Abstract
Many calcifying organisms utilize metabolic CO(2) to generate CaCO(3) 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 CO(2) 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 CO(2) hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO(2) 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 HCO(3)(-) uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO(2) by Cara7 coupled to HCO(3)(-) 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 CO(2) to generate a versatile construction material.