Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct

Dynamics of Co-Transcriptional Pre-mRNA Folding Influences the Induction of Dystrophin Exon Skipping by Antisense Oligonucleotides

Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a “window of analysis” that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered “engaged” if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2) engaged nucleotides at 3′ or 5′ ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotides at 3′ or 5′ ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3′ end of a short target site attenuates AON efficiency more than at 5′ end

Spontaneously opening GABA receptors play a significant role in neuronal signal filtering and integration

Acknowledgements This work was supported by The Rosetrees Trust Research Grant A1066, RS MacDonald Seedcorn Award and Wellcome Trust—UoE ISSF Award to S.S. The authors thank Prof. David Wyllie (University of Edinburgh) and Prof. Dmitri Rusakov (UCL) for their valuable suggestions on paper preparation.Peer reviewedPublisher PD

The circadian clock goes genomic

Staiger D, Shin J, Johansson M, Davis SJ. The circadian clock goes genomic. Genome Biology. 2013;14(6): 208.Large-scale biology among plant species, as well as comparative genomics of circadian clock architecture and clock-regulated output processes, have greatly advanced our understanding of the endogenous timing system in plants

Parathyroid hormone-induced alterations of protein content and phosphorylation in enriched apical membranes of opossum kidney cells.

Parathyroid hormone (PTH) reduces Na/Pi co-transport activity in opossum kidney (OK) cells in a process mediated by protein kinases A and C. Further, inactivation of Na/Pi transport involves irreversible inhibition, possibly via internalization, of the transport system. This study analyzed alterations of concentration and phosphorylation of membrane proteins of an apically enriched preparation induced by short (10 min) and long (3 h) term incubation with 10(-10) M PTH of monolayer cultures of the OK-cell line. To this end, an apically enriched membrane fraction was isolated from cells grown on Petri dishes and analyzed by two-dimensional gel electrophoresis. Long term exposure of the cells to PTH induced changes in apical protein concentration. Four proteins were found to be decreased and one protein was found to be increased in its concentration. Addition of 10(-10) M PTH to the cells led to transient phosphorylation of five proteins. In contrast to transient phosphorylation, phosphorylation of one protein increased over the time period of 3 h. Combined analysis of silver staining and autoradiography led to the detection of an acidic 35-kDa protein in which specific phosphorylation increased over a time period of hours. The results document for the first time alterations in apical membrane protein content and phosphorylation state mediated by PTH when added to an intact cellular system. It is concluded that the identified proteins represent possible candidates for being involved directly or indirectly in PTH alterations of membrane transport

Somatostatin inhibition of hormone release: effects on cytosolic Ca++ and interference with distal secretory events

In normal pituitary cells somatostatin (SRIF) blocks the spontaneous oscillations in [Ca++]i by inhibiting the generation of action potentials. This is sufficient to explain the inhibitory effect on basal, but not entirely that on stimulated pituitary hormone secretion. In insulin secreting cells, which, in contrast to pituitary cells, only fire action potentials on stimulus-evoked depolarization, SRIF hyperpolarization and lowering of [Ca++]i is only transient. The marked inhibition of insulin secretion is suggested to be due to a coordinated action of SRIF on membrane potential and [Ca++]i as well as a direct interference with late secretory events