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Jens Peter Andersen - One of the best experts on this subject based on the ideXlab platform.
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a Darier Disease mutation relieves kinetic constraints imposed by the tail of sarco endo plasmic reticulum ca2 atpase 2b
Journal of Biological Chemistry, 2018Co-Authors: Stine A Mikkelsen, Peter Vangheluwe, Jens Peter AndersenAbstract:The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2b isoform possesses an extended C terminus (SERCA2b tail) forming an 11th transmembrane (TM) helix, which slows conformational changes of the Ca2+-pump reaction cycle. Here, we report that a Darier Disease (DD) mutation of SERCA2b that changes a glutamate to a lysine in the cytoplasmic loop between TM8 and TM9 (E917K) relieves these kinetic constraints. We analyzed the effects of this mutation on the overall reaction and the individual partial reactions of the Ca2+ pump compared with the corresponding mutations of the SERCA2a and SERCA1a isoforms, lacking the SERCA2b tail. In addition to a reduced affinity for Ca2+, caused by the mutation in all three isoforms examined, we observed a unique enhancing effect on the turnover rates of ATPase activity and Ca2+ transport for the SERCA2b E917K mutation. This relief of kinetic constraints contrasted with inhibitory effects observed for the corresponding SERCA2a and SERCA1a (E918K) mutations. These observations indicated that the E917K/E918K mutations affect the rate-limiting conformational change in isoform-specific ways and that the SERCA2b mutation perturbs the interactions of TM11 with other SERCA2b regions. Mutational analysis of an arginine in TM7 that interacts with the glutamate in SERCA1a crystal structures suggested that in wildtype SERCA2b, the corresponding arginine (Arg-835) may be involved in mediating the conformational restriction by TM11. Moreover, the E917K mutation may disturb TM11 through the cytoplasmic loop between TM10 and TM11. In conclusion, our findings have identified structural elements of importance for the kinetic constraints imposed by TM11.
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dissection of the functional differences between sarco endo plasmic reticulum ca2 atpase serca 1 and 2 isoforms and characterization of Darier Disease serca2 mutants by steady state and transient kinetic analyses
Journal of Biological Chemistry, 2003Co-Authors: Leonard Dode, Jens Peter Andersen, Natalie Leslie, Jittima Dhitavat, B Vilsen, Alain HovnanianAbstract:Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier Disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the Disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.
Alain Hovnanian - One of the best experts on this subject based on the ideXlab platform.
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SERCA2 dysfunction in Darier Disease causes endoplasmic reticulum stress and impaired cell-to-cell adhesion strength: Rescue by miglustat
Journal of Investigative Dermatology, 2014Co-Authors: Magali Savignac, Laure Guibbal, Anissa Edir, Marina Simon, Alain HovnanianAbstract:Darier Disease (DD) is a severe dominant genetic skin disorder characterized by the loss of cell-to-cell adhesion and abnormal keratinization. The defective gene, ATP2A2, encodes sarco/endoplasmic reticulum (ER) Ca2+ -ATPase isoform 2 (SERCA2), a Ca2+ -ATPase pump of the ER. Here we show that Darier keratinocytes (DKs) display biochemical and morphological hallmarks of constitutive ER stress with increased sensitivity to ER stressors. Desmosome and adherens junctions (AJs) displayed features of immature adhesion complexes: expression of desmosomal cadherins (desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3)) and desmoplakin was impaired at the plasma membrane, as well as E-cadherin, β-, α-, and p120-catenin staining. Dsg3, Dsc3, and E-cadherin showed perinuclear staining and co-immunostaining with ER markers, indicative of ER retention. Consistent with these abnormalities, intercellular adhesion strength was reduced as shown by a dispase mechanical dissociation assay. Exposure of normal keratinocytes to the SERCA2 inhibitor thapsigargin recapitulated these abnormalities, supporting the role of loss of SERCA2 function in impaired desmosome and AJ formation. Remarkably, treatment of DKs with the orphan drug Miglustat, a pharmacological chaperone, restored mature AJ and desmosome formation, and improved adhesion strength. These results point to an important contribution of ER stress in DD pathogenesis and provide the basis for future clinical evaluation of Miglustat in Darier patients.
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activity of the hspca1 golgi ca2 pump is essential for ca2 mediated ca2 response and cell viability in Darier Disease
Journal of Cell Science, 2006Co-Authors: Lucie Foggia, Alain Hovnanian, Ida Aronchik, Karin M Aberg, Barbara E Brown, Theodora M MauroAbstract:Keratinocyte differentiation, adhesion and motility are directed by extracellular Ca2+ concentration increases, which in turn increase intracellular Ca2+ levels. Normal keratinocytes, in contrast to most non-excitable cells, require Ca2+ release from both Golgi and endoplasmic reticulum Ca2+ stores for efficient Ca2+ signaling. Dysfunction of the Golgi human secretory pathway Ca2+-ATPase hSPCA1, encoded by ATP2C1, abrogates Ca2+ signaling and causes the acantholytic genodermatosis, Hailey-Hailey Disease. We have examined the role of the endoplasmic reticulum Ca2+ store, established and maintained by the sarcoplasmic and endoplasmic reticulum Ca2+-ATPase SERCA2 encoded by ATP2A2, in Ca2+ signaling. Although previous studies have shown acute SERCA2 inactivation to abrogate Ca2+ signaling, we find that chronic inactivation of ATP2A2 in keratinocytes from patients with the similar acantholytic genodermatosis, Darier Disease, does not impair the response to raised extracellular Ca2+ levels. This normal response is due to a compensatory upregulation of hSPCA1, as inactivating ATP2C1 expression with siRNA blocks the response to raised extracellular Ca2+ concentrations in both normal and Darier keratinocytes. ATP2C1 inactivation also diminishes Darier Disease keratinocyte viability, suggesting that compensatory ATP2C1 upregulation maintains viability and partially compensates for defective endoplasmic reticulum Ca2+-ATPase in Darier Disease keratinocytes. Keratinocytes thus are unique among mammalian cells in their ability to use the Golgi Ca2+ store to mediate Ca2+ signaling.
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dissection of the functional differences between sarco endo plasmic reticulum ca2 atpase serca 1 and 2 isoforms and characterization of Darier Disease serca2 mutants by steady state and transient kinetic analyses
Journal of Biological Chemistry, 2003Co-Authors: Leonard Dode, Jens Peter Andersen, Natalie Leslie, Jittima Dhitavat, B Vilsen, Alain HovnanianAbstract:Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier Disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the Disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.
Jakob D. Wikstrom - One of the best experts on this subject based on the ideXlab platform.
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Is Darier Disease a Multi-organ Condition?
Acta dermato-venereologica, 2021Co-Authors: Etty Bachar-wikstrom, Jakob D. WikstromAbstract:Darier Disease is a severe, rare autosomal dominant inherited skin condition caused by mutations in the ATP2A2 gene encoding sarcoendoplasmic reticulum Ca2+-ATPase isoform 2 in the endoplasmic reticulum. Since sarcoendoplasmic reticulum Ca2+-ATPase isoform 2 is expressed in most tissues, and intracellular calcium homeostasis is of fundamental importance, it is conceivable that other organs besides the skin may be involved in Darier Disease. This review focusses on the association of Darier Disease with other organ dysfunctions and Diseases, emphasizing their common molecular pathology. In conclusion, Darier Disease should be considered a systemic condition that requires systemic and Disease mechanism targeted treatments.
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Darier Disease is associated with heart failure: a cross-sectional case-control and population based study
Scientific Reports, 2020Co-Authors: Etty Bachar-wikstrom, Martin Cederlof, Henrik Larsson, Ivone U S Leong, Tara Ahanian, Philip Curman, Jakob D. WikstromAbstract:Human data supporting a role for endoplasmic reticulum (ER) stress and calcium dyshomeostasis in heart Disease is scarce. Darier Disease (DD) is a hereditary skin Disease caused by mutations in the ATP2A2 gene encoding the sarcoendoplasmic-reticulum Ca2^+ ATPase isoform 2 (SERCA2), which causes calcium dyshomeostasis and ER stress. We hypothesized that DD patients would have an increased risk for common heart Disease. We performed a cross-sectional case-control clinical study on 25 DD patients and 25 matched controls; and a population-based cohort study on 935 subjects with DD and matched comparison subjects. Main outcomes and measures were N-terminal pro-brain natriuretic peptide, ECG and heart diagnosis (myocardial infarction, heart failure and arrythmia). DD subjects showed normal clinical heart phenotype including heart failure markers and ECG. The risk for heart failure was 1.59 (1,16-2,19) times elevated in DD subjects, while no major differences were found in myocardial infarcation or arrhythmias. Risk for heart failure when corrected for cardivascular risk factors or alcohol misuse was 1.53 (1.11–2.11) and 1.58 (1,15–2,18) respectively. Notably, heart failure occurred several years earlier in DD patients as compared to controls. We conclude that DD patients show a Disease specific increased risk of heart failure which should be taken into account in patient management. The observation also strenghtens the clinical evidence on the important role of SERCA2 in heart failure pathophysiology.
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Metabolic phenotype in Darier Disease: a cross-sectional clinical study
Diabetology & Metabolic Syndrome, 2020Co-Authors: Tara Ahanian, Etty Bachar-wikstrom, Martin Cederlof, Ivone U S Leong, Philip Curman, Kerstin Brismar, Jakob D. WikstromAbstract:Background Human data supporting a role for endoplasmic reticulum (ER) stress and calcium dyshomeostasis in diabetes is scarce. Darier Disease (DD) is a hereditary skin Disease caused by mutations in the ATP2A2 gene encoding the sarcoendoplasmic-reticulum ATPase 2 (SERCA2) calcium pump, which causes calcium dyshomeostasis and ER stress. We hypothesize that DD patients have a diabetes-like metabolic phenotype and the objective of this study was to examine the association between DD with impaired glucose tolerance and diabetes. Methods Cross-sectional clinical study on 25 DD patients and 25 matched controls. Metabolic status was assessed primarily by fasting blood glucose, oral glucose tolerance test, HOMA2-%S (insulin resistence) and HOMA2-%B (beta cell function). Results DD subjects showed normal oral glucose tolerance test and HOMA2-%S, while fasting blood glucose was lower and c-peptide as well as HOMA2-%B was higher. Conclusion Increased HOMA2-%B values are indicative of increased basal insulin secretion which is a type of beta cell dysfunction associated to diabetes development. These results supports a role of ER stress in diabetes pathophysiology and contribute to the understanding of DD as a multi-organ syndrome.
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novel mutations in Darier Disease and association to self reported Disease severity
PLOS ONE, 2017Co-Authors: Ivone U S Leong, Jakob D. Wikstrom, Martin Cederlof, Alexander Stuckey, Tara AhanianAbstract:Darier Disease is a rare and severe autosomal dominant skin Disease characterised by malodorous keratotic papules in seborrheic areas of the skin. Darier Disease affects up to 1 in 30 000 people and is caused by mutations in the ATP2A2 gene, which encodes to the sarco/endoplasmic reticulum calcium-ATPase isoform 2 that pumps calcium into the endoplasmic reticulum. Although many ATP2A2 variants have been described, it is not known if genotype correlates with phenotype, which could be important for prognosis and treatment. This is the first study to use whole exome sequencing to screen the ATP2A2 gene in a cohort of 28 clinically diagnosed Darier Disease patients. Twenty-one different Disease causing variants were identified and 15 of these were novel. Sixteen of the 21 variants were predicted to be pathogenic using in silico prediction programs. There were seven missense, four intronic/splice-sites, three frameshifts, two in-frame deletions, four nonsense and one synonymous mutations. This study also found ten patients who harbour more than one ATP2A2 variant. The phenotype of the patient cohort was assessed by photography and by patient questionnaires. The genotype-phenotype association was examined for all variants in relation to the patient's Disease severity score, and no correlation could be established.
Hajime Iizuka - One of the best experts on this subject based on the ideXlab platform.
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comprehensive analysis of expression and function of 51 sarco endo plasmic reticulum ca2 atpase mutants associated with Darier Disease
Journal of Biological Chemistry, 2006Co-Authors: Yuki Miyauchi, Akemi Ishidayamamoto, Kazuo Yamasaki, Takashi Daiho, Hidetoshi Takahashi, Hiroshi Suzuki, Stefania Danko, Hajime IizukaAbstract:We examined possible defects of sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier Disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595-35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the Disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.
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distinct types of abnormality in kinetic properties of three Darier Disease causing sarco endo plasmic reticulum ca2 atpase mutants that exhibit normal expression and high ca2 transport activity
Journal of Biological Chemistry, 2004Co-Authors: Katsuhiko Sato, Akemi Ishidayamamoto, Kazuo Yamasaki, Takashi Daiho, Yuki Miyauchi, Hidetoshi Takahashi, Satoshi Nakamura, Hajime Iizuka, Hiroshi SuzukiAbstract:Abstract The possible functional abnormalities in three different Darier Disease-causing Ca2+-ATPase (SERCA2b) mutants, Ile274 → Val at the lumenal end of M3, Leu321 → Phe on the cytoplasmic part of M4, and Met719 → Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca2+ transport activities that were essentially identical (L321F) or slightly lower (I274V by ∼35% and M719I by ∼30%) as compared with those of the wild type. These mutations happened to be in Japanese patients found previously by us. Kinetic analyses revealed that each of the mutants possesses distinct types of abnormalities; M719I and L321F possess the 2–3-fold reduced affinity for cytoplasmic Ca2+, whereas I274V possesses the normal high affinity. L321F exhibited also the remarkably reduced sensitivity to the feedback inhibition of the transport cycle by accumulated lumenal Ca2+, as demonstrated with the effect of Ca2+ ionophore on ATPase activity and more specifically with the effects of Ca2+ (up to 50 mm) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca2+ homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca2+ in L321F likely brings the lumenal Ca2+ to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca2+ homeostasis, but nevertheless all types of perturbation result in Darier Disease. It might be possible that the observed unique feature of L321F could possibly be associated with the specific symptoms in the pedigree with this mutation, neuropsychiatric disorder, and behavior problems. The results also provided further insight into the global nature of conformational changes of SERCAs for ATP-driven Ca2+ transport.
Hiroshi Suzuki - One of the best experts on this subject based on the ideXlab platform.
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comprehensive analysis of expression and function of 51 sarco endo plasmic reticulum ca2 atpase mutants associated with Darier Disease
Journal of Biological Chemistry, 2006Co-Authors: Yuki Miyauchi, Akemi Ishidayamamoto, Kazuo Yamasaki, Takashi Daiho, Hidetoshi Takahashi, Hiroshi Suzuki, Stefania Danko, Hajime IizukaAbstract:We examined possible defects of sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier Disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595-35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the Disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.
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distinct types of abnormality in kinetic properties of three Darier Disease causing sarco endo plasmic reticulum ca2 atpase mutants that exhibit normal expression and high ca2 transport activity
Journal of Biological Chemistry, 2004Co-Authors: Katsuhiko Sato, Akemi Ishidayamamoto, Kazuo Yamasaki, Takashi Daiho, Yuki Miyauchi, Hidetoshi Takahashi, Satoshi Nakamura, Hajime Iizuka, Hiroshi SuzukiAbstract:Abstract The possible functional abnormalities in three different Darier Disease-causing Ca2+-ATPase (SERCA2b) mutants, Ile274 → Val at the lumenal end of M3, Leu321 → Phe on the cytoplasmic part of M4, and Met719 → Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca2+ transport activities that were essentially identical (L321F) or slightly lower (I274V by ∼35% and M719I by ∼30%) as compared with those of the wild type. These mutations happened to be in Japanese patients found previously by us. Kinetic analyses revealed that each of the mutants possesses distinct types of abnormalities; M719I and L321F possess the 2–3-fold reduced affinity for cytoplasmic Ca2+, whereas I274V possesses the normal high affinity. L321F exhibited also the remarkably reduced sensitivity to the feedback inhibition of the transport cycle by accumulated lumenal Ca2+, as demonstrated with the effect of Ca2+ ionophore on ATPase activity and more specifically with the effects of Ca2+ (up to 50 mm) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca2+ homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca2+ in L321F likely brings the lumenal Ca2+ to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca2+ homeostasis, but nevertheless all types of perturbation result in Darier Disease. It might be possible that the observed unique feature of L321F could possibly be associated with the specific symptoms in the pedigree with this mutation, neuropsychiatric disorder, and behavior problems. The results also provided further insight into the global nature of conformational changes of SERCAs for ATP-driven Ca2+ transport.
