Pharmacological inhibitors of cysteine proteases have provideduseful insights into the regulation of calpain activity inerythrocytes. However, the precise biological function of calpainactivity in erythrocytes remains poorly understood. Erythrocytesexpress calpain-1, an isoform regulated by calpastatin, theendogenous inhibitor of calpains. In the present study, weinvestigated the function of calpain-1 in mature erythrocytes usingour calpain-1-null [KO (knockout)] mouse model. The calpain-1gene deletion results in improved erythrocyte deformabilitywithout any measurable effect on erythrocyte lifespan in vivo.The calcium-induced sphero-echinocyte shape transition iscompromised in the KO erythrocytes. Erythrocyte membraneproteins ankyrin, band 3, protein 4.1R, adducin and dematin aredegraded in the calcium-loaded normal erythrocytes but not inthe KO erythrocytes. In contrast, the integrity of spectrin andits state of phosphorylation are not affected in the calciumloadederythrocytes of either genotype. To assess the functionalconsequences of attenuated cytoskeletal remodelling in the KOerythrocytes, the activity of major membrane transporters wasmeasured. The activity of the K+\u2013Cl 12 co-transporter and theGardos channel was significantly reduced in the KO erythrocytes.Similarly, the basal activity of the calcium pump was reducedin the absence of calmodulin in the KO erythrocyte membrane.Interestingly, the calmodulin-stimulated calcium pump activitywas significantly elevated in the KO erythrocytes, implying awider range of pump regulation by calcium and calmodulin. Takentogether, and with the atomic force microscopy of the skeletalnetwork, the results of the present study provide the first evidencefor the physiological function of calpain-1 in erythrocytes withtherapeutic implications for calcium imbalance pathologies suchas sickle cell disease
