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

Abstract

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.

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

“…Surprisingly, mutant P201L fully complemented the pmr1 null phenotypes and was indistinguishable from the wild-type hSPCA1, suggesting that P201L mutation does not disrupt pump function. Although it remains a possibility that small decreases in activity might have been masked by overexpression of the mutant, a recent biochemical analysis of this mutant expressed in COS-1 cells also failed to reveal any defects in formation of the catalytic phosphoenzyme intermediate or in subcellular localization (27). Taken together, data from yeast and mammalian cells suggest that patients with P201L harbor additional disease-causing mutations in introns or promoter regions of ATPC1, as suggested by Li et al (47).…”

“…Pmr1 homologs are now known to be ubiquitous in all higher eukaryotes, with the notable exception of plants, and much of what we know about the structure and physiological role of these transporters comes from studies in yeast. More recently, these studies have extended to SPCA homologs from humans (5,27,83), Caenorhabditis elegans (13,87), and rats (Ref. 69; reviewed in Ref.…”

Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn 2+ efflux and protects against toxicity

“…FACS sorted cells were used for uptake assays using permeabilized cells and for the viability assays using methylthiazolylphenyl-tetrazolium bromide (MTT). Uptake of 54 Mn 2+ in permeabilized cells was done as described (4,45). MTT assay was performed as described (15).…”

“…Haploinsufficiency of SPCA1 results in the development of Hailey-Hailey disease, a blistering dermatosis associated with increased cytosolic Ca 2+ levels and impaired Ca 2+ signaling in keratinocytes (5-7). Whereas much attention has been focused on its Ca 2+ transport activity, SPCA1 can also transport Mn 2+ into the Golgi with the same affinity as Ca 2+ (4,8). The Mn 2+ transport activity of SPCA1 is particularly important because mechanisms regulating Mn 2+ homeostasis in mammalian cells are unknown.…”

“…P-type pumps share a high degree of sequence, structural and functional similarity, and their varied substrate specificities arise from minor changes in their ionbinding residues. During ion transport, autophosphorylation at an invariant aspartic acid residue in the cytoplasmic domain induces a characteristic conformation change from the E1 state, which has high affinity for the ion, to the E2 state, which has low affinity for the ion (1)(2)(3)(4). Transport through these pumps is limited by the rate of this conformational change (2).…”