Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. Here, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1 null (KO) mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin, and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodeling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of K-Cl cotransporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes implying a wider range of pump regulation by calcium and calmodulin. Together, with the atomic force microscopy of the skeletal network, our data provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium-imbalance pathologies such as sickle cell disease.