Abstract
The possible functional abnormalities in three different Darier disease-causing Ca(2+)-ATPase (SERCA2b) mutants, Ile(274) --> Val at the lumenal end of M3, Leu(321) --> Phe on the cytoplasmic part of M4, and Met(719) --> Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca(2+) transport activities that were essentially identical (L321F) or slightly lower (I274V by approximately 35% and M719I by approximately 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 Ca(2+), 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 Ca(2+), as demonstrated with the effect of Ca(2+) ionophore on ATPase activity and more specifically with the effects of Ca(2+) (up to 50 mm) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca(2+) homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca(2+) in L321F likely brings the lumenal Ca(2+) to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca(2+) 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 Ca(2+) transport.
Highlights
Immunofluorescence microscopy for the COS-1 cells transfected with SERCA2b cDNA clearly demonstrated that all the three mutants as well as the wild type are co-localized with the protein disulfide-isomerase (ER marker) and localized on ER, whereas the cells transfected with the vector containing no SERCA2b cDNA exhibited very weak background signals
Possible Causes of DD by the Three Mutations—In the present study, we explored functional abnormalities in the three DD-causing SERCA2b mutants I274V, L321F, and M719I, because they exhibited nearly normal expression and localization in cells and the seemingly high specific ATPase and coupled Ca2ϩ transport activities
Because DD is thought to be caused by haploinsufficiency in Ca2ϩ homeostasis in keratinocytes and the previous analyses on 22 different DD mutations revealed [32, 34] that the mutations resulted in the remarkably reduced or almost completely suppressed protein expression and/or function of SERCA2b, the present results on the three new types of mutants likely provide further insight into the haploinsufficiency and the possible consequence of the insensitivity to lumenal Ca2ϩ found with L321F as discussed below
Summary
We found that three DD-causing SERCA2b mutants that have never been explored previously, Ile274 Val, Leu321 Phe, and Met719 Ile (Fig. 2), are expressed in COS-1 cells at a level as high as that of wild type ( escaping from degradation by the quality control) and possess high Ca2ϩ-ATPase and coupled transport activities that are essentially identical to (L321F) or slightly lower than (I274V by ϳ35% and M719I by ϳ30%) those of the wild type These three mutants happened to be the cases of Japanese DD patients [25]
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