M.P. Møller, A. Cvjetkovic, E. Kosmidis, A. Narducci, M. Isselstein, C.G. Shuttle, B. Justesen, I.L. Jørgensen, S. Basse-Hansen, M. Dyla, J. Holmkvist, M. Kjaergaard, P.A. Pedersen, P. Nissen, T. Günther Pomorski, and D. Stamou (2026).
Long-term single-molecule Ca2+ flux recordings reveal mode-switching regulation of Ca2+-ATPases.
bioRxiv 2026.01.28.701947
doi: 10.64898/2026.01.28.701947
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.