The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca²⁺-microdomains by tuning PMCA3 activity

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca²⁺ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca²⁺ pumps and exchangers maintains the fluctuation of cytosolic Ca²⁺ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca²⁺ATPase (PMCA pump) selectively regulate cytosolic Ca²⁺ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca²⁺]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca²⁺] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca²⁺ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca²⁺ extrusion activity in the sub-PM Ca²⁺ microdomains. These results highlight the importance to set a precise threshold of [Ca²⁺] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.