Wesley Tien ChiangYao-Kai ChangWei-Han HuiShu-Wei ChangChen-Yi LiaoYi-Chuan ChangChun-Jung ChenWei-Chen WangChien-Chen LaiChun-Hsiung WangSiou-Ying LuoShan-Ho ChouYa-Ping HuangTzyy-Leng HorngMing-Hon HouStephen P. MuenchRen-Shiang ChenMing-Daw TsaiNien-Jen Hu2024-07-022024-07-022024-12https://www.scopus.com/record/display.uri?eid=2-s2.0-85192356343&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/719558The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous systemtruejournal article10.1038/s41467-024-48057-y2-s2.0-85192356343