The alkali cation potassium is the main osmolyte in the cytoplasm of prokaryotes (1). It binds close to the active center of the ribosome (2) and participates actively in pH homeostasis (3) and osmoadaptation (4, 5) by transport across the cell membrane. Hence, prokaryotes regulate their K ϩ contents and adapt them rapidly in response to changes in the environment. For this purpose, they possess a number of K ϩ channels, pumps, and transporters (1, 6). Several of the K ϩ -uptake systems contain a K ϩ -translocating subunit belonging to the superfamily of K ϩ transporters (7, 8) (termed SKT proteins (9)). These proteins may have evolved from simple K ϩ channels of the M 1 PM 2 type, like KcsA (10, 11) or Kir (12), by multiple gene duplications and gene fusions (7). Whereas the channels form homotetramers from four identical M 1 PM 2 subunits, SKT proteins consist of four covalently linked M 1 PM 2 motifs connected by cytoplasmic loops. The four p-loops (P) are thought to fold back from the external medium to the middle of the membrane, where they form a part of the permeation pathway for K ϩ through the channel center (7-9, 13). Within each p-loop, most SKT proteins contain one conserved glycine residue, which is part of their K ϩ selectivity filter (9, 14 -17). With single conserved glycine residues in SKT proteins, this filter appears to have a simpler structure than in K ϩ channels, in which the filter is formed by the well conserved p-loop sequence TVGYG from each subunit (18).The SKT-protein KtrB forms the K ϩ -translocating subunit of the Na ϩ -dependent K ϩ -uptake system KtrAB from bacteria (9, 14, 19 -22). KtrA, the other subunit from KtrAB, is located at the cytoplasmic side of the membrane and is a member of the RCK/KTN protein family (1, 23). KtrA may regulate K ϩ transport by binding ATP (24,25). It confers velocity, Na ϩ dependence, and K ϩ selectivity to the complex (9). KtrB alone transports K ϩ slowly in a process that is independent of Na ϩ . In addition, it transports Na ϩ with relatively low affinity (K m value of ϳ3 mM Na ϩ (9)). The exact structure of KtrB is unknown, but it has been modeled based on the structure of KcsA (11,13). Most of the KtrB structure was similar to that of KcsA. However, in particular, the C termini from the membrane spans M 2C and M 2D deviated from that of KcsA-M 2 . This may reflect the difference in function between the channel KcsA and the transporter KtrB (13). Subsequent cross-linking studies showed that the external half of KtrB is very similar to that of the KcsA tetramer, whereas its cytoplasmic half deviates. In addition, KtrB may form dimers (26). In their modeling studies, Durell and Guy (13) focused on membrane span M 2C . They divided it into three regions, from M 2C1 to M 2C3 (see Fig. 1A). M 2C1 and M 2C3 can form hydrophobic ␣-helices. However, M 2C2 contains many conserved small and polar residues (Ala, Gly, and Ser, Thr, Lys, respectively; see Fig. 1B). It may form a random coil or ␤-turn structure (13). According to the first Durell and Guy model ...