scholarly journals Receptor-mediated activation of recombinant Trpl expressed in Sf9 insect cells

1995 ◽  
Vol 305 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Y Hu ◽  
W P Schilling
Keyword(s):  
Photoreceptor Cell ◽  
Receptor Potential ◽  
Photoreceptor Cells ◽  
Sharp Contrast ◽  
Membrane Receptors ◽  
Cation Channels ◽  
Sf9 Cells ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Stimulation Of

The Drosophila proteins, Trp and Trpl, are suggested to be cation channels responsible for depolarization of the receptor potential associated with stimulation of insect photoreceptor cells by light. Consistent with this hypothesis, we recently showed that recombinant Trpl forms Ca(2+)- and Ba(2+)-permeable non-selective cation channels when expressed in Sf9 cells using the baculovirus expression vector. As Trpl may be activated in the photoreceptor cell after stimulation of phospholipase C, we hypothesized that a similar regulation of recombinant Trpl may be observed in the Sf9 cell after activation of heterologous membrane receptors linked to Ca(2+)-signal-transduction pathways. To test this hypothesis, Ca2+ signalling was examined in Fura-2-loaded Sf9 cells infected with baculovirus containing cDNA for the M5 muscarinic receptor alone (M5 cells) or in cells co-infected with both M5 and Trpl-containing baculoviruses (M5-Trpl cells). Addition of carbachol (100 microM) to M5 cells produced an increase in cytosolic free Ca2+ concentration ([Ca2+]i) (mean +/- S.D.; n = 17) from 101 +/- 20 to 762 +/- 178 nM which declined to a sustained elevated level of 384 +/- 102 nM after 3 min. The sustained component was eliminated by removal of extracellular Ca2+ or by addition of La3+ or Gd3+ (10 microM). In M5-Trpl cells, basal [Ca2+]i increased as a function of time after infection. To evaluate the contribution of Ca2+ influx to the overall profile observed, Ba2+, a Ca2+ surrogate that is not a substrate for the Ca2+ pump, was used. The increase in basal [Ca2+]i seen in M5-Trpl cells was associated with an increase in basal Ba2+ influx. Addition of carbachol to M5-Trpl cells at 30-36 h after infection produced a large increase in [Ca2+]i to a sustained value of 677 +/- 143 nM. This change in [Ca2+]i was (1) blocked by atropine, (2) attenuated in the absence of extracellular Ca2+, and (3) relatively insensitive to La3+, but blocked by Gd3+ in the 0.1-1 mM range. In the presence of 10 microM Gd3+ to block the endogenous-receptor-mediated Ca(2+)-influx in M5-Trpl cells. In sharp contrast increase in Ba2+ influx in M5-Trpl cells. In sharp contrast, neither Ca2+ nor Ba2+ influx through Trpl was affected by thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase pump.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Stimulation of Drosophila TrpL by capacitative Ca2+ entry

1999 ◽  
Vol 341 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Mark ESTACION ◽  
William G. SINKINS ◽  
William P. SCHILLING
Keyword(s):  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Photoreceptor Cells ◽  
Channel Activity ◽  
Terminal Domain ◽  
Binding Domains ◽  
Single Channel Activity ◽  
Transient Receptor ◽  
Stimulation Of

Trp-like protein (TrpL, where Trp is transient receptor-potential protein) of Drosophila, a non-selective cation channel activated in photoreceptor cells by a phospholipase C-dependent mechanism, is thought to be a prototypical receptor-activated channel. Our previous studies showed that TrpL channels are not activated by depletion of internal Ca2+ stores when expressed in Sf9 cells. Using fura-2 to measure cation influx via TrpL, and cell-attached patch recordings to monitor TrpL single-channel activity directly, we have found a thapsigargin-induced increase in TrpL activity in the presence of extracellular bivalent cations, with Ca2+ > Sr2+ Ba2+. The increase in TrpL channel activity was blocked by concentrations of La3+ that completely inhibited endogenous capacitative Ca2+ entry (CCE), but have no effect on TrpL, suggesting that TrpL exhibits trans-stimulation by cation entry via CCE. TrpL has two putative calmodulin (CaM)-binding domains, designated CBS-1 and CBS-2. To determine which site may be required for stimulation of TrpL by the cytosolic free Ca2+ concentration ([Ca2+]i), a chimaeric construct was created in which the C-terminal domain of TrpL containing CBS-2 was attached to human TrpC1, a short homologue of Trp that is not activated by depletion of internal Ca2+ stores or by a rise in [Ca2+]i. This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca2+ entry via CCE. Examination of CaM binding in gel-overlay experiments showed that TrpL and the TrpC1-TrpL chimaera bound CaM, but TrpC1 or a truncated version of TrpL lacking CBS-2 did not. These results suggest that only CBS-2 binds CaM in native TrpL and that the C-terminal domain containing this site is important for trans-stimulation of TrpL by CCE.

scholarly journals Induction of Robust and Specific Humoral and Cellular Immune Responses by Bovine Viral Diarrhea Virus Virus-Like Particles (BVDV-VLPs) Engineered with Baculovirus Expression Vector System

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 350
Author(s):  
Zhanhui Wang ◽  
Mengyao Liu ◽  
Haoran Zhao ◽  
Pengpeng Wang ◽  
Wenge Ma ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vector System ◽  
Baculovirus Expression Vector System ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Cellular Immune Responses ◽  
Baculovirus Expression ◽  
Baculovirus Expression Vector ◽  
Cellular Immune ◽  
Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV) is an important animal pathogen that affects cattle. Infections caused by the virus have resulted in substantial economic losses and outbreaks of BVDV are reported globally. Virus-like particles (VLPs) are promising vaccine technology largely due to their safety and strong ability to elicit robust immune responses. In this study, we developed a strategy to generate BVDV-VLPs using a baculovirus expression vector system (BEVS). We were able to assemble BVDV-VLPs composed of dimerized viral proteins E2 and Erns, and the VLPs were spherical particles with the diameters of about 50 nm. Mice immunized with 15 μg of VLPs adjuvanted with ISA201 elicited higher levels of E2-specific IgG, IgG1, and IgG2a antibodies as well as higher BVDV-neutralizing activity in comparison with controls. Re-stimulation of the splenocytes collected from mice immunized with VLPs led to significantly increased levels of CD3+CD4+T cells and CD3+CD8+T cells. In addition, the splenocytes showed dramatically enhanced proliferation and the secretion of Th1-associated IFN-γ and Th2-associated IL-4 compared to that of the unstimulated control group. Taken together, our data indicate that BVDV-VLPs efficiently induced BVDV-specific humoral and cellular immune responses in mice, showing a promising potential of developing BVDV-VLP-based vaccines for the prevention of BVDV infections.

scholarly journals The Ventral Photoreceptor Cells of Limulus

1969 ◽  
Vol 54 (3) ◽  
pp. 310-330 ◽  
Author(s):  
Ronald Millecchia ◽  
Alexander Mauro
Keyword(s):  
Receptor Potential ◽  
Photoreceptor Cells ◽  
Regenerative Process ◽  
Resting Potential ◽  
Transient Phase ◽  
External Concentration ◽  
Slow Potential ◽  
Retinular Cells ◽  
Convenient Model ◽  
State Component

The ventral photoreceptors of Limulus polyphemus are unipolar cells with large, ellipsoidal somas located long both "lateral olfactory nerves." As a consequence of their size and location, the cells are easily impaled with microelectrodes. The cells have an average resting potential of -48 mv. The resting potential is a function of the external concentration of K. When the cell is illuminated, it gives rise to the typical "receptor potential" seen in most invertebrate photoreceptors which consists of a transient phase followed by a maintained phase of depolarization. The amplitude of the transient phase depends on both the state of adaptation of the cell and the intensity of the illumination, while the amplitude of the maintained phase depends only on the intensity of the illumination. The over-all size of the receptor potential depends on the external concentration of Na, e.g. in sodium-free seawater the receptor potential is markedly reduced, but not abolished. On the other hand lowering the Ca concentration produces a marked enhancement of both components of the response, but predominantly of the steady-state component. Slow potential fluctuations are seen in the dark-adapted cell when it is illuminated with a low intensity light. A spike-like regenerative process can be evoked by either the receptor potential or a current applied via a microelectrode. No evidence of impulse activity has been found in the axons of these cells. The ventral photoreceptor cell has many properties in common with a variety of retinular cells and therefore should serve as a convenient model of the primary receptor cell in many invertebrate eyes.

scholarly journals TRP and the PDZ Protein, Inad, Form the Core Complex Required for Retention of the Signalplex in Drosophila Photoreceptor Cells

2000 ◽  
Vol 150 (6) ◽  
pp. 1411-1422 ◽  
Author(s):  
Hong-Sheng Li ◽  
Craig Montell
Keyword(s):  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Light Response ◽  
Receptor Potential ◽  
Photoreceptor Cells ◽  
Macromolecular Complex ◽  
Pdz Domains ◽  
The Core ◽  
Transient Receptor ◽  
Drosophila Photoreceptor

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is inactivation no afterpotential D (INAD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind INAD, including the transient receptor potential (TRP) channel, which depends on INAD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of INAD are not known. In this work, we showed that INAD was required for retention rather than targeting of TRP to the rhabdomeres. In addition, we demonstrated that TRP bound to INAD through the COOH terminus, and this interaction was required for localization of INAD. Other proteins that depend on INAD for localization, phospholipase C and protein kinase C, also mislocalized. However, elimination of any other member of the signalplex had no impact on the spatial distribution of INAD. A direct interaction between TRP and INAD did not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the TRP/ INAD complex is to form the core unit required for localization of the signalplex to the rhabdomeres.

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