Signaling microdomains regulate inositol 1,4,5-trisphosphate-mediated intracellular calcium transients in cultured neurons

Author Posting. © Society for Neuroscience, 2005. This article is posted here by permission of Society for Neuroscience for personal use, not for redistribution. The definitive version was published in Journal of Neuroscience 25 (2005): 2853-2864, doi:10.1523/JNEUROSCI.4313-04.2005.Ca2+ signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular Ca2+ signals, we correlated the subcellular distribution of components of the InsP3 pathway with measurements of agonist-induced intracellular Ca2+ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKIγ) and chromogranin B (CGB), proteins acting synergistically to increase InsP3 receptor (InsP3R) activity and sensitivity. In contrast, Ca2+ pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum as well as buffers that antagonize the rise in intracellular Ca2+ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKIγ or disrupting the CGB-InsP3R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca2+ signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca2+ signaling in different types of neurons.This work was supported by grants from the National Institutes of Health (GM63496, DK61747 to B.E.E., and MH67830 to M.F.Y.), Whitehall Foundation (M.F.Y.), German National Merit Foundation (S.N.J. and C.-U.C.), and Vetenskapsrådet, the Swedish Research Council (P.U.)

Neuronal calcium sensor-1 enhancement of InsP3 receptor activity is inhibited by therapeutic levels of lithium

Author Posting. © American Society for Clinical Investigation, 2006. This article is posted here by permission of American Society for Clinical Investigation for personal use, not for redistribution. The definitive version was published in Journal of Clinical Investigation 116 (2006): 1668-1674, doi:10.1172/JCI22466.Regulation and dysregulation of intracellular calcium (Ca2+) signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) has been linked to many cellular processes and pathological conditions. In the present study, addition of neuronal calcium sensor-1 (NCS-1), a high-affinity, low-capacity, calcium-binding protein, to purified InsP3R type 1 (InsP3R1) increased the channel activity in both a calcium-dependent and -independent manner. In intact cells, enhanced expression of NCS-1 resulted in increased intracellular calcium release upon stimulation of the phosphoinositide signaling pathway. To determine whether InsP3R1/NCS-1 interaction could be functionally relevant in bipolar disorders, conditions in which NCS-1 is highly expressed, we tested the effect of lithium, a salt widely used for treatment of bipolar disorders. Lithium inhibited the enhancing effect of NCS-1 on InsP3R1 function, suggesting that InsP3R1/NCS-1 interaction is an essential component of the pathomechanism of bipolar disorder.This work was supported by a grant from the NIH (GM63496 to B.E. Ehrlich), German National Merit Foundation scholarships (C. Schlecker and W. Boehmerle), and a National Kidney Foundation Fellowship (A. Varshney)

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Analysis of the cytoplasmic interaction between polycystin-1 and polycystin-2

Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, polycystin-1 (PC1) and polycystin-2 (PC2), form a signaling complex using direct intermolecular interactions. Two distinct domains in the C-terminal tail of PC2 have recently been identified, an EF-hand and a coiled-coil domain. Here, we show that the PC2 coiled-coil domain interacts with the C-terminal tail of PC1, but that the PC2 EF-hand domain does not. We measured the K0.5 of the interaction between the C-terminal tails of PC1 and PC2 and showed that the direct interaction of these proteins is abrogated by a PC1 point mutation that was identified in ADPKD patients. Finally, we showed that overexpression of the PC1 C-terminal tail in MDCK cells alters the Ca2+ response, but that overexpression of the PC1 C-terminal tail containing the disease mutation does not. These results allow a more detailed understanding of the mechanism of pathogenic mutations in the cytoplasmic regions of PC1 and PC2

InsP(3)-mediated intracellular calcium signalling is altered by expression of synaptojanin-1

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] plays an important physiological role as a precursor for the InsP(3)-mediated intracellular calcium (Ca(2+)) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (synaptojanin-1), a phosphoinositide phosphatase, regulates the turnover of a PtdIns(4,5)P(2) pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P(2) pools involved in Ca(2+) signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-phosphatase) domain. SJ-1 significantly attenuated the generation of Ca(2+) oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P(2) phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P phosphatase Sac1 did not affect Ca(2+) oscillations, although it increased the Ca(2+) efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca(2+) signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P(2) pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca(2+) signals