The topological similarity of voltage-gated proton channels (HV1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether HV1s have a similar structure. We present the construction and validation of a homology model of the human HV1 (hHV1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hHV1 by exploiting the homology of hHV1 with VSDs of K+ and Na+ channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hHV1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp112. Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hHV1. Each Arg residue in the S4 helix of hHV1 was replaced by His to test accessibility using Zn2+ as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg211, the third Arg residue in the S4 helix in hHV1, remains accessible to the internal solution and is located near the charge transfer center, Phe150.

Water transport channels in membrane proteins of the aquaporin superfamily are impermeable to ions, including H+ and OH-. We examine the molecular basis for the blockage of proton translocation through the single-file water chain in the pore of a bacterial aquaporin, GlpF. We compute the reversible thermodynamic work for the two complementary steps of the Grotthuss "hop-and-turn" relay mechanism: consecutive transfers of H+ along the hydrogen-bonded chain (hop) and conformational reorganization of the chain (turn). In the absence of H+, the strong preference for the bipolar orientation of water around the two Asn-Pro-Ala (NPA) motifs lining the pore over both unidirectional polarization states of the chain precludes the reorganization of the hydrogen-bonded network. Inversely, translocation of an excess proton in either direction is opposed by a free-energy barrier centered at the NPA region. Both hop and turn steps of proton translocation are opposed by the electrostatic field of the channel.

Determination of a high-resolution 3D structure of voltage-gated sodium channel Na V Ab opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na + through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of Na V Ab in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na + through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na + between the extracellular medium and the central cavity of the channel. Analysis of Na + dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na + ions, with a computed rate of translocation of (6 ± 1) × 10 6 ions·s −1 that is consistent with expectations from electrophysiological studies. The binding of Na + is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na + ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquidlike energy landscape propitious to Na + diffusion. By stabilizing multiple ionic occupancy states while helping Na + ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na + permeation.T he rapid passage of cations in and out of excitable cells through selective pathways underlies the generation and regulation of electrical signals in all living organisms (1-4). The metazoan cell membrane is exposed to a high-Na + , low-K + concentration on the extracellular (EC) side, and to a low-Na + , high-K + concentration on the intracellular (IC) side. Selective voltage-gated Na + and K + channels control the response of the cell to changes in the membrane potential. In particular, voltagegated Na + channels (Na V ) are responsible for the initiation and propagation of action potentials in cardiac and skeletal myocytes, neurons, and endocrine cells (1-4). Mutations in Na V channel genes are responsible for a wide range of debilitating channelopathies, including congenital epilepsy, paramyotonia, erythromelalgia, familial hemiplegic migraine, paroxysmal extreme pain disorder, and periodic paralyses (5, 6), underlining the importance of deciphering the relationship between the structure and function of Na V channels. Here, we use molecular simulations to study the binding and permeation of Na + in bacterial sodium channel Na V Ab.Although several atomic structures of K + -selective channels have been solved over the past decade (7-12), the atomic structure of an Na + -selective channel from the bacterium Arcobacter butzleri, Na V Ab, wa...

Magnesium ions (Mg 2+ ) are essential for life, but the mechanisms regulating their transport into and out of cells remain poorly understood. The CorA-Mrs2-Alr1 superfamily of Mg 2+ channels represents the most prevalent group of proteins enabling Mg 2+ ions to cross membranes. Thermotoga maritima CorA (TmCorA) is the only member of this protein family whose complete 3D fold is known. Here, we report the crystal structure of a mutant in the presence and absence of divalent ions and compare it with previous divalent ion-bound TmCorA structures. With Mg 2+ present, this structure shows binding of a hydrated Mg 2+ ion to the periplasmic Gly-Met-Asn (GMN) motif, revealing clues of ion selectivity in this unique channel family. In the absence of Mg 2+ , TmCorA displays an unexpected asymmetric conformation caused by radial and lateral tilts of protomers that leads to bending of the central, pore-lining helix. Molecular dynamics simulations support these movements, including a bell-like deflection. Mass spectrometric analysis confirms that major proteolytic cleavage occurs within a region that is selectively exposed by such a bell-like bending motion. Our results point to a sequential allosteric model of regulation, where intracellular Mg 2+ binding locks TmCorA in a symmetric, transport-incompetent conformation and loss of intracellular Mg 2+ causes an asymmetric, potentially influx-competent conformation of the channel.crystallography | gating | limited proteolysis | pentamer C ompared with other common biological ions (Na + , K + , Ca 2+ , Cl − ), very little is known on a molecular level about the cellular homeostasis of Mg 2+ . As the most abundant intracellular divalent cation, it stabilizes phosphate compounds (DNA, RNA, ATP) and their synthesis, is essential for the function of over 300 enzymes, and is central to photosynthesis in plants (1). In addition to antagonizing Ca 2+ signaling (2), Mg 2+ has recently been implicated as a key second messenger in T-cell activation through the MagT1 Mg 2+ channel (3). The CorA protein is the primary transport system for Mg 2+ in Bacteria and Archaea and is required for bacterial pathogenesis (4, 5). It can functionally substitute for its eukaryotic homologs Alr1 and Mrs2 (6, 7), suggesting that CorA represents an important model system for these eukaryotic Mg 2+ channels. Alr1 is the major Mg 2+ uptake system in the plasma membrane of yeast (8), and Mrs2 is present in the inner mitochondrial membranes of yeast (6), plants (9), and mammals (10). Despite Mrs2 being essential for normal mitochondrial function (11), its expression is a hallmark of embryonic stem cells (12), and Mrs2 overexpression has been linked to a multidrug resistance phenotype in cancer (13,14). Patch-clamp analysis established Mrs2 as a high-conductance (155 pS) Mg 2+ -selective channel (15), and a similar conductivity has been indicated for CorA (16).Three crystal structures of wild-type Thermotoga maritima CorA (TmCorA-WT), obtained in the presence of divalent cations (17-19), revealed a symmetric ho...

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