Highlights d The structure of the cardiac sodium channel reveals key functional features d The antiarrhythmic drug flecainide blocks the pore below the selectivity filter d The ion selectivity filter and inactivation gate are revealed in atomic detail d An arrhythmia mutation creates a pathogenic gating pore 2 Å in diameter

The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energycoupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of Escherichia coli YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of "sequence motif A." This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.T ransporters are a type of membrane protein essential for all living cells that actively up-take nutrition and export metabolic substances and toxic materials across cellular membranes. Transporters are divided into two major types based on their energy sources. Although primary active transporters directly consume energy from ATP hydrolysis to drive substrate transport, secondary active transporters use energy derived from the electrochemical potential across the cell membrane. The major facilitator superfamily (MFS) is the largest class of secondary transporters and is present in all life kingdoms (1). For example, 25% of prokaryotic transporters belong to the MFS family (2), and the human genome contains over 110 MFS proteins (3). Currently, 3D crystal structures of nine bacterial MFS transporters (4-13) and one from fungi (9) have been reported at medium-high resolution. These studies have shown that MFS proteins contain a 12-transmembrane (TM) helix core composed of two six-helix rigid domains forming a central TM channel, which transports substrates using a rocker-switch mechanism (5). In such a mechanism, MFS proteins are believed to switch between two major conformations, inward and outward, which differ by an ∼40°rotation of one domain relative to the other. Both conformations have been captured in MFS crystal structures. However, many questions remain to be addressed, particularly those related to energy coupling and functional roles of conserved motifs.YajR, a 49-kDa transporter of the MFS family, has putatively been classified as a drug efflux protein solely on the basis of amino acid sequence analysis (14). In Escherichia coli, YajR consists of 454 amino acid residues. Besides containing 12 TM helices, YajR is predicted to possess an extra domain of about 65 residues at the C-terminal. Of the MFS proteins with reported 3D structures, the TM core of YajR shares highest sequence homology (21% identity) with EmrD (SI Appendix, Fig. S1A), which belongs to the 12-TM drug-resistance H + -driven antiporter (DHA12) subfamily (15). The YajR gene is found in a number of Gram-negative bacteria, and it shows high ...

Major facilitator superfamily (MFS) is a large class of secondary active transporters widely expressed across all life kingdoms. Although a common 12-transmembrane helix-bundle architecture is found in most MFS crystal structures available, a common mechanism of energy coupling remains to be elucidated. Here, we discuss several models for energy-coupling in the transport process of the transporters, largely based on currently available structures and the results of their biochemical analyses. Special attention is paid to the interaction between protonation and the negative-inside membrane potential. Also, functional roles of the conserved sequence motifs are discussed in the context of the 3D structures. We anticipate that in the near future, a unified picture of the functions of MFS transporters will emerge from the insights gained from studies of the common architectures and conserved motifs.Keywords: : MFS transporters; energy coupling mechanisms; membrane potential; motif A MFS TransportersMajor facilitator superfamily (MFS) is the largest class of secondary active transporters and is present in cells across all life kingdoms. 1 So far, over 15,000 genes have been identified as encoding MFS transporters. 2 For example, 25% of prokaryotic transporters belong to the MFS family, 3 whereas in the human genome, genes coding for at least 110 MFS proteins have been identified. 4 One of the defining features of transporters is that they commonly use the electrochemical potential of one substance, for example, from ATP hydrolysis (for primary active transport) or an ion gradient (for secondary active transport), to drive the transport of another substance (i.e., the substrate). Many MFS transporters utilize proton-motive force (PMF) to drive the transport process. 5,6 In such cases, an MFS transporter often consumes one proton to transport one molecule of an electroneutral substrate. Thus, MFS transporters appear to be more energy-efficient in terms of the stoichiometric ratio of substrate to protons, compared to the other major class of active transporters, the ATP-Binding Cassette (ABC) transporters. The ABC transporters consume two ATP molecules (equivalent to about 6 protons) per transport cycle. On the basis of transport direction of the substrate relative to that of the driving substance, MFS transporters can be classified into symporters, antiporters, and, in case of a driving substance being absent, uniporters. Considering the countless types of molecules that a cell encounters during its lifespan, the Published by Wiley-Blackwell. V C 2015 The Protein Society number of transporter genes is quite small. Therefore, many transporters have to accommodate numerous types of ligands, and such poly-specificity is likely to be a common property of MFS transporters. 7,8 This review discusses common energy-coupling mechanisms in MFS transporters, and uses PMFdriven electrogenic transporters as an example. Many parts of the discussion are, however, applicable to other types of secondary active transporters. Tw...