Got Oxygen? the Impact of Band 3 on RBC Function during Exercise

Introduction: Band 3, or anion exchanger 1 (AE1), facilitates chloride-bicarbonate exchange across the membrane of red blood cells (RBCs), where it acts as critical regulator of oxygen delivery and red blood cell metabolism. The N-terminal domain of AE1 (residues 12-23) also serves as a binding site to deoxygenated hemoglobin, while AE1 residues 1-11 bind glycolytic enzymes (PFK, ALDOA, GAPDH) at high oxygen saturation. Therefore, the AE1-hemoglobin interaction acts as an oxygen sensor for metabolic regulation, a mechanism that ensures RBCs prioritize generation of energy and allosteric regulators that favor oxygen off-loading under hypoxia (e.g., ATP, 2,3-bisphosphoglycerate (BPG)), while favoring NADPH production by redirecting metabolism to the Pentose Phosphate Pathway during high oxidative stress. This allows RBC function to adapt to varied physiological conditions, such as high altitude or high oxygen demand during exercise. Under these hypoxic conditions, increasing the glycolytic rate fuels the Rapoport-Luebering shunt and BPG synthesis to favor O2 off-loading to tissues in the face of exercise-induced increase in oxygen demand. Dysregulation of AE1 activity in the setting of aging, exercise, or cell damage compromises metabolic flexibility and leads to RBC degradation, emphasizing AE1's role in maintaining RBC health. Methods: Here we generated novel humanized mouse models carrying either (i) human canonical band3 (HUB3) or two different band3 knock out variations: (ii) high affinity deletion (first 11 amino acids deleted, HA-Del) or (iii) binding site knock out (amino acid residues 12-23, BS-KO). The original AE1 mice were generated by the Low lab on a 129 background, prior to backcrossing for 6-7 generations to C57BL6. As we recently noted, some residual 129 mouse polymorphisms were carried over, particularly on chromosome 1, coding for genes like Steap3 that affect RBC lipid peroxidation. To overcome these limitations, we generated new mouse models by introducing identical AE1 (HUB3, HA-DEL and BS-KO) on a clean C57BL6/J background. We then leveraged these mouse models to assess the effects of AE1 regulatory activity on critical speed (CS), a functional measurement of exercise tolerance to exhaustion. To expand the molecular resolution of genetically associated adaptations to exercise, CS tests were combined with hemodynamic measurements, imaging (e.g., scanning electron microscopy of RBCs before and after exercise) and multi-omics (mass spectrometry-based metabolomics, lipidomics and proteomics) characterization of plasma and RBCs pre and post run to exhaustion. Results: The BS-KO mice had a significantly slower median CS compared to both HA-Del and HUB3 (20.9% and 11.2%). Complete blood count and hemodynamic data showed no significant difference between the three groups, aside from significantly decreased cardiovascular efficiency in the BS-KO mice (p=0.004). SO2 was significantly decreased in BS-KO mice at 85%. RBC metabolomics analysis highlighted changes pre and post exercise, showing increased Land's cycle activity and pantothenate levels in BS-KO mice, which are critical for membrane repair. Plasma metabolomics reflected the systemic hypoxia the KO animals experienced during exercise, showing significant increases of Kreb's cycle metabolites such as malate, fumarate, and succinate (p=0.001). Conclusion: Using novel humanized mouse models with modification of Band 3 regulatory protein binding sites, we observed that the deoxyhemoglobin BS-KO mice had significantly slower CS and cardiovascular efficiency compared to other models, indicating impaired metabolic and physiological adaptability to the high physiological demand of exercise. Metabolomics and proteomics analyses revealed that BS-KO mice experienced systemic hypoxia and membrane damage, with significant changes in metabolites linked to hypoxia and stress responses. These findings underscore the importance of AE1 in maintaining RBC metabolic flexibility particularly under stressful conditions such as exercise. The results suggest potential therapeutic targets for mitigating RBC-related pathologies in conditions of oxidative stress and metabolic dysregulation.