摘要
Cells tightly regulate calcium because it controls processes ranging from signaling to survival. We show that the membrane protein BsYetJ uses two distinct electrostatic gates to couple proton sensing to calcium leak: One gate controls channel opening, whereas the other tunes conductance and selectivity once the pore is open. We also establish a nanodisc-based delivery strategy that inserts purified membrane proteins directly into live mammalian cells, enabling functional testing without genetic manipulation. Together, these results define a molecular mechanism linking protonation to ion transport and provide a versatile platform for studying membrane proteins in living cells. Proton-coupled ion transport is a fundamental chemical process underlying membrane physiology, yet how local electrostatics are transduced into gated Ca2+ permeation remains poorly defined. Here, we combine single-channel planar bilayer electrophysiology, nanodisc-based double electron–electron resonance spectroscopy, atomistic modeling, and a nanodisc nano-delivery strategy that enables direct functional insertion of purified membrane proteins into live mammalian cells. Applying this integrated toolkit to the bacterial transmembrane Bax-inhibitor-1–containing motif prototype BsYetJ, we resolve a hierarchical electrostatic gating mechanism governed by two salt bridges with distinct physical roles. A periplasmic E49–R205 interaction functions as a proton-sensitive latch that drives transmembrane helix 2 displacement and controls opening probability, while a cytoplasmic E182–R15 pair operates as a local electrostatic determinant of Ca2+ self-block that tunes conductance and selectivity without large-scale conformational change. Quantitative separation of these effects reveals how protonation reshapes the energy landscape of ion permeation. Live-cell Ca2+ imaging following nano-delivery recapitulates this gating logic in a cellular membrane setting. Together, this work establishes dual salt-bridge electrostatics as a chemical principle for graded Ca2+ leak and introduces nano-delivery as a powerful platform for connecting molecular electrostatics to cellular ion transport.