Modeling of Open, Closed, and Open-Inactivated States of the hERG1 Channel: Structural Mechanisms of the State-Dependent Drug Binding

The human ether-a-go-go related gene 1 (hERG1) K ion channel is a key element for the rapid component of the delayed rectified potassium current in cardiac myocytes. Since there are no crystal structures for hERG channels, creation and validation of its reliable atomistic models have been key targets in molecular cardiology for the past decade. In this study, we developed and vigorously validated models for open, closed, and open-inactivated states of hERG1 using a multistep protocol. The conserved elements were derived using multiple-template homology modeling utilizing available structures for Kv1.2, Kv1.2/2.1 chimera, and KcsA channels. Then missing elements were modeled with the ROSETTA <i>De Novo</i> protein-designing suite and further refined with all-atom molecular dynamics simulations. The final ensemble of models was evaluated for consistency to the reported experimental data from biochemical, biophysical, and electrophysiological studies. The closed state models were cross-validated against available experimental data on toxin footprinting with protein–protein docking using hERG state-selective toxin BeKm-1. Poisson–Boltzmann calculations were performed to determine gating charge and compare it to electrophysiological measurements. The validated structures offered us a unique chance to assess molecular mechanisms of state-dependent drug binding in three different states of the channel

Characterization of radioisotope substrate uptake in HeLa cells lentivirally transduced with HA-human NIS, HA-minke whale NIS, or HA-zebrafish NIS.

Raw uptake value of (A-B) 125I-, (C) 99mTcO4-, or (D) B18F4- in the absence or presence of 50 μM perchlorate (ClO4) in transfected or transduced cells. (E-H) Uptake of each radioisotope normalized to hNIS uptake via cell surface protein expression. (I-L) The percentage of maximum 125I- uptake activity (uptake at 0 μM ClO4) maintained in the presence of 50 μM perchlorate (ClO4). Values are averages of triplicate assays with standard deviation. n.s. indicates not significant; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001; Untrans. = untransfected or untransduced.</p

Motion Planning of Multi-Agent Systems under Temporal Logic Specifications

In this thesis, a top-down hierarchical framework is proposed to deal with multiagentsymbolic motion planning and control. Agents are assigned a distributable globalmission, and are partitioned into leaders and followers. The global mission is distributedamong the followers and each one synthesizes a discrete motion plan. By exploitingthe concept of network controllability, an open-loop optimal control law is synthesizedcentrally and executed in a distributed manner. This control law guarantees the concurrentexecution and fulfillment of the followers’ discrete motion plans. Simulations areperformed to verify the proposed control law both for single- and multi-leader, leaderfollowernetworks

Amino acid sequence alignment between the NIS proteins of <i>H</i>. <i>sapiens</i> (human), <i>B</i>. <i>acutorostrata scammoni</i> (minke whale), and <i>D</i>. <i>rerio</i> (zebrafish).

Cyan highlighting indicates absolute conservation to human NIS. Yellow indicates similar residue to human NIS. Underline indicates putative transmembrane domain in human NIS, only TM1-12 are indicated. Closed circles indicate site of a mutation known to cause a transport defect in humans [22]. Open circles indicate site of a mutation known to cause membrane trafficking defect in humans [22]. Black triangles indicate a charged residue where mutation to alanine significantly reduces iodide uptake in human NIS [71]. Red triangles indicate a charged residue where mutation to alanine significantly reduces iodide uptake in human NIS and this residue is not charged in zebrafish NIS [71]. Open diamonds indicate additional positively charged residues in zebrafish NIS. Blue diamonds indicate additional negatively charged residues in minke whale NIS. Bold red lettering indicates residue reported to be involved in stoichiometry control and translocation dynamics [52–53]. Numbering follows human NIS.</p

Molecular models of human-, minke whale-, and zebrafish NIS.

(A) Overlap of hNIS, wNIS, and zNIS. The thickness of the alpha-helices and loops represent per-residue RMSD values calculated between the corresponding residues of hNIS, wNIS, and zNIS with hNIS as a reference. Thick alpha helices and loops signify low RMSD values (regions with high structural similarity). Color coding: red–fully conserved residues between hNIS, wNIS, and zNIS; yellow–substitution with a chemically similar residue in wNIS and zNIS; blue–substitution with non-similar residue; grey spheres–Na+ ions, black sphere–I- ion. (B-C) Two different projections of protein areas within 10Å of Na+ and within 15Å of I-. Dashed lines indicate some or all of residues in the alpha helix are outside the 10/15Å cutoff. (D-E) Two different projections of residues from the ion coordination spheres (within 5Å) identified from MD simulations. The residue numbers of the non-conserved residues are also shown.</p

Perchlorate IC₅₀ determination for HA-human-, HA- minke whale-, and HA-zebrafish NIS.

Lentivirally transduced HeLa cells were incubated with 125I- with increasing concentration of perchlorate (ClO4-) for 50 minutes prior to two washes with cold buffer. IC50 values were determined via non-linear regression using the equation setting: {inhibitor} vs. normalized response—variable slope–inhibition in Prism 8. Human NIS ClO4- IC50 = 1.566 μM. Minke whale NIS ClO4- IC50 = 4.566 μM. Zebrafish NIS ClO4- IC50 = 0.081 μM. Values are average of triplicate assays with standard deviation.</p

Kinetic analysis of HA-human NIS, HA-minke whale NIS, and HA-zebrafish NIS.

Picomoles of iodide concentrated into HeLa cells lentivirally transduced with HA-NIS proteins after 4 minutes at 37°C. Uptake values were normalized to cell surface protein expression. Solid lines and symbols indicate experimentally derived uptake values. Dashed lines indicate predicted Michaelis-Menten equation curves determined with the least squares (ordinary) fit method in Prism. Km and Vmax values are represented in the table. Units for Km are μM; units for Vmax are pmol I-/4 minutes. Values are averages of triplicate assays with standard error. p-values calculated with experimental values at 50 μM KI; **** indicates p < 0.0001.</p

Characterization of HA-NIS protein expression and iodide transport.

Species are ordered in ascending evolutionary proximity to humans as determined by TimeTree (pig, mouse, rat, and dog diverged equidistantly) [73]. (A) Percentage of 1.25 μg/ml puromycin-selected cells which were positively-stained with α-HA-AlexaFluor647 antibody. (B) Median fluorescence intensity of 1.25 μg/ml puromycin-selected cells which were stained with α-HA-AlexaFluor647 antibody. Values are averages of duplicate assays. (C) Absolute uptake value of each NIS protein in the absence or presence of 100 μM perchlorate (ClO4), which has not been equalized to protein expression. Values are averages of triplicate assays with standard deviation. (D) Percentage of maximum 125I- uptake maintained in the presence of 100 μM perchlorate. Untrans. = untransduced, Ab = antibody.</p

Competitive substrate inhibition assays.

125I- uptake with competitive substrates in HeLa cells transduced with (A) HA-human NIS, (B) HA-whale NIS, or (C) HA-zebrafish NIS. Data shown as the percentage of maximum 125I- uptake activity (0 μM compound) maintained in the presence of increasing concentrations of substrate or inhibitor. Circular markers indicate naturally occurring anions. Triangular markers indicate anions which may occur naturally at low levels or are generated inside the organism. Square markers indicate anions not found naturally. Values are averages of duplicate assays with standard deviation.</p

Properties of NIS proteins investigated.

Properties of NIS proteins investigated.</p