External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

Association mapping reveals gene action and interactions in the determination of flowering time in barley

The interaction between members of a gene network has an important impact on the variation of quantitative traits, and can influence the outcome of phenotype/genotype association studies. Three genes (Ppd-H1, HvCO1, HvFT1) known to play an essential role in the regulation of flowering time under long days in barley were subjected to an analysis of nucleotide diversity in a collection of 220 spring barley accessions. The coding region of Ppd-H1 was highly diverse, while both HvCO1 and HvFT1 showed a rather limited level of diversity. Within all three genes, the extent of linkage disequilibrium was variable, but on average only moderate. Ppd-H1 is strongly associated with flowering time across four environments, showing a difference of five to ten days between the most extreme haplotypes. The association between flowering time and the variation at HvFT1 and HvCO1 was strongly dependent on the haplotype present at Ppd-H1. The interaction between HvCO1 and Ppd-H1 was statistically significant, but this association disappeared when the analysis was corrected for the geographical origin of the accessions. No association existed between flowering time and allelic variation at HvFT1. In contrast to Ppd-H1, functional variation at both HvCO1 and HvFT1 is limited in cultivated barley.     

Welcome to this second edition of the JoCB Bulletin containing items of information for the Chemical Biology Community

JOCB Bulletin

Welcome to this second edition of the JoCB Bulletin containing items of information for the Chemical Biology Community     

Quantitative Imaging of Human Red Blood Cells Infected with Plasmodium falciparum

During its 48 h asexual reproduction cycle, the malaria parasite Plasmodium falciparum ingests and digests hemoglobin in excess of its metabolic requirements and causes major changes in the homeostasis of the host red blood cell (RBC). A numerical model suggested that this puzzling excess consumption of hemoglobin is necessary for the parasite to reduce the colloidosmotic pressure within the host RBC, thus preventing lysis before completion of its reproduction cycle. However, the validity of the colloidosmotic hypothesis appeared to be compromised by initial conflicts between model volume predictions and experimental observations. Here, we investigated volume and membrane area changes in infected RBCs (IRBCs) using fluorescence confocal microscopy on calcein-loaded RBCs. Substantial effort was devoted to developing and testing a new threshold-independent algorithm for the precise estimation of cell volumes and surface areas to overcome the shortfalls of traditional methods. We confirm that the volume of IRBCs remains almost constant during parasite maturation, suggesting that the reported increase in IRBCs' osmotic fragility results from a reduction in surface area and increased lytic propensity on volume expansion. These results support the general validity of the colloidosmotic hypothesis, settle the IRBC volume debate, and help to constrain the range of parameter values in the numerical model.     

Physical aspects of COPI vesicle formation

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Unpaired 5′ ppp-Nucleotides,as Found in Arenavirus Double-stranded RNA Panhandles,Are Not Recognized by RIG-I

Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position +2 of the template rather than at the precise 3′ end (position +1). After formation of a dinucleotide, 5′ pppGpC_OH is then realigned on the template before this primer is extended. The net result of this “prime and realign” mechanism of genome initiation is that 5′ pppG is found as an unpaired 5′ nucleotide when the complementary genome ends anneal to form a double-stranded (dsRNA) panhandle. Using 5′ pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5′ nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5′ ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.