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The mechanism by which the ammonium transporter,AmtB,conducts NH4 +/NH3 into the cytoplasm was investigated using conventional molecular dynamics (MD) simulations.These simulations revealed that the neutral molecule,NH3,passes automatically through the channel upon its arrival at the Am2 site,and that the function of the channel as a one-way valve for passage of NH3 is not determined by the cytoplasmic exit gate but,rather,by the periplasmic entrance gate of the channel.The NH3,produced by deprotonation of NH4 + at the entrance gate,is spontaneously conveyed to the central region of the channel via a hydrogen bond network comprising His168,His318,Tyr32 and the NH3 molecule.Finally,the NH3 molecule exits the channel through the exit gate formed by Phe31,Ile266,Val314 and His318.In addition,Ser263 is shown to play a critical role in the final stages,acting as a pivoting arm to shunt the NH3 molecule from the cytoplasmic exit gate of the channel out into the cytoplasm.This finding is further supported by another simulation which shows that NH3 fails to be translocated through the channel formed by the Ser263Ala mutation.Thus,this study casts new insights on the mechanism of AmtB-mediated passage of NH3 across cellular membranes.