Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).     

Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP(3)R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GXGXXG (amino acids 1773-1780 and 2016-2021, respectively). In IP(3)R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920-1925), which is conserved in the three IP(3)R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP(3)-induced Ca(2+) release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.     

Receptor-induced Ca(2+) signaling in cultured myenteric neurons

We studied the effect of excitatory neurotransmitters (10(-5) M) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of cultured myenteric neurons. ACh evoked a response in 48.6% of the neurons. This response consisted of a fast and a slow component, respectively mediated by nicotinic and muscarinic receptors, as revealed by specific agonists and antagonists. Substance P evoked a [Ca(2+)](i) rise in 68.2% of the neurons, which was highly dependent on Ca(2+) release from intracellular stores, since after thapsigargin (5 microM) pretreatment only 8% responded. The responses to serotonin, present in 90.7%, were completely blocked by ondansetron (10(-5) M), a 5-HT(3) receptor antagonist. Specific agonists of other serotonin receptors were not able to induce a [Ca(2+)](i) rise. Removing extracellular Ca(2+) abolished all serotonin and fast ACh responses, whereas substance P and slow ACh responses were more persistent. We conclude that ACh-induced signaling involves both nicotinic and muscarinic receptors responsible for a fast and a more delayed component, respectively. Substance P-induced signaling requires functional intracellular Ca(2+) stores, and the 5-HT(3) receptor mediates the serotonin-induced Ca(2+) signaling in cultured myenteric neurons.     

Calcium in the Golgi apparatus

The secretory-pathway Ca2+-ATPases (SPCAs) represent a recently recognized family of phosphorylation-type ATPases that supply the lumen of the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this structure. Mutations of the human SPCA1 gene (ATP2C1) cause Hailey-Hailey disease, an autosomal dominant skin disorder in which keratinocytes in the suprabasal layer of the epidermis detach. We will first review the physiology of the SPCAs and then discuss how mutated SPCA1 proteins can lead to an epidermal disorder.     

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.     

Dissection of the functional differences between human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 isoenzymes by steady-state and transient kinetic analyses

Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 microm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1 approximately PCa to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.     

Endogenously bound calmodulin is essential for the function of the inositol 1,4,5-trisphosphate receptor

Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional 45Ca2+ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP3-induced Ca2+ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3-induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.     

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.     

Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains

Although the details of the structural involvement of histone H1 in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD–CTD to the nucleosome.     

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.