The Secretory Pathway Ca ATPases SPCA1 and SPCA2 transport Ca and Mn into the Golgi and Secretory Pathway. SPCA2 mediates store-independent Ca entry (SICE) via STIM1-independent activation of Orai1, inducing constitutive Ca influx in mammary epithelial cells during lactation. Here, we show that like SPCA2, also the overexpression of the ubiquitous SPCA1 induces cytosolic Ca influx, which is abolished by Orai1 knockdown and occurs independently of STIM1. This process elevates the Ca concentration in the cytosol and in the non-endoplasmic reticulum (ER) stores, pointing to a functional coupling between Orai1 and SPCA1. In agreement with this, we demonstrate via Total Internal Reflection Fluorescence microscopy that Orai1 and SPCA1a co-localize near the plasma membrane. Interestingly, SPCA1 overexpression also induces Golgi swelling, which coincides with translocation of the transcription factor TFE3 to the nucleus, a marker of Golgi stress. The induction of Golgi stress depends on a combination of SPCA1 activity and SICE, suggesting a role for the increased Ca level in the non-ER stores. Finally, we tested whether impaired SPCA1a/Orai1 coupling may be implicated in the skin disorder Hailey-Hailey disease (HHD), which is caused by SPCA1 loss-of-function. We identified HHD-associated SPCA1a mutations that impair either the Ca transport function, Orai1 activation, or both, while all mutations affect the Ca content of the non-ER stores. Thus, the functional coupling between SPCA1 and Orai1 increases cytosolic and intraluminal Ca levels, representing a novel mechanism of SICE that may be affected in HHD.

Dysregulation of the Golgi/Secretory Pathway Ca transport ATPase SPCA2 is implicated in breast cancer. During lactation and in luminal breast cancer types, SPCA2 interacts with the plasma membrane Ca channel Orai1, promoting constitutive Ca influx, which is termed store independent Ca entry (SICE). The mechanism of SPCA2/Orai1 interaction depends on the N- and C-termini of SPCA2. These extensions may play a dual role in activating not only Orai1, but also Ca transport into the Golgi/secretory pathway, which we tested by investigating the impact of various SPCA2 N- and/or C-terminal truncations on SICE and Ca transport activity of SPCA2. C-terminal truncations impair SICE and SPCA2 activity, but also affect targeting, whereas N-terminal truncations affect targeting and inactivate SPCA2, but remarkably, SICE activation remains unaffected. Importantly, overexpression of SPCA2 increases the Ca content of non-ER stores, which depends on Orai1 and SPCA2 activity. Thus, Orai1-mediated Ca-influx and SPCA2-mediated Ca uptake activity into the Golgi/secretory pathway might be coupled possibly in a microdomain. This channel/pump complex may efficiently transfer Ca into the secretory pathway, which might play a role in SPCA2-expressing secretory cells, such as mammary gland during lactation.

The sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA‐stabilized E2−AlF4− form (3.3 Å) and the Ca2+‐occluded [Ca2]E1‐AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform‐specific residues are acceptor sites for post‐translational modifications. In addition, molecular dynamics simulations predict that isoform‐specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform‐specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.