Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells

Many calcifying organisms utilize metabolic CO₂ to generate CaCO₃ minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO₂ pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H⁺-selective electrodes were used to demonstrate eCA-catalyzed CO₂ hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO₂ hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pH_i regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO₃^- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO₂ by Cara7 coupled to HCO₃^- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO₂ to generate a versatile construction material.