Project description:Human two-pore channels (TPCs) are endolysosomal cation channels and play an important role in NAADP-evoked Ca2+ release and endomembrane dynamics. We found that YM201636, a PIKfyve inhibitor, potently inhibits PI(3,5)P2-activated human TPC2 with an IC50 of 0.16 μM. YM201636 also effectively inhibits NAADP-activated TPC2 and a constitutively-open TPC2 L690A/L694A mutant channel; whereas it exerts little effect when applied in the channel's closed state. PI-103, a YM201636 analog and an inhibitor of PI3K and mTOR, also inhibits human TPC2 with an IC50 of 0.64 μM. With mutational, virtual docking, and molecular dynamic simulation analyses, we found that YM201636 and PI-103 directly block the TPC2's open-state channel pore at the bundle-cross pore-gate region where a nearby H699 residue is a key determinant for channel's sensitivity to the inhibitors. H699 likely interacts with the blockers around the pore entrance and facilitates their access to the pore. Substitution of a Phe for H699 largely accounts for the TPC1 channel's insensitivity to YM201636. These findings identify two potent TPC2 channel blockers, reveal a channel pore entrance blockade mechanism, and provide an ion channel target in interpreting the pharmacological effects of two commonly used phosphoinositide kinase inhibitors.
Project description:In all resolved structures of complex I, there exists a tunnel-like Q-chamber for ubiquinone binding and reduction. The entrance to the Q-chamber in ND1 subunit forms a narrow bottleneck, which is rather tight and requires thermal conformational changes for ubiquinone to get in and out of the binding chamber. The substitution of alanine with threonine at the bottleneck (AlaThr MUT), associated with 3460/ND1 mtDNA mutation in human complex I, is implicated in Leber's Hereditary Optic Neuropathy (LHON). Here, we show the AlaThr MUT further narrows the Q-chamber entrance cross-section area by almost 30%, increasing the activation free energy barrier of quinone passage by approximately 5 kJ mol-1. This severely disrupts quinone binding and reduction as quinone passage through the bottleneck is slowed down almost tenfold. Our estimate of the increase in free energy barrier is entirely due to the bottleneck narrowing, leading to a reduction of the transition state entropy between WT and MUT, and thus more difficult quinone passage. Additionally, we investigate details of possible water exchange between the Q-chamber and membrane. We find water exchange is dynamic in WT but may be severely slowed in MUT. We propose that LHON symptoms caused by 3460/ND1 mtDNA mutation are due to slowed quinone binding. This leads to an increased production of reactive oxidative species due to upstream electron backup at the FMN site of complex I, thus resulting in a mt bioenergetic defect.
Project description:Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10(-13) cm(3)/s (mean ± SE), 3.0 ± 0.3 × 10(-13) cm(3)/s, 2.5 ± 0.2 × 10(-13) cm(3)/s, and 0.9 ± 0.1 × 10(-13) cm(3)/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10(-13) cm(3)/s and 2.5 ± 0.1 × 10(-13) cm(3)/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and α-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon thickness, could explain the observed differences in water permeability with changes in bilayer thickness.
Project description:The association between the myrmecophyte Triplaris and ants of the genus Pseudomyrmex is an often-reported example of mutualism in the Neotropics. The ants colonize the hollow stems of their hosts, and in exchange, the plants benefit from a reduced degree of herbivory. The previous studies have shown that workers can discriminate their host from other plants, including a closely related species. Little is known about how queens locate their host during the colonization process, but it has been suggested that host recognition is mediated by volatiles. Since queens of Pseudomyrmex mordax colonize their hosts during the seedling stage, we hypothesized that queens would discriminate leaves of seedlings from adult plants. To evaluate our hypothesis, we used a two-sided olfactometer, to test the preference of queens towards different leaf and plant ages of Triplaris americana. Virgin queens of Pseudomyrmex mordax preferred seedlings over adult plants, as well as plant leaves over empty controls, showing no discrimination for leaf age. Our results suggest that the volatiles virgin queens recognize are either produced or are more abundant at the early growing stage of the host when colonization is crucial for the host's survival.
Project description:Membrane rejection models generally neglect the effect of the pore entrance on intrapore particle transport. However, entrance effects are expected to be particularly important with ultrathin membranes, where membrane thickness is typically comparable to pore size. In this work, a 2D model was developed to simulate particle motion for spherical particles moving at small Re and infinite Pe from the reservoir outside the pore into a slit pore. Using a finite element method, particles were tracked as they accelerated across the pore entrance until they reached a steady velocity in the pore. The axial position in the pore where particle motion becomes steady is defined as the particle entrance length (PEL). PELs were found to be comparable to the fluid entrance length, larger than the pore size and larger than the thickness typical of many ultrathin membranes. Results also show that, in the absence of particle diffusion, hydrodynamic particle-membrane interactions at the pore mouth result in particle "funneling" in the pore, yielding cross-pore particle concentration profiles focused at the pore centerline. The implications of these phenomena on rejection from ultrathin membranes are examined.
Project description:Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels have the largest conductance (250-300 pS) of all K(+)-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA(-) increased currents and MTSET(+) decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K(+)](i). Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.
Project description:Ryanodine (Ryd) irreversibly targets ryanodine receptors (RyRs), a family of intracellular calcium release channels essential for many cellular processes ranging from muscle contraction to learning and memory. Little is known of the atomistic details about how Ryd binds to RyRs. In this study, we used all-atom molecular dynamics simulations with both enhanced and bidirectional sampling to gain direct insights into how Ryd interacts with major residues in RyRs that were experimentally determined to be critical for its binding. We found that the pyrrolic ring of Ryd displays preference for the R4892AGGG-F4921 residues in the cavity of RyR1, which explain the effects of the corresponding mutations in RyR2 in experiments. Particularly, the mutant Q4933A (or Q4863A in RyR2) critical for both the gating and Ryd binding not only has significantly less interaction with Ryd than the wild-type, but also yields more space for Ryd and water molecules in the cavity. These results describe clear binding modes of Ryd in the RyR cavity and offer structural mechanisms explaining functional data collected on RyR blockade.
Project description:Mutualism benefits partner species, and theory predicts these partnerships can affect the abundance, diversity, and composition of partner and non-partner species. We used 16 years of monitoring data to determine the ant partner species of tree cholla cacti (Cylindropuntia imbricata), which reward ants with extrafloral nectar in exchange for anti-herbivore defense. These long-term data revealed one dominant ant partner (Liometopum apiculatum) and two less common partners (Crematogaster opuntiae and Forelius pruinosus). We then used short-term characterization of the terrestrial ant community by pitfall trapping to sample partner and non-partner ant species across ten plots of varying cactus density. We found that the dominant ant partner tended a higher proportion cacti in plots of higher cactus density, and was also found at higher occurrence within the pitfall traps in higher density plots, suggesting a strong positive feedback that promotes ant partner occurrence where plant partners are available. Despite the strong association and increased partner occurrence, ant community-wide effects from this mutualism appear limited. Of the common ant species, the occurrence of a single non-partner ant species was negatively associated with cactus density and with the increased presence of L. apiculatum. Additionally, the composition and diversity of the ant community in our plots were insensitive to cactus density variation, indicating that positive effects of the mutualism on the dominant ant partner did not have cascading impacts on the ant community. This study provides novel evidence that exclusive mutualisms, even those with a strong positive feedback, may be limited in the scope of their community-level effects.
Project description:Ant-plant interactions are diverse and abundant and include classic models in the study of mutualism and other biotic interactions. By estimating a time-scaled phylogeny of more than 1,700 ant species and a time-scaled phylogeny of more than 10,000 plant genera, we infer when and how interactions between ants and plants evolved and assess their macroevolutionary consequences. We estimate that ant-plant interactions originated in the Mesozoic, when predatory, ground-inhabiting ants first began foraging arboreally. This served as an evolutionary precursor to the use of plant-derived food sources, a dietary transition that likely preceded the evolution of extrafloral nectaries and elaiosomes. Transitions to a strict, plant-derived diet occurred in the Cenozoic, and optimal models of shifts between strict predation and herbivory include omnivory as an intermediate step. Arboreal nesting largely evolved from arboreally foraging lineages relying on a partially or entirely plant-based diet, and was initiated in the Mesozoic, preceding the evolution of domatia. Previous work has suggested enhanced diversification in plants with specialized ant-associated traits, but it appears that for ants, living and feeding on plants does not affect ant diversification. Together, the evidence suggests that ants and plants increasingly relied on one another and incrementally evolved more intricate associations with different macroevolutionary consequences as angiosperms increased their ecological dominance.
Project description:Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices α4 and α7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix α4 is stabilized by the hydrogen bond between Glu67 (helix α4) and Gln130 (helix α7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix α4. This local conformational switch of helix α4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.