2.A.38 The K⁺ Transporter (Trk) Family

The proteins of the Trk family are derived from Gram-negative and Gram-positive bacteria, yeast and plants. The proteins of E. coli K12 (TrkH and TrkG) as well as several yeast and plant proteins have been functionally and topologically characterized. While TrkH is generally present in E. coli and other enteric bacteria, TrkG is encoded by a foreign gene located within the prophage rac region of the E. coli K12 chromosome. TrkG is not present in several other E. coli strains. The sizes of the Trk family members vary from 423 residues to 1235 residues. The bacterial proteins are of 423-558 residues, the Triticum aestivum protein is 533 residues, and the yeast proteins vary between 841 and 1241 residues. These proteins possess 8 putative transmembrane α-helical spanners. An 8 TMS topology with N- and C-termini on the inside, has been established for AtHKT1 of A. thaliana (Kato et al., 2001) and Trk2 of S. cerevisiae (Zeng et al., 2004). This folding pattern resembles quadruplicated primitive K⁺ channels of the VIC superfamily (TC #1.A.1) instead of typical 12 TMS carriers (Matsuda et al., 2004). As homology has been established between Trk carriers and VIC family channels, the latter were presumably the precursors of the former.

The phylogenetic tree reveals that the proteins cluster according to phylogeny of the source organism with (1) the Gram-negative bacterial Trk proteins, (2) the Gram-negative and Gram-positive bacterial Ktr proteins, (3) the yeast proteins and (4) the plant proteins comprising four distinct clusters (Saier et al., 1999). S. cerevisiae possesses two paralogues, high- and low-affinity K⁺ transporters.

The KtrAB system of Vibrio alginolyticus consists of a cytoplasmic ATP-binding regulatory subunit, KtrA (Kroning et al., 2007), shared by several other K⁺ transporters and channels including an integral membrane protein, KtrB, which is very similar to NtpJ of E. hirae and distantly related to TrkH of E. coli (Nakamura et al., 1998b). KtrA binds ATP to a β-α-β-Rossman fold. Other nucleotides bound, but only ATP bound with high affinity and changed the conformation of KtrA so that KtrA and KtrB associate (Kroning et al., 2007) in vivo, both ATP and ΔΨ were required for transport activity. All of these systems may be multicomponent. However, the integral membrane constituents have been proposed to exhibit a basic structure of four consecutive M1-P-loop-M2 motifs analogous to the KcsA K⁺ channel of Streptomyces lividans (see TC #1.A.1). Supporting this model, modification of the four putative selectivity filter glycine residues (one in each P-loop) in KtrB of V. alginolyticus altered the K⁺ affinity (100 fold) and selectivity, altering the K⁺/Na⁺ selectivity (Tholema et al., 2005). Two homologues of the V. alginolyticus KtrAB are found in B. subtilis. Both systems catalyze K⁺ uptake but with differing Kms: 1 mM for KtrAB and 10 mM for KtrCD (Holtmann et al., 2003).

The E. coli TrkH and TrkG proteins are complexed to two peripheral membrane proteins, TrkA, the ATP/NAD⁺-binding protein, and TrkE, an ATP-binding protein. The peripheral membrane proteins are thought to function in regulation rather than energy coupling. TrkE maps to the sapABCDF operon which encodes an ABC transporter (TC #3.A.1.5.5), and the SapD ATP binding cassette (ABC) protein of E. coli can stimulate K⁺ uptake via either Trk^H or Trk^G (Harms et al., 2001). Therefore, SapD is probably TrkE. ATP binding to SapD, rather than ATP hydrolysis, appears to activate. Thus, the pmf drives transport while ATP binding activates transport. At least one other ATP-activating protein is probably present in the E. coli cell (Harms et al., 2001).

TrkA family members regulate various K⁺ transporters in all three domains of life. These regulatory subunits are generally called K⁺ transport/nucleotide binding subunits (Bateman et al., 2000). Systems regulated include TrkG/H (TC #2.A.38.1.1) regulated by TrkA (with two KTN domains) and KtrB (TC #2.A.38.4.2) regulated by KtrA (Roosild et al., 2002). In addition, homologous regulatory domains are sometimes incorporated into the transporter polypeptide chains as in the case of the KefC and KefB proteins of E. coli (TC #2.A.37.1.1 and 2.A.37.1.2, respectively).

TrkA domains can bind NAD⁺ and NADH, possibly allowing K⁺ transporters to be responsive to the redox state of the cell. The ratio of NADH/NAD⁺ may control gating. Multiple crystal structures of two KTN domains complexed with NAD⁺ or NADH reveal that these ligands control the oligomeric (tetrameric) state of KTN. The results suggest that KTN is inherently flexible, undergoing a large conformational change through a hinge motion (Roosild et al., 2002). The KTN domains of Kef channels interact dynamically with the transporter. The KTN conformation then controls permease activity (Roosild et al., 2002). 

Both yeast transport systems are believed to function by K⁺:H⁺ symport, but the wheat protein functions by K⁺:Na⁺ symport. It is possible that some of these proteins can function by a channel-type mechanism. Positively charged residues in TMS8 of several ktr/Trk/HKT transporters probably face the channel and block a conformational change that is essential for channel activity while allowing secondary active transport (Kato et al., 2007).

KtrB is the K⁺-translocating subunit of the K⁺-uptake system KtrAB. It is a member of the superfamily of K⁺transporters (SKT proteins) with other sub-families occurring in archaea, bacteria, fungi, plants and trypanosomes (Hänelt et al., 2011). SKT proteins may have originated from small K⁺ channels by at least two gene duplication and two gene fusion events. They contain four covalently linked M(1)PM(2) domains, in which M(1) and M(2) stand for transmembrane stretches, and P for a P-loop, which folds back from the external medium into the membrane. SKT proteins distinguish themselves in two important aspects from K⁺ channels: first, with just one conserved glycine residue in their P-loops they contain a much simpler K⁺-selectivity filter sequence than K⁺ channels with their conserved Thr-Val-Gly-Tyr-Gly sequence. Secondly, the middle part M(2C2) from the long transmembrane stretch M(2C) of KtrB from the bacterium Vibrio alginolyticus forms a gate inside the membrane, which prevents K⁺ permeation to the cytoplasm. Hänelt et al. (2011) discuss the mechanism of K⁺ transport via KtrB and other SKT proteins and how the KtrA protein regulates the transport activity.

The generalized transport reaction catalyzed by the Trk family members is therefore probably:

K⁺ (out) + H⁺ (out) ⇌ K⁺ (in) + H⁺ (in)