The serotonergic system plays an important role in many psychiatric disorders. Its role in depression is well established (1). The majority of antidepressants, including TCAs, 6 cause increased synaptic serotonin (5-HT) levels via blockade of 5-HT reuptake into the presynaptic neuron (2-4) by competitive inhibition of hSERT. TCAs have been in clinical use since the 1950s, with imipramine being the first and most prominent compound (5). In severely depressed hospitalized patients, TCAs appear to be more efficacious than selective serotonin reuptake inhibitors (6). TCAs remain in widespread clinical use, especially for treatment-resistant depression (7).hSERT belongs to the neurotransmitter sodium symporter family (2, 8). These transporters utilize the electrochemical gradient of sodium and chloride ions to accumulate 5-HT against its own gradient (9 -11). No experimentally solved structures of the monoamine transporters exist, including hSERT and the dopamine and norepinephrine transporters. However, the structure of LeuT, a bacterial homolog of the neurotransmitter sodium symporters, in a substrate-occluded conformation, was reported in 2005 (12). Two sodium ions (12) and a chloride ion bind near the central substrate site (13-14) structurally and functionally coupling sodium and chloride binding to substrate binding. Recently, different transport mechanisms have been suggested (15,16).Subsequently, a low affinity noncompetitive binding site for TCAs in the extracellular vestibule of the LeuT 11 Å above the central binding site was identified (17,18). The relevance of the LeuT vestibular site for TCA binding to the physiologically relevant target, hSERT, is a matter of debate. This study identifies the central binding site, not the putative vestibular site, as relevant for TCA binding to hSERT and furthermore describes and validates the orientation of TCAs within this site.In this paper, we present induced fit docking studies of imipramine and selected analogs in the previously described homology models of hSERT (19). We present binding affinity studies of 10 imipramine analogs (Fig. 1 Copenhagen Ø, Denmark. 4 To whom correspondence may be addressed. E-mail: birgit@chem.au.dk. 5 To whom correspondence may be addressed. E-mail: owiborg@post.tele.dk. 6 The abbreviations used are: TCA, tricyclic antidepressant; 5-HT, serotonin; hSERT, human SERT; WT, wild type; PaMLAC, paired mutation ligand analog complementation; MD, molecular dynamics; r.m.s., root mean square.

The sarco/endoplasmic reticulum Ca 2؉ -ATPase (SERCA) is a transmembrane ion transporter belonging to the P II -type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca 2؉ to the sarco/endoplasmic reticulum store, thereby also terminating Ca 2؉ -induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca 2؉ and H ؉ ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca 2؉ entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane ␣-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ionbinding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca 2؉ -free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P II -ATPases including not one, but two cytoplasmic pathways working in concert.P-type ATPases form a large family of transmembrane transporters that couple the energy from ATP hydrolysis to active transport of key cations across biological membranes. These so-called ion pumps are multidomain enzymes that contain polar and charged residues within their transmembrane (TM) 4 domain that mediate binding of the transported ions. For the most well studied members, the sarco/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) and the Na ϩ /K ϩ -ATPase (NKA), the binding sites have been thoroughly described by structural and mutational studies (1-5), whereas the inherently dynamic interactions along the routes of ion entry and exit remain less clear.P-type ATPases generally function according to an alternating access model (6 -8) (also described as an E1/E2 scheme (9 -11)) in which the ion-binding sites are accessible from either the cytoplasmic or extracytoplasmic side, interspersed by occluded states coupled with phosphorylation or dephosphorylation (12-14). Therefore, there must be at least one ion access pathway at each side of the membrane, but it has also been suggested that the Ca 2ϩ exit and proton entry pathways on the luminal side of SERCA are separate (15). This article provides a focused review of the ion pathways in SERCA, pointing in particular to the possibility of two cytoplasmic pathways, one for proton exit and another for Ca 2ϩ binding. The Luminal/Extracellular Pathway: The "Exit Path"The structure of SERCA in the E2P conformation (trapped as a phosphoenzyme intermediate mimicked by BeF 3 Ϫ ) revealed a luminal Ca 2ϩ exit pathway (Exit path) (Fig. 1a) encompassed by TM segments M1-M6 (16, 17). This structu...

P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca 2 + -ATPase (sERCA) in its phosphorylated and dephosphorylated Ca 2 + -free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that sERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of sERCA, a strategy that may be of general nature for many membrane proteins.