Effect of Hailey-Hailey Disease Mutations on the Function of a New Variant of Human Secretory Pathway Ca²⁺/Mn²⁺-ATPase (hSPCA1)*

ATP2C1, encoding the human secretory pathway Ca²⁺/Mn²⁺ 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 Ca²⁺ and Mn²⁺ with equally high affinity into the Golgi of COS-1 cells. Ca²⁺- and Mn²⁺-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (P_i-fuelled) 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 E₁∼P → E₂-P conformational transition. D742Y and G309C were devoid of Ca²⁺- and Mn²⁺-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 Ca²⁺ and Mn²⁺ in D742Y and a preferential loss of sensitivity to Mn²⁺ 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 Mn²⁺ transport selectivity.