Ca²⁺-pumping by PMCA-neuroplastin complexes operates in the kiloHertz-range

Ca²⁺-ATPases in the plasma membrane extrude Ca²⁺ ions from the cytosol to the extracellular space thereby terminating Ca²⁺-signals and controlling Ca²⁺-homeostasis in any type of cell. Recently, these Ca²⁺-pumps have been identified as protein complexes of the transporting subunits PMCAs1-4 and the single-span membrane proteins Neuroplastin (NPTN) or Basigin that are obligatory for efficient trafficking of the pump complexes to the surface membrane. Quantitative investigation of the pumping velocity controlling the time course of Ca²⁺-signals, however, has remained unresolved. Here we show, using Ca²⁺-activated K⁺ channels as fast native reporters of intracellular Ca²⁺ concentration(s) together with membrane-tethered fluorescent Ca²⁺-indicators, that under cellular conditions PMCA2-NPTN complexes can clear Ca²⁺ in the low millisecond-range. Computational modeling exploiting EM-derived densities of Ca²⁺-source(s) and Ca²⁺-transporters in freeze-fracture replicas translated these fast kinetics into transport rates for individual PMCA2-NPTN pumps of more than 5000 cycles/s. Direct comparison with the Na⁺/Ca²⁺-exchanger NCX2, an alternate-access transporter with established cycling rates in the kHz range, indicated similar efficiencies in Ca²⁺-transport. Our results establish PMCA2-NPTN complexes, the most abundant Ca²⁺-clearing tool in the mammalian brain, as transporters with unanticipated high cycling rates and demonstrate that under cellular conditions ATPases may operate in the kHz-range.