fetal bovine calf serum [heat-inactivated]).
At culture day 6 (DIV 6), the neurons were transiently transfected by lipofectamine 3000 with Bicistronic vectors coding for mCherry and aSN or mCherry under synapsin promotor according to manufacturer's instructions yielding varying expression levels in individual neurons. At culture day 8, cytosolic Ca2+ levels in primary neurons were determined using the Ca2+-sensitive fluorescent indicator Fura-2-AM (Molecular Probes/Invitrogen). Cells were loaded with Fura-2 in sterile-filtered HEPES-buffered saline (HBS: 20 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, pH 7.4) containing 2.5 μM Fura-2 AM, 0.04% pluronic acid, F127 for 30 min at 37°C, 5% CO2. The Fura-2-containing medium was replaced with fresh HBS without Fura-2 and incubated additionally for 30 min. After dye loading and prior microscopic analysis, the culture was moved into a recording buffer with thapsigargin to inhibit contribution from SERCA for 5 min., and an area containing 1–3 transfected neurons was found. The fluorescence was measured on an Olympus Scan^R high-content microscope using excitation wavelengths at 340 and 380 nm and emission at 510 nm. The cytosolic Ca2+ levels in single cells were measured by placing a region of interest (ROI) outside the nucleus. The recording was started and at the recording time of 2 min KCl was added to depolarize the neurons. The calcium response was followed over time until 12 min. The response upon K+-induced depolarization was quantified as the area under curve (AUC) from each measured neuron from the 2–12 min. measurement interval.
Cytosolic Ca2+ measurements in SH-SY5Y cells
A doxycycline controlled SH-SY5Y human cell line that stably overexpresses wt human aSN (SH-SY5Y ASYN) was used (Vekrellis et al, 2009). In the presence of doxycycline, aSN expression is suppressed. SH-SY5Y ASYN cells were cultured in the presence of 1 μM doxycycline similarly to previously described (Reimer et al, 2022).
SH-SY5Y ASYN cells were split and incubated with or without 1 μM doxycycline for 3 days, then split and seeded onto polylysine-coated coverslips still with or without 1 μM doxycycline and cultured for an additional 2 days, whereafter calcium extrusion experiments were performed using the fluorescent calcium binding dye Fluo-8 AM (ATT Bioquest).
The cells on the coverslips were loaded with 2.5 μM Fluo-8 in loading buffer (140 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.8 mM CaCl2, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 10 mM Hepes pH 7.4, 2.5 μM Fluo-8 AM) for 30–60 min. at 37°C, 5% CO2. Following Fluo-8 loading, coverslips were washed once in experiment buffer (140 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.8 mM CaCl2, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 10 mM Hepes pH 7.4).
Coverslips were then mounted in an imaging chamber and covered with 300 μl pre-heated (37°C) recording buffer (140 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.8 mM CaCl2, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 10 mM Hepes pH 7.4, 4 μM thapsigargin) and placed in a 37°C pre-heated OkoLab imaging chamber on the microscope stage.
Imaging was performed on a Nikon Ti Eclipse inverted microscope equipped with an OkoLab heating chamber, Perfect Focus 3 system, a Plan Apo 60× (NA 1.40) oil objective, a Zyla sCMOS5.5 Megapixel camera (Andor), fluorescence illumination system CoolLED-pE-300white, and the fluorescence filter set for GFP. The system was controlled by NIS Elements software from Nikon. Imaging was performed similar to previously described (Ernstsen et al, 2022). Images were acquired every second for 8 min in total. Binning of 2 was used. Following baseline imaging for 30 s, 600 μl depolarization buffer (140 mM NaCl, 11.5 mM glucose, 132 mM mM KCl, 5.7 mM CaCl2, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 10 mM Hepes pH 7.4, 4 μM thapsigargin) was added into the center of the imaging chamber, while imaging continued. Two technical replicates were performed. A square region of interest was placed in the cytoplasm of cells (2–4 cells per replicate) for all frames in the time-lapse sequences (481 frames) of the different conditions and mean fluorescence intensity was measured using ImageJ (Schindelin et al, 2012). The response to K+ induced depolarization was quantified as the Area Under Curve (AUC) for each cell.
Proximity ligation assay (PLA)
Proximity ligation assay (PLA) enables in situ detection of protein–protein interaction using single-stranded oligonucleotides conjugated to specie-specific antibodies. If the oligonucleotides are in close proximity (within 40 nm), they can be ligated to form circular DNA, which can be amplified by multiplying the binding site for fluorescently labeled complementary oligonucleotides and thus multiplying the fluorescent readout.
Primary hippocampal neurons were cultured from newborn (P0) C57BL/6 mice (Janvier Labs) as described for aSN-KO. At culture day 14 (DIV14), the neurons were fixed in 4% paraformaldehyde for 30 min at room temperature (RT) followed by a wash in PBS and 10 min permeabilization in 0.1% Triton X-100, 50 mM glycine, 3 mM CaCl2, 2 mM MgCl2, pH 7.4. Unspecific binding was blocked by 3% bovine serum albumin in PBS for 1 h followed by incubation with the primary antibody for 1.5 h [anti-AS (1 μg/ml, ASY-1, Lindersson et al, 2004; Kragh et al, 2009), and anti-PMCA 5F10 (1 μg/ml, Abcam, ab2825)]. The Duolink procedure was conducted according to the manufacturer's instructions with the Duolink® In Situ Red Starter Kit Mouse/Rabbit (Duolink®, Sigma-Aldrich), with secondary antibodies contained in the kit. After PLA staining, the neurons were labeled with synaptophysin (primary—antibody—guinea pig anti-synaptophysin 1, (Synaptic Systems #101004), secondary: goat anti-guinea pig, Alexa Fluor® 488 nm (Abcam, ab150185) to visualize the synapses where synuclein normally is located and DAPI for nuclei. Images were obtained using a Zeiss Observer Z1 inverted microscope equipped with ApoTome.2.
Expression and purification of α-synuclein
Recombinant human aSN wild type and variants were produced in Escherichia coli, and purified as previously described (Huang et al, 2005). Monomeric and oligomeric forms of aSN were produced and isolated as previously described (Betzer et al, 2015). Concentration of all aSN preparations was confirmed by BCA assay. The proteins were stored in −80°C in a buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM KCl. Recombinant 15N-aSN for NMR experiments and aSN(Δ1-14) was prepared as previously described (Cholak et al, 2020) and aSN(1–61) as in (Skaanning et al, 2020). Prior to the experiments, proteins were subjected to size exclusion chromatography on Superdex 200 increase 10/300 column (GE Healthcare) in the buffer containing 50 mM Tris–HCl pH 7.2, 150 mM KCl. Fractions were pooled, concentrated to 1–1.5 mg/ml 5 kDa MWCO Vivaspin protein concentrator (Sartorius), flash-frozen in liquid nitrogen, and stored in −80°C for further experiments.
Size exclusion chromatography profiles and SDS–PAGE analysis are presented in Appendix Fig S2.
PMCA expression constructs and site-directed mutagenesis
Codon-optimized genes encoding for human PMCA1d and PMCA2w/a (Genscript) were cloned into pEMBL-yex4 expression plasmids by homologous recombination in S. cerevisiae as described (Drew et al, 2008).
PMCA1d and PMCA2w/a constructs were subjected to site-directed mutagenesis using QuikChange site-directed mutagenesis kit according to the manufacturer's protocol (Agilent). To obtain an inactive variant D475N (the autophosphorylation site), mutagenetic primers were designed using PrimerX online tool (https://www.bioinformatics.org/primerx). To obtain C-terminally truncated PMCA1, an internal TEV cleavage site was introduced to the original construct. Primers for the insertion of codon-optimized TEV site sequence and the large deletions in PMCA2 were designed according to described methods (Liu & Naismith, 2008). The integrity of all constructs was verified by DNA sequencing (Eurofins).
Expression of recombinant PMCAs in S. cerevisiae
All circular vectors were introduced into yeast cells using PEG/LiAc/ssDNA-mediated transformation (Gietz & Schiestl, 2007). All recombinant PMCAs were expressed in S. cerevisiae strain K616, depleted of the native calcium ATPases (MATα pmr1::HIS3 pmc1::TRP1 cnb::LEU2 ura3-1) (Cunningham & Fink, 1994). All culture media were supplemented with 10 mM CaCl2, as it is a necessary survival condition for the strain to maintain internal stores. For expression culture, 100 ml of uracil-deficient 2% glucose SD medium was inoculated with a colony of transformed cells and allowed to grow for 24 h in 30°C in a 120 rpm shaking incubator. The preculture was used for inoculation of 9 L of -Ura SD medium with 1% glucose and 40 mg/L adenine hemisulfate, starting from OD450 of 0.05. After 22 h, when all glucose in the medium was used and the culture reached OD450 of ~ 5, the expression was induced by 2% galactose, and the culture was supplemented with YP medium and 40 mg/L adenine hemisulfate. Cells were harvested after 18–20 h by centrifugation, washed with water and TEKS buffer (50 mM Tris–HCl pH 7.6, 2 mM EDTA, 100 mM KCl, 0.6 M D-sorbitol), and resuspended in TESin buffer (50 mM Tris–HCl, 2 mM EDTA, 0.6 M D-sorbitol with the addition of PMSF and protease inhibitor. The typical yield was ~ 15 g of cells per liter of culture.
Purification of PMCAs
The cells were disrupted in a pulverisette grinder with an equal volume of 0.5 mm glass beads (4°C, 450 rpm, 4 cycles of 3 min millings, and 1 min pause). The material was centrifuged for 20 min at 2,000 g to remove beads and uncracked cells. The supernatant (S1) was centrifuged for 30 min at 20,000 g for further removal of cell debris. The supernatant (S2) was subjected to ultracentrifugation for 3 h at 150,000 g. Pelleted membranes were suspended in solubilization buffer (20% glycerol, 50 mM Tris–HCl pH 7.2, 150 mM KCl, BME, PMSF, protease inhibitors) and homogenized in a Dounce glass homogenizer. Membranes were flash-frozen in liquid nitrogen and stored at −80°C until further processing.
Membranes were diluted with solubilization buffer to 5 mg/ml total protein concentration (measured with Bradford assay) and solubilized in 1% DDM and 0.2% cholesteryl hemisuccinate for 1 h by stirring in 4°C, followed by ultracentrifugation at 150,000 g for 1 h to pellet unsolubilized material. The supernatant was supplemented with 4 mM CaCl2 and stirred for 2 h in 4°C with CaM-Sepharose beads (GE Healthcare), pre-equilibrated with binding buffer (50 mM Tris–HCl pH 7.2, 150 mM KCl, 0.017% w/v DDM, 10% glycerol, 2 mM CaCl2). The beads were packed onto a gravity flow column and washed with 10–15 column volumes of binding buffer. The protein was eluted a buffer containing 50 mM Tris–HCl pH 7.2, 150 mM KCl, 0.017% w/v DDM, 10% glycerol, 2 mM EGTA. EGTA stock solution (200 mM) was beforehand pH-adjusted with KOH to ~ 7.5. The fractions containing the protein of interest were pooled and concentrated to ~ 3 mg/ml using a 100 kDa MWCO Vivaspin protein concentrator (GE Healthcare). The concentrated protein was subjected to size exclusion chromatography on Superose 6 increase 10/300 column (GE Healthcare) in the buffer containing 50 mM Tris–HCl pH 7.2, 150 mM KCl, 0.017% w/v DDM, 10% glycerol. Fractions were pooled, concentrated to 1–1.5 mg/ml, flash-frozen in liquid nitrogen, and stored in −80°C for further experiments. Size exclusion chromatography profiles and SDS–PAGE analysis of all PMCA variants used in the study are presented the Appendix Fig S2.
To produce C-terminally truncated PMCA1, the PMCA1d construct with a tobacco etch virus (TEV) protease cleavage site inserted next to H1074 just before the autoinhibitory domain was used. The protein was cleaved with TEV protease while bound to the CaM sepharose beads. After overnight incubation with the protease at 4°C, the C-terminally truncated PMCA was collected in the flow-through fraction and run over a Ni2+ affinity column to bind the His-tagged protease. The flow-through was concentrated to ~ 3 mg/ml (100 kDa MWCO) and subjected to size exclusion chromatography on Superdex 200 increase 10/300 column (GE healthcare) in size exclusion buffer containing 50 mM Tris–HCl pH 7.2, 150 mM KCl, 0.017% w/v DDM, 10% glycerol. Peak fractions of PMCAs were pooled, concentrated to 1–1.5 mg/ml using a 100 kDa MWCO Vivaspin protein concentrator (Sartorius), aliquoted, and flash-frozen in liquid N2 and stored at −80°C for further procedures. The purity of the proteins was analyzed by SDS–PAGE (Appendix Fig S2).
CaM-Sepharose beads (GE Healthcare) were pre-equilibrated with binding buffer (40 mM BisTris/HEPES pH 7.2, 100 mM KCl, 0.017% w/v DDM, 3 mM MgCl2, 2 mM CaCl2). PMCA was preincubated with aSN in the mixture of 80 μl total volume containing 0.6 mg/ml PMCA2w/a, 1.5 mg/ml aSN, and 2.5 mg/ml brain lipid extract (BE). Both PMCA and aSN stock solutions were in their final storage buffers, BE stock solution contained 50 mM Tris–HCl pH 7.2, 150 mM KCl, 1% DDM. Simultaneously, the preincubation mixture without lipids was also prepared. Control samples without PMCA or aSN were prepared. After 30 min. preincubation in room temperature, pre-equilibrated CaM-sepharose beads were added and the mixture was diluted to 1 ml with the binding buffer. For the samples where lipids were present, the binding buffer contained 100 μg/ml brain lipid extract. Samples were incubated with agitation in the room temperature for 60 min and washed four times with respective binding buffer. Each wash cycle consisted of 2 min. centrifugation at 900 g to pellet the beads, removal of the supernatant, and addition of 1 ml of the fresh buffer. Finally, 40 μl of the elution buffer (40 mM BisTris/HEPES pH 7.2, 100 mM KCl, 0.017% w/v DDM, 3 mM MgCl2, 2 mM EGTA) was added to the beads and after short centrifugation, supernatant was collected, and analyzed by SDS–PAGE and western blotting. We used primary antibodies: rabbit ASY-R1 (Jensen et al, 2000), mouse 5F10 anti-pan-PMCA (Abcam, b2825); secondary antibodies: anti-rabbit-HRP (DAKO–P0217), anti-mouse-HRP (DAKO–P0260).
Expression and purification of CaM
Mammalian CaM in the pET15b vector was transformed into E. coli strain C41 and grown in LB media at 37°C. Induction was performed with IPTG at an OD600 of 0.6. After 16 h of growth at 21°C, cells were harvested by centrifugation at 3,000 g, resuspended in lysis buffer (20 mM Tris–HCl pH 7.5, 1 mM EDTA, complete protease inhibitor), and lysed using sonication. The lysate was centrifuged for 1 h at 20,000 g. The supernatant was mixed 1:1 with wash buffer (20 mM Tris–HCl pH 7.5, 10 mM CaCl2) and loaded onto a pre-equilibrated (wash buffer) gravity flow phenyl sepharose column. The column was washed with 5 CV wash buffer, 5 CV high salt wash buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 10 mM CaCl2), and 5 CV wash buffer. CaM was eluted in elution buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10 mM EGTA). Before use in activity assays, CaM buffer was exchanged to 20 mM Tris–HCl pH 7.5, 150 mM KCl using PD MiniTrap G25 desalting column (GE Healthcare), aliquoted, and stored in −80°C.
Expression and purification of ACA8
ACA8 expression and membrane isolation was performed similarly to the PMCA isoforms. ACA8 was solubilized in a buffer containing 50 mM Tris–HCl pH 7.6, 100 mM KCl, 20% glycerol, 3 mM MgCl2, 5 mM β-mercaptoethanol (βME), 7.5 mg/ml DDM, 1 mM PMSF, and 1 μg/ml of chymostatin, pepstatin A, and leupeptin. Purification was achieved through Ni2+ affinity followed by size exclusion chromatography. ACA8 was bound to a Ni2+-affinity column in low imidazole buffer (50 mM Tris–HCl pH 7.5, 10 mM imidazole, 200 mM KCl, 10% glycerol, 3 mM MgCl2, 5 mM βME, 0.2 mM LMNG). On the column, the ACA8 His-tag was cleaved off with thrombin and ACA8 was eluted in low imidazole buffer. ACA8 was subjected to size exclusion chromatography on a Superose 6 increase 10/300 column in a SEC buffer (50 mM Tris–HCl pH 7.5, 200 mM KCl, 3 mM MgCl2, 10% glycerol, 0.02 mM LMNG, 5 mM βME). The protein was stored in −80°C in SEC buffer for further experiments.
Expression and purification of PMCA2wA-TM3 loop
Escherichia coli cells (BL21 DE3) were transformed with a modified pET24b plasmid containing N-terminal His6-SUMO tagged PMCA2wA-TM3 loop (L289-K383) using heat shock transformation. Pre-cultures (10 ml LB medium, 50 μg/ml kanamycin) were grown overnight and used to inoculate 1 L of ZYM-5052 media (Studier, 2005), supplemented with 50 μg/ml kanamycin. Cells were grown for 3 h at 37°C with shaking, then transferred to a shaking incubator at 16°C, and grown for a further 24 h. The cells were pelleted at 4,000 g and kept at −20°C for at least 24 h prior to lysis in column binding buffer (50 mM Tris pH8, 150 mM NaCl, 10 mM Imidazole), supplemented with complete protease inhibitor (Sigma), at 20 kpsi using a French pressure cell disruptor (Constant Systems, Daventry, UK). Lysate was cleared by centrifugation at 20,000 g, then passed through a 0.45 μm filter before being incubated with equilibrated Ni-Sepharose fast flow resin (GE Healthcare) for 30 min. Lysate was passed through the column via gravity flow, followed by wash buffer (5× CV) (50 mM Tris pH8, 1 M NaCl, 10 mM Imidazole) 5× CV binding buffer, and eluted with elution buffer (10 ml; 50 mM Tris pH 8, 150 mM NaCl, 250 mM Imidazole). Purified His6-SUMO-PMCA2w was made up to 50 ml (with 50 mM Tris pH 8, 150 mM NaCl) and cleaved with ULP1 (0.1 mg) ON at 4°C. The cleaved protein was purified by collecting the flow-through from a second Ni-Sepharose column and concentrated using Amicon spin filters (Millipore). Protein was stored at −20°C.
All NMR spectra were recorded at 5°C on a Bruker 800 MHz spectrometer equipped with a cryogenic probe and Z-field gradient using Bruker Topspin v4.0.7, recording 15N,1H-HSQC spectra to address interaction and using the backbone assignments obtained in (Cholak et al, 2020). 15N-HSQC spectra were obtained in HEPES buffer (20 mM HEPES pH 7.4, 150 mM NaCl) of 50 μM 15N-aSN alone and in the presence of PMCA2wA-TM3 loop (molar ratio 1:5) and with further addition of 25 mM CaCl2 (molar ratios 1:4:500). Spectra of 9.5 μM 15N-aSN alone and with full-length PMCA2w/a (molar ratio 1:1) were obtained in Tris buffer (50 mM Tris pH 7.5, 150 mM KCl, 1.5% (w/v) DDM). Combined amide (N, HN) chemical shift perturbations (CSPs) were measured by CCP-analysis version 2.5. The spectral analyses were done in CcpNMR analysis (Vranken et al, 2005).
Far-UV CD spectropolarimetry
Far UV CD spectra were recorded at 5°C on a JASCO J-815 spectropolarimeter on 12.8 μM PMCA2wA-TM3 loop dissolved in 10 mM Na2HPO4, 200 mM NaF pH 7.4. The spectrum was recorded from 260 to 190 nm, averaging 10 scans (D.I.T 2 s; data pitch 0.2 nm; band width 1 nm; scan rate 50 mm/min; path length 1 mm). A spectrum of the buffer was recorded using identical settings and subtracted.
SERCA and Na, K-ATPase preparations
SERCA1a from rabbit skeletal muscle was kindly donated by Thomas Lykke-Møller Sørensen, Anne Lillevang, and Claus Olesen. The protein preparation was performed according to previously established procedures (Andersen et al, 1985; Sorensen et al, 2004). Na, K-ATPase was kindly donated by Michael Habeck and Natalya Fedosova. The protein was expressed and purified as described earlier (Habeck et al, 2017).
ATPase activity assay
The ATPase activity was tested by monitoring the release of free inorganic phosphate from ATP in a molybdenum blue assay (Baginski et al, 1967).
Purified PMCA requires relipidation to regain activity; therefore, the samples were preincubated with phospholipids before activity assay. Lipid stock solutions were prepared from powdered lipids as follows: brain lipid extract (from bovine brain Type I, Folch Fraction I, Sigma-Aldrich), brain PC (Avanti), or soy PC (Avanti) was dissolved to a concentration of 20 mg/ml in a buffer containing 50 mM Tris pH 7.5, 150 mM KCl, and 1.5% (w/v) DDM. After cycles of heating (45–50°C), sonicating, and vortexing, a translucent gel was obtained. Stocks were aliquoted and stored in −20°C. Shortly before the assay lipid solution was added to PMCA sample in PMCA:lipid w/w ratio 1:5 and mixed by gentle pipetting. After 15 min, the relipidated protein was added to a final concentration of 2.5 μg/ml to a reaction buffer containing 50 mM KNO3, 5 mM NaN3, 0.25 mM NaMob, 40 mM BisTris/HEPES pH 7.2, 3 mM MgSO4, 1 mM EGTA, and CaCl2 in concentration needed for obtaining the desired concentration of free calcium ions. The free calcium concentration was calculated using Maxchelator Ca/Mg/ATP/EGTA Calculator v1.0 using constants from NIST database (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator). aSN and/or CaM were added to the reaction mixture in desired concentrations and corresponding protein storage buffer was added to control samples in equal volume. The reactions were started by adding 3 mM ATP, carried out at 37°C, and stopped after 7 or 11 min by adding a stop solution. Stop solution was prepared shortly before every assay by mixing ice-cold solutions A (17 μM ascorbic acid, 0.1% SDS in 0.5 M HCl) and B (0.7 μM ammonium molybdate VI tetrahydrate) in 5:1 proportion. The absorbance was measured with Victor 3 96-well plate reader (PerkinElmer) at 860 nm.
Mathematical modeling of aSN-dependent calcium regulation
The solve_ivp function from the scipy (Virtanen et al, 2020) package was used for numerical integration of the model equations to simulate the model behavior. Simulation results were visualized using the matplotlib (Hunter, 2007) library to create informative plots and figures. To optimize the parameters of the kinetic equation used for PMCA, the scipy.minimize function was employed, minimizing the discrepancy of the equation to the experimental data. With respect to the modeling work presented here, data handling tasks were performed using the pandas (McKinney, 2010; Data ref: Reback et al, 2022) and numpy (Harris et al, 2020) libraries.
Transcriptomics data analysis
Data on gene expression for PMCA1-4 and aSN were used. Data on tissue expression and exon incorporation from VastDB for the genes ATP2B1, ATP2B2, ATP2B3, ATP2B4, and SNCA were imported in R. For the PMCA exons of relevance, the “percent spliced-in” (PSI) was correlated to aSN tissue expression. For tissues in specified aSN expression intervals (ranging from 0 to 190 interval size of 10), the average incorporation of the exons was determined, and the mean PSI was plotted as a function of the aSN expression interval. The code written in R for the analysis is included in the Appendix S1. Links to analyzed VastDB datasets are provided in the Expanded View Table EV1.
Protein disorder prediction
Protein disorder prediction was performed using NetSurfP 2.0 software developed by the Technical University of Denmark and available at https://services.healthtech.dtu.dk/service.php?NetSurfP-2.0.
Statistics were performed using GraphPad Prism and P values < 0.05 were considered significant. The statistical method used is described in the figure legends.
All plasmids and DNA primers used in this study are available from the Lead Contact (Poul Nissen) without restriction. Reagents used in the study were of general use and from commercial sources. This study includes no data deposited in external repositories. The transcriptomics data are publicly available at https://vastdb.crg.eu/. All original code written in R for the analysis is available in this paper's Appendix S1. The mathematical modeling data are available at https://ford.biologie.hu-berlin.de/jorin/pmca_asn/-/tree/main/. The remaining data reported in this paper, including DNA sequences used in the study, will be shared by the lead contact upon request.
This work was supported by a “Mobility plus, 3rd edition” fellowship grant of the Polish Ministry of Science and Higher Education to AK, a postdoctoral research grant from the Extremadura Province (PO17009) to MCB, by a PhD stipend from the Aarhus Graduate School of Science at Aarhus University to STL, by a collaborative grant from H. Lundbeck A/S to PHJ, by Lundbeck Foundation grants R223-2015-4222 for PHJ and R248-2016-2518 for Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular to PN and PHJ, and by funds from a project 2 research grant from the Independent Research Fund Denmark (7014-00328B) and the Brainstruc research center (R155-2015-2666) and a professorship grant (R310-2018-3713) of the Lundbeck Foundation to PN. Further support was given by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 101023654, awarded to EAN. and the Novo Nordisk Foundation (#NNF18OC0033926 to BBK). All NMR data were recorded at cOpenNMR, an infrastructure facility funded by the Novo Nordisk Foundation (#NNF18OC0032996). The mathematical modeling work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy—The Berlin Mathematics Research Center MATH+ (EXC-2046/1, project ID: 390685689) to EK.
We thank Torben Heick Jensen and Thomas Gonatopoulos-Pournatzis for advice on transcriptomics data analysis. We are grateful for biosamples and valuable discussion to Michael Habeck for Na,K-ATPase, Thomas Lykke-Møller Sørensen, Anne Lillevang, and Claus Olesen for SERCA. We thank Joseph A Lyons and Magnus Kjærgaard for valuable discussions on membrane proteins and intrinsically disordered proteins. We are grateful to Anna Marie Nielsen, Tetyana Klymchuk, Benedicte Vestergaard, and Jacob H Martinsen for technical assistance, and we thank Mogens Høgh Jensen, Jesper Ferkinghoff-Borg, and Mathias S. Heltberg for valuable input on enzymatic network modeling.
Antoni Kowalski: Data curation; formal analysis; validation; investigation; visualization; methodology; writing – original draft; writing – review and editing. Cristine Betzer: Validation; investigation; visualization; writing – review and editing. Sigrid Thirup Larsen: Data curation; software; formal analysis; validation; investigation; visualization; writing – review and editing. Emil Gregersen: Validation; investigation; visualization; writing – review and editing. Estella A Newcombe: Investigation; visualization; methodology; writing – review and editing. Montaña Caballero Bermejo: Investigation. Annette Eva Langkilde: Resources; formal analysis; supervision; writing – review and editing. Birthe B Kragelund: Resources; data curation; formal analysis; supervision; funding acquisition; validation; methodology; writing – review and editing. Poul Henning Jensen: Conceptualization; resources; data curation; formal analysis; supervision; funding acquisition; validation; methodology; project administration; writing – review and editing. Poul Nissen: Conceptualization; resources; data curation; formal analysis; supervision; funding acquisition; validation; methodology; writing – original draft; project administration; writing – review and editing. Shweta Jain: Investigation. Robert Edwards: Resources; data curation; formal analysis; funding acquisition; investigation; methodology. Lene N Nejsum: Resources; data curation; formal analysis; funding acquisition; investigation; methodology. Christina V Ernstsen: Investigation. Viktor Wisniewski Bendtsen: Software; formal analysis; investigation; methodology. Jorin Diemer: Data curation; software; formal analysis; investigation; methodology; writing – review and editing. Edda Klipp: Resources; data curation; software; formal analysis; supervision; funding acquisition; validation; methodology; project administration. Alicia Espiña Bou: Investigation.
Disclosure and competing interests statement
Poul Nissen is a member of the Advisory Editorial Board of The EMBO Journal. This has no bearing on the editorial consideration of this article for publication.
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