Structural Basis for Silicic Acid Uptake by Higher Plants

Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.     

The influence of different lipid environments on the structure and function of the hepatitis C virus p7 ion channel protein

Abstract

The hepatitis C virus (HCV) encodes the p7 protein that oligomerizes to form an ion channel. The 63 amino acid long p7 monomer is an integral membrane protein predominantly found in the endoplasmic reticulum (ER). Although it is currently unknown whether p7 is incorporated into secreted virions, its presence is crucial for the release of infectious virus. The molecular and biophysical mechanism employed by the p7 ion channel is largely unknown, but in vivo it is likely to be embedded in membranes undergoing changes in lipid composition. In this study we analyze the influence of the lipid environment on p7 ion channel structure and function using electrophysiology and synchrotron radiation circular dichroism (SRCD) spectroscopy. We incorporated chemically synthesized p7 polypeptides into artificial planar membranes of various lipid compositions. A lipid bilayer composition comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (4:1 PC:PE) led to burst-like patterns in the channel recordings with channel openings lasting up to 0.5 s. The reverse ratio of PC:PE (1:4) gave rise to individual channels continuously opening for up to 8 s. SRCD spectroscopy of p7 embedded into liposomes of corresponding lipid compositions suggests there is a structural effect of the lipid composition on the p7 protein.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Fairy wrasses perceive and respond to their deep red fluorescent coloration

Fluorescence enables the display of wavelengths that are absent in the natural environment, offering the potential to generate conspicuous colour contrasts. The marine fairy wrasse Cirrhilabrus solorensis displays prominent fluorescence in the deep red range (650–700 nm). This is remarkable because marine fishes are generally assumed to have poor sensitivity in this part of the visual spectrum. Here, we investigated whether C. solorensis males can perceive the fluorescence featured in this species by testing whether the presence or absence of red fluorescence affects male–male interactions under exclusive blue illumination. Given that males respond aggressively towards mirror-image stimuli, we quantified agonistic behaviour against mirrors covered with filters that did or did not absorb long (i.e. red) wavelengths. Males showed significantly fewer agonistic responses when their fluorescent signal was masked, independent of brightness differences. Our results unequivocally show that C. solorensis can see its deep red fluorescent coloration and that this pattern affects male–male interactions. This is the first study to demonstrate that deep red fluorescent body coloration can be perceived and has behavioural significance in a reef fish.     

Novel strategies for assessing and managing the risks posed by invasive alien species to global crop production and biodiversity

International actions to combat the threat posed by invasive alien species (IAS) to crops and biodiversity have intensified in recent years. The formulation of 15 guiding principles on IAS by the Convention on Biological Diversity (CBD) stimulated the International Plant Protection Convention (IPPC) to review its role in protecting biodiversity. IPPC standards now demonstrate clearly that the risks posed by any organism that is directly or indirectly injurious to cultivated or uncultivated plants can be assessed and managed under the IPPC. Since the IPPC, unlike the CBD, constitutes an international legal instrument recognised by the World Trade Organization, greater protection from the introduction of IAS is now available. However, phytosanitary measures can only be enacted if they can be justified by risk analysis and we outline some novel strategies to improve the assessment and management of the risks posed by IAS, highlighting some of the key challenges which remain.     

Single-channel activity in sea urchin sperm revealed by the patch-clamp technique

Ionic fluxes are deeply involved in the response of spermatozoa to the egg. Using the patch-clamp technique, we show for the first time single ion channel activity in sea urchin spermatozoa and spermatozoa heads. Due to their small size gigaseals were obtained in suspended cells by applying suction through the pipette. The rate of gigaseal formation was very low and improved to 6% (n = 1145) when flagella were detached from sperm. Current-voltage curves created from single-channel events showed conductances of approx. 65 and 170 pS, suggesting the presence of two types of channels. At least one appears to be a K⁺ channel.