Long-term single-molecule Ca ²⁺ flux recordings reveal mode-switching regulation of Ca ²⁺ -ATPases

Abstract Calcium (Ca²⁺) is a universal second messenger that governs processes ranging from muscle contraction and secretion to gene expression and cell fate. Ca²⁺-ATPases establish and maintain steep Ca²⁺ gradients across intracellular membranes, yet how regulatory inputs modulate the underlying single-pump Ca²⁺ currents has remained inaccessible. Here we develop a non-saturating, self-regenerating single-vesicle assay that monitors over hours the zeptoampere (10⁻²¹ A) currents produced by individual Ca²⁺-ATPases. In parallel, we establish a workflow to record single-molecule currents from human sarco/endoplasmic reticulum Ca²⁺-ATPases (hSERCA) in native endoplasmic reticulum vesicles. Using reconstituted LMCA1, a bacterial SERCA homologue, we observe stochastic switching between minute-long pumping and inactive modes, as well as uncoupled Ca²⁺ leakage events that are suppressed by vanadate. Extravesicular pH controls a previously unrecognized dormant pre-activation mode that delays the onset of pumping, without measurably altering pumping rates or active-mode lifetimes. Extending the assay to endogenous hSERCA reveals delayed activation and ultraslow pumping/inactive mode-switching without detectable transprotein Ca²⁺ leakage. ATP and Ca²⁺ regulate the probability of hSERCA activation by modulating dormant-mode occupancy. Together, these results extend ultraslow mode-switching, previously observed only for proton pumps, to Ca²⁺-ATPases and identify probability-gated entry into productive cycling as a distinct regulatory axis of human Ca²⁺-ATPase regulation that can modulate the timing and heterogeneity of Ca²⁺ store refilling without changing on-cycle kinetics.