Annexin 2 is a ubiquitous Ca 2 þ -binding protein that is essential for actin-dependent vesicle transport. Here, we show that in spontaneously motile cells annexin 2 is concentrated in dynamic actin-rich protrusions, and that depletion of annexin 2 using siRNA leads to the accumulation of stress fibres and loss of protrusive and retractile activity. Cells co-expressing annexin 2-CFP and actin-YFP exhibit Ca 2 þ -dependent fluorescense resonance energy transfer throughout the cytoplasm and in membrane ruffles and protrusions, suggesting that annexin 2 may directly interact with actin. This notion was supported by biochemical studies, in which we show that annexin 2 reduces the polymerisation rate of actin monomers in a dose-dependent manner. By measuring actin polymerisation rates in the presence of barbed-end and pointed-end cappers, we further demonstrate that annexin 2 specifically inhibits filament elongation at the barbed ends. These results show that annexin 2 has an essential role in maintaining the plasticity of the dynamic membraneassociated actin cytoskeleton, and that its activity in this context may be at least partly explained through direct interactions with polymerised and monomeric actin.

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.

No abstract

We have investigated the contribution of lipid rafts to activation of the NADPH oxidase enzyme system in neutrophils. Membrane-bound NADPH oxidase subunits are present in the lipid raft compartment of neutrophils. Cytosolic NADPH oxidase components are mainly absent from but are recruited to rafts upon FcQ Q receptor activation. In parallel, protein kinase C isotypes are recruited to the rafts. Kinetic analysis of NADPH oxidase activation revealed that rafts determine the onset but not the maximal rate of enzyme activity. Thus lipid rafts serve to physically juxtapose the NADPH oxidase e¡ector, protein kinase C and FcQ Q receptor, resulting in e⁄cient coupling. ß