scholarly journals A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eder Gambeta ◽  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
Gerald W. Zamponi
Keyword(s):  
Trigeminal Neuralgia ◽  
Missense Mutation ◽  
Neurotransmitter Release ◽  
Trigeminal System ◽  
Wild Type ◽  
Calcium Dependent ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

scholarly journals Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in Yersinia enterocolitica

2004 ◽  
Vol 186 (16) ◽  
pp. 5366-5375 ◽  
Author(s):  
Peter Burghout ◽  
Frank Beckers ◽  
Emmie de Wit ◽  
Ria van Boxtel ◽  
Guy R. Cornelis ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Yersinia Enterocolitica ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Protein Secretion System ◽  
Membrane Lipoprotein ◽  
Type Iii Protein Secretion

ABSTRACT The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ∼30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

scholarly journals The structural biology of voltage-gated calcium channel function and regulation

2006 ◽  
Vol 34 (5) ◽  
pp. 887-893 ◽  
Author(s):  
F. Van Petegem ◽  
D.L. Minor
Keyword(s):  
Neurotransmitter Release ◽  
Action Potentials ◽  
Molecular Switches ◽  
Voltage Gated ◽  
Calcium Dependent ◽  
Voltage Gated Calcium Channels ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Soluble Calcium ◽  
Molecular Framework

Voltage-gated calcium channels (CaVs) are large (∼0.5 MDa), multisubunit, macromolecular machines that control calcium entry into cells in response to membrane potential changes. These molecular switches play pivotal roles in cardiac action potentials, neurotransmitter release, muscle contraction, calcium-dependent gene transcription and synaptic transmission. CaVs possess self-regulatory mechanisms that permit them to change their behaviour in response to activity, including voltage-dependent inactivation, calcium-dependent inactivation and calcium-dependent facilitation. These processes arise from the concerted action of different channel domains with CaV β-subunits and the soluble calcium sensor calmodulin. Until recently, nothing was known about the CaV structure at high resolution. Recent crystallographic work has revealed the first glimpses at the CaV molecular framework and set a new direction towards a detailed mechanistic understanding of CaV function.

The Role of the cAMP-Response Element in the Bcl-2 Promoter in the Regulation of Endogenous Bcl-2 Expression and Apoptosis in Murine B Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2987-2987
Author(s):  
Hong Xiang ◽  
Linda M. Boxer
Keyword(s):  
B Cells ◽  
Wild Type ◽  
Camp Response Element ◽  
Response Element ◽  
Calcium Dependent ◽  
Flow Cytometric ◽  
Immature B Cells

Abstract We have previously shown in B cell lines that the cAMP-response element (CRE) is a major positive regulatory site in the bcl-2 promoter. This element is not only essential for bcl-2 deregulation in t(14;18) cells, but it is also responsible for the positive regulation of bcl-2 expression during the activation of mature B cells and the rescue of immature B cells from calcium-dependent apoptosis in vitro. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutant CRE site was introduced into the bcl-2 promoter region. The mutant CRE reduced the expression of bcl-2 mRNA in several tissues, including thymus, kidney, lung, liver, brain and heart. The levels of bcl-2 mRNA and protein were also significantly lower in splenic B cells from the knock-in mice. Consistent with these results, the activation of B cells from the knock-in mice by anti-CD 40, lipopolysaccharide (LPS) or anti-IgM was reduced as compared to B cells from wild-type littermates. B cells with the mutant CRE were more susceptible to the induction of apoptosis with several different agents consistent with the decreased expression of bcl-2. Preliminary flow cytometric studies suggest that the number of B cells is decreased in the knock-in mice at 8 weeks of age. Quantitative chromatin immunoprecipitation assays revealed essentially no binding of CREB or ATF-2 and decreased binding of CBP and c-Rel to the mutant CRE site in the bcl-2 promoter. Our previous studies have shown that the CRE site in the bcl-2 promoter is linked to the mediation of signal transduction pathways in B cells, so we investigated the effect of forskolin, a cAMP-elevating agent. We found that treatment of the B cells from the knock-in mice with forskolin led to significantly more cell death than observed with wild-type B cells. Taken together, these findings indicate that the CRE site in the bcl-2 promoter has a functional role in the regulation of endogenous bcl-2 expression and plays an important role in the regulation of apoptosis in B cells.

scholarly journals Gelsolin Mediates Collagen Phagocytosis through a Rac-dependent Step

2004 ◽  
Vol 15 (2) ◽  
pp. 588-599 ◽  
Author(s):  
Pamela D. Arora ◽  
Michael Glogauer ◽  
Andras Kapus ◽  
David J. Kwiatkowski ◽  
Christopher A. McCulloch
Keyword(s):  
Actin Filaments ◽  
Calcium Ionophore ◽  
Initial Contact ◽  
Β1 Integrin ◽  
Wild Type ◽  
Collagen Binding ◽  
Calcium Dependent ◽  
Collagen Phagocytosis ◽  
Α2β1 Integrin

The role of gelsolin, a calcium-dependent actin-severing protein, in mediating collagen phagocytosis, is not defined. We examined α2β1 integrin-mediated phagocytosis in fibroblasts from wild-type (WT) and gelsolin knockout (Gsn-) mice. After initial contact with collagen beads, collagen binding and internalization were 60% lower in Gsn- than WT cells. This deficiency was restored by transfection with gelsolin or with β1 integrin-activating antibodies. WT cells showed robust rac activation and increased [Ca2+]i during early contact with collagen beads, but Gsn- cells showed very limited responses. Transfected gelsolin in Gsn- cells restored rac activation after collagen binding. Transfection of Gsn- cells with active rac increased collagen binding to WT levels. Chelation of intracellular calcium inhibited collagen binding and rac activation, whereas calcium ionophore induced rac activation in WT and Gsn- cells. We conclude that the ability of gelsolin to remodel actin filaments is important for collagen-induced calcium entry; calcium in turn is required for rac activation, which subsequently enhances collagen binding to unoccupied α2β1 integrins.

scholarly journals Single-Channel Mechanism of Modulation of Calcium-Dependent Inactivation of the Voltage-Gated Calcium Channel Cav1.3 by its C-Terminus

2013 ◽  
Vol 104 (2) ◽  
pp. 459a
Author(s):  
Elena Novikova ◽  
Elza Kuzmenkina ◽  
Wanchana Jangsangthong ◽  
Jan Matthes ◽  
Alexandra Koschak ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Single Channel ◽  
Voltage Gated ◽  
Calcium Dependent ◽  
C Terminus ◽  
Dependent Inactivation ◽  
Voltage Gated Calcium Channel

scholarly journals Calcium-dependent inactivation controls cardiac L-type Ca2+ currents under β-adrenergic stimulation

2019 ◽  
Vol 151 (6) ◽  
pp. 786-797 ◽  
Author(s):  
Danna Morales ◽  
Tamara Hermosilla ◽  
Diego Varela
Keyword(s):  
Calcium Current ◽  
Calcium Currents ◽  
Adrenergic Stimulation ◽  
Sodium Calcium Exchanger ◽  
Adrenergic Agonist ◽  
Rat Cardiomyocytes ◽  
Calcium Dependent ◽  
Sodium Calcium ◽  
Dependent Inactivation

The activity of L-type calcium channels is associated with the duration of the plateau phase of the cardiac action potential (AP) and it is controlled by voltage- and calcium-dependent inactivation (VDI and CDI, respectively). During β-adrenergic stimulation, an increase in the L-type current and parallel changes in VDI and CDI are observed during square pulses stimulation; however, how these modifications impact calcium currents during an AP remains controversial. Here, we examined the role of both inactivation processes on the L-type calcium current activity in newborn rat cardiomyocytes in control conditions and after stimulation with the β-adrenergic agonist isoproterenol. Our approach combines a self-AP clamp (sAP-Clamp) with the independent inhibition of VDI or CDI (by overexpressing CaVβ2a or calmodulin mutants, respectively) to directly record the L-type calcium current during the cardiac AP. We find that at room temperature (20–23°C) and in the absence of β-adrenergic stimulation, the L-type current recapitulates the AP kinetics. Furthermore, under our experimental setting, the activity of the sodium–calcium exchanger (NCX) does not affect the shape of the AP. We find that hindering either VDI or CDI prolongs the L-type current and the AP in parallel, suggesting that both inactivation processes modulate the L-type current during the AP. In the presence of isoproterenol, wild-type and VDI-inhibited cardiomyocytes display mismatched L-type calcium current with respect to their AP. In contrast, CDI-impaired cells maintain L-type current with kinetics similar to its AP, demonstrating that calcium-dependent inactivation governs L-type current kinetics during β-adrenergic stimulation.

p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

IGF Binding Protein 2 Supports the Cycling of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1604-1604
Author(s):  
HoangDinh Huynh ◽  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Ex Vivo ◽  
Bone Marrow Stroma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
Marrow Stroma ◽  
Igf Signaling

Abstract Abstract 1604 Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). The role of IGFBP2 in HSCs and cancer is very intriguing. IGFBP2 can bind to insulin-like growth factor (IGF) ligands and displays IGF-dependent growth inhibitory effects on many cell types. On the other hand, IGFBP2 is capable of stimulating growth of certain cancer cells, and is overexpressed in many cancer patients and its expression is correlated with cancer progression. Here we sought to study the role of IGFBP2 in regulation of the activity of normal HSCs. We showed that IGFBP2 was expressed in differentiated hematopoietic cells and bone marrow stroma but not in HSCs. Consistent with its gene expression pattern, IGFBP2-/- HSCs had similar repopulation activity as their wild-type counterparts. By contrast, when we transplanted HSCs into IGFBP2-/- or wild-type recipient mice, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-/- recipients, suggesting that the environmental IGFBP2 positively supports HSC activity. Further co-culture of HSCs with IGFBP2-/- or wild-type bone marrow stromal cells indicated that IGFBP2 produced by bone marrow stroma indeed supports HSC expansion. Consistently, HSCs in IGFBP2-/- mice showed decreased frequency and cell cycling, and had upregulated expression of cell cycle inhibitors of p21, p16, and p19. To determine whether IGFBP2's effect on HSCs depends on IGF signaling, we compared the repopulation of donor cells deficient for the IGF type I receptor in wild-type and IGFBP2-/- recipients. These HSCs that are defective in IGF signaling still have decreased repopulation in IGFBP2-/- recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF signaling. To identify the functional domain of IGFBP2 in regulation of HSC activity, we constructed IGFBP2 with mutated RGD domain or deleted c-terminus and used the mutant IGFBP2 proteins in ex vivo culture of HSCs. We found that the c-terminus of IGFBP2 is essential to support HSC activity. We are currently in the process of identifying the potential receptor of IGFBP2 on HSCs. In summary, we found that IGFBP2 supports the cycling of normal HSCs, and this effect is independent of IGF signaling. Our study is important in revealing the relationship among environmental cues and cell fates of stem cells and opens up a new avenue in investigation of the roles of IGFBP2 in stem cells and cancer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle

2004 ◽  
Vol 123 (4) ◽  
pp. 377-386 ◽  
Author(s):  
Guangju Ji ◽  
Morris E. Feldman ◽  
Kai Su Greene ◽  
Vincenzo Sorrentino ◽  
Hong-Bo Xin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Wild Type ◽  
Calcium Dependent ◽  
Outward Currents ◽  
Associated Proteins ◽  
Kinetics Of ◽  
Spontaneous Transient Outward Currents

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

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