In general, for the spontaneous incorporation process associated with the apparent rate constant kspon, it can be expected that the better the charge of the cation is screened by EB molecules, the lower the activation barrier for the incorporation into the membrane core will be. Among the possible structures, the 2:1 sandwich complex provides the highest degree of charge screening being surrounded by a 5 -7 Å organic layer created by two EB molecules. Since EB and K + adopt such a EB2:K + complex according to our and previous assignments (as it is the case for Cs + ), it is not surprising that the spontaneous incorporation occurs efficiently on the similar time scales as observed previously for peptides forming transmembrane channels. In this light, it is therefore also comprehensible that in presence of Na + this process is hindered, since a uniform 2:1 sandwich complex cannot be formed according to DFT results. Accordingly, our data excludes the presence of belted 1:1 EB:Na + in complex, and points to the predominant presence of the distorted EB2:Na + and the EB:Na + out as main membrane-associated structures. Since both structures will screen the ion's charge less efficiently than an "ideal" EB2:M + complex, direct incorporation will be hindered, and thus the considerable difference in activation barrier is observed. Importantly, despite its asymmetric structure, only the distorted EB2:Na + would provide sufficient charge screening to incorporate into the hydrophobic membrane interior.