Quantifying Ca²⁺ and Mg²⁺ Transport by Annexins A5 and A6 in Proteoliposomes Using Ultrasensitive Diamond Microelectrodes

Matrix vesicles (MVs) initiate physiological mineralization through Ca²⁺ and phosphate (Pi) nucleation, to produce hydroxyapatite (HA) crystals. Annexins A5 (AnxA5) and A6 (AnxA6) mediate Ca²⁺ influx into the MV lumen. For the first time, this study has used ultrasensitive boron-doped nanocrystalline diamond microelectrode arrays (BNCD-MEA) to quantify Ca²⁺ and Mg²⁺ transport kinetics (Kt) in AnxA5 and AnxA6 reconstituted proteoliposomes. The proteoliposomes (9:1 dipalmitoyl-phosphatidylcholine/dipalmitoylphosphatidylserine) incorporated annexins with 54–64% efficiency, as confirmed by annexin quantification, dynamic light scattering, zeta potential, and reported atomic force microscopy data. BNCD-MEA calibration yielded linear ΔV responses for up to 0–2 mM Ca²⁺/Mg²⁺ (r > 0.97). Our data revealed that AnxA5 had higher affinity for Ca²⁺ (Kt = 7.6 mM) than AnxA6 (Kt = 11.2 mM) and marked selectivity toward Mg²⁺. Adding EDTA halted Ca²⁺ internalization by MVs, which confirmed that annexin-facilitated transport was reversible. Compared to Ca²⁺ uptake by the annexins, smaller Mg²⁺ uptake highlighted that AnxA5 and AnxA6 were selective. These innovations enabled Kt to be precisely calculated via double reciprocal plots, to elucidate the AnxA5 channel-like efficiency versus the role played by AnxA6 in MVs. Overall, these findings advance the BNCD-MEA applications for investigating ion dynamics in physiological biomineralization and pathological calcification.