Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage dependent manner, but unlike many other TRP channels is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3Å resolution as determined by single particle electron cryo-microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium binding site within the intracellular side of the S1–S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.

Zinc is an essential micronutrient for all living organisms, required for signaling and proper function of a range of proteins involved in e.g. DNA-binding and enzymatic catalysis 1 . In prokaryotes and photosynthetic eukaryotes Zn 2+ -transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn 2+ and related elements 2,3 . Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.P i ) of ZntA from Shigella sonnei, determined at 3.2 and 2.7 Å resolution, respectively. The structures reveal a similar fold as the Cu + -ATPases with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn 2+ ions. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including Cys392, Cys394 and Asp714. The pathway closes in the E2.P i state where Asp714 interacts with the conserved Lys693, which possibly stimulates Zn 2+ release as a built-in counter-ion, as also proposed for H + -ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter-

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.