Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca 2؉ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 2 (SERCA2) isoforms, SERCA2a Sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) 1 are single-subunit integral membrane P-type ATPases that mediate the ATP-driven transport of cytosolic Ca 2ϩ against a concentration gradient into the lumen of intracellular Ca 2ϩ -releasable stores such as sarcoplasmic and endoplasmic reticulum (1-4). SERCAs belong to the P-type ATPase family distinguished by the obligatory formation of an aspartyl-phosphorylated intermediate as part of their catalytic cycle. The enzyme cycles reversibly between several states (Scheme 1), of which at least E 1 Ca 2 , E 1 ϳP(Ca 2 ), E 2 -P, and E 2 can be experimentally distinguished. The transfer of the ␥-phosphoryl group of ATP to the aspartate (Asp 351 ) of the phosphorylation domain, leading to the ADP-sensitive high energy phosphoenzyme intermediate E 1 ϳP(Ca 2 ), is activated by conformational changes associated with the binding of two calcium ions in exchange for protons (E 2 to E 1 Ca 2 transition). The conversion of this intermediate to the ADP-insensitive low-energy E 2 -P phosphoenzyme intermediate constitutes a crucial rate-limiting step in Ca 2ϩ translocation (1-3). High resolution models for the atomic structure, generated by x-ray crystallography of crystals in E 1 Ca 2 (5) and Ca 2ϩ -free E 2 (6) forms, and extensive mutational studies (1, 7-11) of the 110-kDa SERCA1a enzyme (994 amino acids) have shown that Ca 2ϩ translocation and ATP utilization are coupled through long range intramolecular interactions between the 10-helix membrane-spanning domain, harboring the Ca 2ϩ binding sites, and the large cytosolic head consisting of actuator (A), phosphorylation (P), and nucleotide-binding (N) domains (Fig. 1).The three human SERCA genes (ATP2A1, ATP2A2, and ATP2A3) encode up to a total of 10 isoforms as a result of the alternative splicing of their pre-mRNA (12-16). Mutations in SERCA genes have been recently detected in human diseases such as Brody disease (muscle disorder) for SERCA1 (17) and

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ATP2C1, encoding the human secretory pathway Ca 2؉ /Mn 2؉ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca 2؉ and Mn 2؉ with equally high affinity into the Golgi of COS-1 cells. Ca 2؉ -and Mn 2؉ -dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (P ifuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E 1 ϳP 3 E 2 -P conformational transition. D742Y and G309C were devoid of Ca 2؉ -and Mn 2؉ -dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from P i was retained in these mutants but with a loss of sensitivity to both Ca 2؉ and Mn 2؉ in D742Y and a preferential loss of sensitivity to Mn 2؉ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn 2؉ transport selectivity.