title: Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.
abstract: 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. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] 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 remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter 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. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study 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.
authors.affiliation: Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Wieschhaus
authors.forename: Anwar
authors.initials: A
authors.lastname: Khan
authors.forename: Asma
authors.initials: A
authors.lastname: Zaidi
authors.forename: Henry
authors.initials: H
authors.lastname: Rogalin
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Hanada
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.forename: Lucia
authors.initials: L
authors.lastname: De Franceschi
authors.forename: Carlo
authors.initials: C
authors.lastname: Brugnara
authors.forename: Alicia
authors.initials: A
authors.lastname: Rivera
authors.forename: Athar H
authors.initials: AH
authors.lastname: Chishti
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002154:Calpain / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004905:Erythrocyte Aging / Q000187:drug effects / Q000502:physiology; D004907:Erythrocyte Deformability / Q000187:drug effects / Q000502:physiology*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000097:blood; D008565:Membrane Proteins / Q000097:blood; D051379:Mice; D018345:Mice, Knockout; D018625:Microscopy, Atomic Force; D009996:Osmotic Fragility / Q000187:drug effects / Q000502:physiology; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000097:blood; D013102:Spherocytes / Q000187:drug effects / Q000502:physiology
chemical_list: D001798:Blood Proteins; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496925:Kcnn4 protein, mouse; D008565:Membrane Proteins; D000001:Calcimycin; D003994:Bucladesine; D002154:Calpain; C112682:Capn1 protein, mouse; D053499:Plasma Membrane Calcium-Transporting ATPases