Regulation of Drosophila TRPL channels by immunophilin FKBP59.

Transient receptor potential and transient receptor potential-like (TRPL) are Ca(2+)-permeable cation channels found in Drosophila photoreceptor cells associated with large multimeric signaling complexes held together by the scaffolding protein, INAD. To identify novel proteins involved in channel regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein, FKBP52 (previously known as FKBP59). To determine the function of dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca(2+) influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunoprecipitation resulted in a temperature-dependent dissociation of dFKBP59 and TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lysates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in vivo. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lysates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutagenesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709) eliminated interaction of the TRPL with dFKBP59. These results provide strong support for the hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an important role in modulation of channel activity via interaction with conserved leucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel.

Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells.

BACKGROUND: Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells. RESULTS: Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation. CONCLUSIONS: These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.

Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

Maitotoxin activates a nonselective cation channel and a P2Z/P2X(7)-like cytolytic pore in human skin fibroblasts.

Maitotoxin (MTX), a potent cytolytic agent, activates Ca(2+) entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca(2+) concentration that depended on extracellular Ca(2+) and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-D-glucamine (167 Da). Thus MTX initially activates Ca(2+)-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X(7) receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore.

Maitotoxin and P2Z/P2X(7) purinergic receptor stimulation activate a common cytolytic pore.

The effects of maitotoxin (MTX) on plasmalemma permeability are similar to those caused by stimulation of P2Z/P2X(7) ionotropic receptors, suggesting that 1) MTX directly activates P2Z/P2X(7) receptors or 2) MTX and P2Z/P2X(7) receptor stimulation activate a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in 1) THP-1 monocytic cells before and after treatment with lipopolysaccharide and interferon-gamma, a maneuver known to upregulate P2Z/P2X(7) receptor, 2) wild-type HEK cells and HEK cells stably expressing the P2Z/P2X(7) receptor, and 3) BW5147.3 lymphoma cells, a cell line that expresses functional P2Z/P2X(7) channels that are poorly linked to pore formation. In control THP-1 monocytes, addition of MTX produced a biphasic increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)); the initial increase reflects MTX-induced Ca(2+) influx, whereas the second phase correlates in time with the appearance of large pores and the uptake of ethidium. MTX produced comparable increases in [Ca(2+)](i) and ethidium uptake in THP-1 monocytes overexpressing the P2Z/P2X(7) receptor. In both wild-type HEK and HEK cells stably expressing the P2Z/P2X(7) receptor, MTX-induced increases in [Ca(2+)](i) and ethidium uptake were virtually identical. The response of BW5147.3 cells to concentrations of MTX that produced large increases in [Ca(2+)](i) had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and Bz-ATP-induced pores activate with similar kinetics and exhibit similar size exclusion. Last, MTX-induced pore formation, but not channel activation, is greatly attenuated by reducing the temperature to 22 degrees C, a characteristic shared by the P2Z/P2X(7)-induced pore. Together, the results demonstrate that, although MTX activates channels that are distinct from those activated by P2Z/P2X(7) receptor stimulation, the cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.

Stimulation of Drosophila TrpL by capacitative Ca2+ entry.

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.

Functional expression of TrpC1: a human homologue of the Drosophila Trp channel.

TrpC1 appears to be a store-operated channel (SOC) when expressed in mammalian cells. In the present study, TrpC1 was expressed in Sf9 insect cells using the baculovirus expression system. Expression of TrpC1 caused an increase in basal cytosolic free Ca2+ concentration ([Ca2+]i) as a function of post-infection time. Basal Ba2+ influx, an index of plasmalemmal Ca2+ permeability, was also increased and was blocked by La3+. Although the thapsigargin-induced change in [Ca2+]i was greater in TrpC1-expressing cells than controls, Ba2+ influx was unaffected by thapsigargin. Whole-cell membrane currents recorded in TrpC1-expressing cells increased as a function of post-infection time and were (1) inwardly rectifying in symmetrical sodium gluconate solutions, (2) non-selective with respect to Na+, Ca2+ and Ba2+, and (3) blocked by La3+. Furthermore TrpC1 currents were unaffected by (1) thapsigargin, (2) dialysis of the cell with Ins(1,4,5)P3 or (3) dialysis of the cell with solutions containing high concentrations of the Ca2+ chelator, EGTA. These results suggest that TrpC1 forms non-selective cation channels that are constitutively active when expressed in Sf9 cells, but insensitive to depletion of the internal Ca2+ stores. Thus TrpC1 may be a subunit of a SOC which alone can form functional channels in Sf9 cells, but which requires additional subunits or cytoplasmic factors present in mammalian cells for expression of SOC activity.

Concomitant and hormonally regulated expression of trp genes in bovine aortic endothelial cells.

Recent findings have suggested that the vertebrate trp family of channel proteins is the structural basis for Ca2+ influx through the capacitative calcium entry (CCE) pathway. We have discerned, in bovine aortic endothelial cells, the concomitant expression of four such vertebrate genes: trp-1 (two splice variants), trp-3, trp-4 and trp-5. Exogenous hormones rendered dynamic effects on the transcript levels of these genes. Most notably, beta-estradiol significantly down-regulated trp-4 while trans-retinoic acid dramatically up-regulated trp-5; yet these hormones rendered little change in CCE. These findings suggest that the extent of a given trp channel's participation in CCE is not reflected in alterations of its transcript level.

Differential expression of mammalian TRP homologues across tissues and cell lines.

Mammalian homologues of the Drosophila trp gene have been invoked as the structural basis for the currents associated with capacitative Ca2+ entry (CCE) in many cell types. Trp homologues are members of a large protein family that may associate as channel subunits providing an explanation for the functional diversity of store-operated channels observed in these cells. However, there is little information as to which of these genes are co-expressed at the cellular level. We have examined the tissue specific expression of five mammalian trp genes and determined which are co-expressed in five different cell lines. The results show tissue- and cell-specific co-expression of multiple trp forms. This implies that the subunit composition of a particular CCE channel may vary depending on the cell type.

Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.

The transient receptor potential (trp)-like (trpl) gene is thought to encode an ion channel important for signal transduction in Drosophila photoreceptor cells. Consistent with this hypothesis, heterologous expression of the trpl-encoded protein (Trpl) is associated with the appearance of an outwardly rectifying, nonselective cation current. In the present study, single channels were recorded in cell-attached, inside-out, and outside-out membrane patches from Sf9 insect cells infected with recombinant baculovirus-containing trpl cDNA under control of the polyhedrin promoter. The single-channel current-voltage relationship was linear from -100 to +80 mV with a slope conductance of 89-110 pS. The probability of opening was voltage sensitive, increasing at positive potentials contributing to the outwardly rectifying properties of the whole cell currents. The single channels 1) were never observed in Sf9 cells infected with recombinant baculovirus containing the B2 bradykinin receptor cDNA or in noninfected Sf9 cells; 2) appear at the same time postinfection as the Trpl whole cell current; 3) were nonselective with respect to Na+, Ca2+, and Ba2+; 4) were blocked by 1-2 mM La3+ and Gd3+ (but not 10 microM); and 5) were blocked by 4-8 mM Mg2+. The single Trpl channel activity increased spontaneously with time after patch formation, and the activity was further increased by application of bradykinin to cells expressing both the B2 bradykinin receptor and the Trpl protein. These results suggest that this single-channel activity reflects expression of the Trpl protein and provides conclusive evidence that trpl encodes a nonselective cation channel consistent with its proposed role in Drosophila phototransduction.

Functional expression of a human thrombin receptor in Sf9 insect cells: evidence for an active tethered ligand.

Desensitization of recombinant human thrombin receptors expressed in Sf9 insect cells was compared with native thrombin receptors in megakaryoblast erythroleukaemia (HEL) cells. Addition of thrombin (2 units/ml) or agonist peptide SFLLRN (10 microM) to HEL cells, or to Sf9 cells infected with recombinant baculovirus containing the thrombin receptor cDNA, produced an increase in the free cytosolic Ca2+ concentration ([Ca2+]i) as measured by fura-2. The response in HEL cells was transient, reflecting a rapid homologous desensitization. In contrast, [Ca2+]i in Sf9 cells expressing the thrombin receptor increased rapidly to a peak value that slowly declined, but remained elevated for at least 12 min following stimulation by thrombin. The sustained [Ca2+]i response to thrombin was not reversed by washout of thrombin or by any subsequent addition of hirudin. Pretreatment of Sf9 cells with either thrombin (2 units/ml) or SFLLRN (10 or 50 microM) for 5 min produced a shift in the ED50 for SFLLRN (added 10 min after washout) from 0.4 microM to 20 and 7 microM, respectively. Thus, desensitization of thrombin receptors expressed in Sf9 cells occurs slowly and reflects a decrease in receptor affinity. The sustained [Ca2+]i response in Sf9 cells stimulated by thrombin may reflect continuous activation by the tethered ligand. To test this hypothesis, the effect of protease treatment during the sustained phase of the response was examined. Addition of either aminopeptidase M or thermolysin reversed the sustained response to SFLLRN, but only thermolysin reversed the sustained response to thrombin. Thermolysin had no effect on the change in [Ca2+]i observed following carbachol stimulation of Sf9 cells expressing the M5 muscarinic receptor. Furthermore, following thermolysin treatment, the cells remained responsive to a subsequent application of SFLLRN. These results demonstrate that the tethered ligand remains active for extended periods of time after thrombin stimulation and suggests that further hydrolysis by extracellular proteases may represent an important mechanism of rapid receptor deactivation.

The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity.

Previous studies have shown that the Drosophila cation channels designated Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.

Ins(1,4,5)P3 activates Drosophila cation channel Trpl in recombinant baculovirus-infected Sf9 insect cells.

The trp-like (trpl) gene product (Trpl) is thought to form a nonselective cation channel important for signal transduction in Drosophila photoreceptor cells. This channel may be the insect homologue of mammalian channels involved in Ca2+ signal transduction. To determine the mechanism of receptor-mediated activation of Trpl, whole cell membrane currents were examined in Sf9 insect cells after infection with recombinant baculovirus. Stimulation by bradykinin increased whole cell Trpl currents three- to fivefold. Similar activation of Trpl was observed by inclusion of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the pipette solution during whole cell recordings. These currents were 1) not seen in noninfected cells or in cells expressing only the B2 receptor, 2) mimicked by D-myo-inositol 2,4,5-trisphosphate, and 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate, 3) not seen with D-myo-inositol 1,4-bisphosphate or D-myo-inositol 1,3,4,5-tetrakisphosphate, and 4) blocked by heparin, but not by de-N-sulfated heparin. In contrast, Trpl currents were unaffected by thapsigargin. These results demonstrate that the Trpl cation channel is activated by Ins(1,4,5)P3 in a heparin-sensitive fashion. Regulation of channel activity by Ins(1,4,5)P3 may occur by a number of mechanisms, including direct binding of Ins(1,4,5)P3 to the Trpl channel or direct physical interaction between the Ins(1,4,5)P3 receptor/Ca(2+)-release channel of the endoplasmic reticulum and the Trpl protein.

The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum.

We previously reported that expression of rotavirus nonstructural glycoprotein NSP4 is responsible for an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells (P. Tian, Y. Hu, W. P. Schilling, D. A. Lindsay, J. Eiden, and M. K. Estes, J. Virol. 68:251-257, 1994). The purpose of the present study was to determine the mechanism by which NSP4 causes an increase in [Ca2+]i by measuring the permeability of the cytoplasmic and endoplasmic reticulum (ER) membranes in recombinant-baculovirus-infected Sf9 cells. No obvious change in plasmalemma permeability to divalent cations was observed in cells expressing NSP4 compared with that in cells expressing another rotaviral glycoprotein (VP7) when the influx of Ba2+, a Ca2+ surrogate, was monitored. The basal Ca2+ permeability of the internal Ca2+ store was evaluated by measuring the release of Ca2+ induced by ionomycin, a Ca2+ ionophore, or thapsigargin, an inhibitor of the ER Ca(2+)-ATPase pump, following suspension of the cells in Ca(2+)-free extracellular buffer. Releasable Ca2+ decreased with time to a greater extent in cells expressing NSP4 compared with that in cells expressing VP7, suggesting that NSP4 increases the basal Ca2+ permeability of the ER membrane. To determine the possible mechanism by which NSP4 increases ER permeability, purified NSP4 protein or a 22-amino-acid synthetic peptide consisting of residues 114 to 135 (NSP4(114-135) was added exogenously to noninfected Sf9 cells during measurement of [Ca2+]i. Both NSP4 and the NSP4(114-135 peptide produced a time-dependent increase in [Ca2+]i that was attenuated by prior inhibition of phospholipase C with U-73122. Pretreatment of the cells with thapsigargin completely blocked the increase in [Ca2+]i produced by NSP4(114-135, but the peptide only partially reduced the change in [Ca2+]i produced by thapsigargin. No changes in [Ca2+]i were seen in cells treated with control peptides. These results suggest that (i) exogenous NSP4 increases [Ca2+]i through the activation of phospholipase C, (ii) Ca2+ release by exogenous NSP4 is from a store that is a subset of the thapsigargin-sensitive compartment, and (iii) amino acid residues 114 to 135 of NSP4 are sufficient for this activity. In contrast to exogenous NSP4, the mechanism by which endogenously expressed NSP4 increases [Ca2+]1 appears to be unrelated to phospholipase C, since no effect of U-73122 was seen on the elevated [Ca2+]1 in cells expressing NSP4 and exogenously applied NSP4(114-135) caused a further increase in [Ca2+]1 in cells expressing NSP4 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of vascular tone: cross-talk between sarcoplasmic reticulum and plasmalemma.

Selected topics on the roles of sarcoplasmic reticulum (SR) in the control of vascular smooth muscle (VSM) tone are briefly reviewed with particular reference to the regulation of cytosolic concentration of free calcium ions, [Ca2+]i. Although morphological evidence and subcellular membrane studies indicate a relatively meager quantity of SR in VSM and of endoplasmic reticulum (ER) in endothelial cells (ECs) compared with skeletal muscle and cardiac muscle, contractility studies suggest that vascular tone is, to a large extent, regulated by the intracellular Ca2+ stores in smooth muscle and endothelial cells. Cytosolic Ca2+ levels control myosin light chain phosphorylation and contraction in VSM and activation of NO synthase and phospholipase A2 in ECs to regulate nitric oxide (NO) and prostaglandin I2 formation. Understanding of the importance of SR or ER in modulating the [Ca2+]i in VSM and ECs has been further advanced as a result of the new development and refinement of biophysical techniques in the measurement of cellular Ca2+ concentrations and ion currents, such as fluorescent Ca2+ indicators and patch-clamp techniques. Experimental evidence has accumulated in support of the existence of cross-talk between SR-ER and the plasma membrane (PM). Novel pharmacological tool drugs selective for the SR-ER Ca2+ pump, such as thapsigargin and cyclopiazonic acid, as well as for SR-ER Ca2+ channels, such as ryanodine (for the Ca(2+)-induced Ca2+ release channel) and inositol polyphosphates and heparin (for the inositol-1,4,5-trisphosphate activated Ca2+ channel), together with the use of blockers for selective PM Ca2+ channels have enabled better formulation and elucidation of the mechanisms of cross-talk between SR-ER and PM.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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)

Activation of recombinant trp by thapsigargin in Sf9 insect cells.

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor.

The purpose of the present study was to examine Ca2+ signaling mechanisms in Sf9 cells and to demonstrate expression and functional linkage of a mammalian receptor to changes in cytosolic free Ca2+ concentration ([Ca2+]i). Addition of p-octopamine (50 microM to fura 2-loaded Sf9 cells produced a small transient increase in [Ca2+]i from a basal level of 58 +/- 10 to 194 +/- 7.6 (SD) nM. The response to octopamine was inhibited by both cyproheptadine and chlorpromazine and was mimicked by clonidine. In contrast, [Ca2+]i did not change in response to dopamine (50 microM), substance P (50 nM), histamine (50 microM), ATP (50 microM), acetylcholine (10 or 100 microM), carbachol (10 or 100 microM), serotonin (50 microM), epinephrine (10 microM), or bradykinin (50 nM). The Ca(2+)-adenosinetriphosphatase inhibitors thapsigargin (200 nM) and 2,5-di-tert-butylhydroquinone (BHQ; 10 microM) increased [Ca2+]i to 307 +/- 13 and 137 +/- 20 nM, respectively. In contrast to BHQ, the response to thapsigargin was attenuated by La3+ or removal of extracellular Ca2+ and increased by elevation of extracellular Ca2+. These results suggest that thapsigargin but not BHQ stimulates Ca2+ influx. The rat brain muscarinic receptor (subtype M5) was incorporated into the baculovirus by homologous recombination. Addition of carbachol (100 microM) increased [Ca2+]i from 92.7 +/- 6.4 to 480 +/- 26 nM in Sf9 cells infected with recombinant virus containing the M5 receptor cDNA. The effect of carbachol on [Ca2+]i was concentration dependent with a 50% effective concentration of approximately 30 microM and was blocked by atropine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Appearance of a novel Ca2+ influx pathway in Sf9 insect cells following expression of the transient receptor potential-like (trpl) protein of Drosophila.

Activation of phospholipase C, elevation of free cytosolic Ca2+ concentration ([Ca2+]i) and stimulation of Ca2+ influx have been implicated in Drosophila phototransduction. Electrophysiological studies suggest that trp and trpl proteins may be important for the light-activated Ca2+ current found in Drosophila photoreceptor cells. Although these proteins exhibit homologies to voltage-gated Ca2+ and Na+ channels, their actual function in insect cells and their relation to proteins involved in mammalian cell Ca2+ signaling remains unknown. In the present study, [Ca2+]i was examined in fura-2-loaded Sf9 insect cells infected with recombinant baculovirus containing cDNA for the trpl protein. Ca2+ influx was examined by use of Ba2+, a Ca2+ surrogate that is not a substrate for Ca(2+)-pumps or carriers and by measurement of whole-cell membrane currents. The results suggest that expression of trpl is associated with appearance of a Ca2+ permeable, non-selective cation channel formed by the trpl protein.