Dual salt bridges govern proton gating and calcium leak in BsYetJ across bilayers and live cells

• 成果类型: Article
• 署名作者: Cheng, Chu-Chun; Li, Chieh-Chin; Wang, Yun-Shan; Lin, Chun-Wei; Chiang, Yun-Wei
• 署名单位: National Tsing Hua University; Cornell University; Weill Cornell Medicine
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2536595123
• 发表日期: 2026-07-07
• 页码: e2536595123
• 关键词: calcium channel; nanodisc; salt bridge; deer; electrophysiology; BAX INHIBITOR-1; suppressor; apoptosis; channel; TMBIM

摘要

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.