The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH2-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10−5 or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.

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Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.

Summary Potassium channels activated by intracellular Na+ ions (KNa) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode KNa channels. We have now found that Slick and Slack subunits co-assemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization and their response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of two alternative splice variants the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric KNa channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are co-expressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal KNa channels.

Biodegradable poly(lactide)/poly(butylene adipate-co-terephthalate) (PLA/PBAT) blends were prepared by reactive blending in the presence of chain-extenders. Two chain-extenders with multi-epoxy groups were studied. The effect of chain-extenders on the morphology, mechanical properties, thermal behavior, and hydrolytic degradation of the blends was investigated. The compatibility between the PLA and PBAT was significantly improved by in situ formation of PLA-co-PBAT copolymers in the presence of the chain-extenders, results in an enhanced ductility of the blends, e.g., the elongation at break was increased to 500% without any decrease in the tensile strength. The differential scanning calorimeter (DSC) results reveal that cold crystallization of PLA was enhanced due to heterogeneous nucleation effect of the in situ compatibilized PBAT domains. As known before, PLA is sensitive to hydrolysis and in the presence of PBAT and the chain-extenders, the hydrolytic degradation of the blend was evident. A three-stage hydrolysis mechanism for the system is proposed based on a study of weight loss and molecular weight reduction of the samples and the pH variation of the degradation medium.

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7-or 9-residue sequences from Shab , Shaw , or Shal . As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three-to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels. The high sensitivity of voltage-gated potassium and sodium channels arises from a large charge displacement, ‫ف‬ 13 e 0 in magnitude, as a channel moves from its resting state to the open state (Schoppa et al., 1992;Hirschberg et al., 1995;Aggarwal and Mackinnon, 1996;Seoh et al., 1996). It is widely thought that this charge displacement involves the S4 region of the protein sequence, with contributions from other regions (Sigworth, 1994;Seoh et al., 1996). The S4 region of the Shaker potassium channel has seven basic residues which could contribute to the mobile charge, and recent experiments have shown changes in the accessibility of S4 residues upon voltage-dependent channel activation (Larsson et al., 1996;Yang et al., 1996). According to some theories, the S4 segment is an alpha helix that undergoes a large, helical screw movement normal to the lipid bilayer in order to transfer the necessary gating charge (Catterall, 1986;Durell et al. 1992). If indeed this is how the S4 region moves, then the S3-S4 linker, to which it is tethered at its NH 2 -terminal end, must be flexible to support the large displacement.There has been little study of the S3-S4 linker of voltage-gated channels, apart from work on L-type calcium channels by Nakai et al. (1994). These authors showed that exchange of the S3-S4 region in Domain 1 changed the channel from the skeletal muscle (slowly activating) to the cardiac (rapidly activating) kinetic behavior. A comparison of potassium channel S3-S4 linkers is shown in Fig. 1. The apparent length of the linker is variable between subfamilies, ranging from 25 residues for Drosophila Shaker to only 7 residues in Shaw . The members of the Shaker subfamily show a moderate degree of sequence identity, but within the Shab and Shal subfamilies there is a remarkable degree of identi...

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Summary Ferulate 5‐hydroxylase (F5H) is a limiting enzyme involved in biosynthesizing sinapyl (S) monolignol in angiosperms. Genetic regulation of F5H can influence S monolignol synthesis and therefore improve saccharification efficiency and biofuel production. To date, little is known about whether F5H is post‐transcriptionally regulated by endogenous microRNAs (miRNAs) in woody plants. Here, we report that a microRNA, miR6443, specifically regulates S lignin biosynthesis during stem development in Populus tomentosa. In situ hybridization showed that miR6443 is preferentially expressed in vascular tissues. We further identified that F5H2 is the direct target of miR6443. Overexpression of miR6443 decreased the transcript level of F5H2 in transgenic plants, resulting in a significant reduction in S lignin content. Conversely, reduced miR6443 expression by short tandem target mimics (STTM) elevated F5H2 transcripts, therefore increasing S lignin composition. Introduction of a miR6443‐resistant form of F5H2 into miR6443‐overexpression plants restored lignin ectopic composition, supporting that miR6443 specifically regulated S lignin biosynthesis by repressing F5H2 in P. tomentosa. Furthermore, saccharification assays revealed decreased hexose yields by 7.5–24.5% in miR6443‐overexpression plants compared with the wild‐type control, and increased hexoses yields by 13.2–14.6% in STTM6443‐overexpression plants. Collectively, we demonstrate that miR6443 modulates S lignin biosynthesis by specially regulating F5H2 in P. tomentosa.