Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells.

The endocytosis of GABA(A) receptors was investigated in HEK 293 cells expressing receptor alpha1beta2- and alpha1beta2gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1beta2- and alpha1beta2gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degrees C. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABA(A) receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABA(A) receptor internalization, while the endocytosis of beta(2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

Kinetic properties of GABA rho1 homomeric receptors expressed in HEK293 cells

The rho1 subunit of the ionotropic GABA receptors is thought to contribute to the formation of the GABA(C) receptors with pharmacological and physiological properties distinct from those of GABA(A) receptors. Previous characterization of this subunit expressed in the Xenopus oocytes revealed an ion channel with slow activation and deactivation and no desensitization, quite different from the properties of GABA(C) receptors observed in native cells. We expressed the human rho1 subunit in human embryonic kidney (HEK) 293 cells and quantitatively characterized the kinetic properties of these receptors using a rapid drug application device. The rho1 subunit expressed in HEK293 cells exhibited pharmacological and kinetic properties qualitatively identical to those described when rho1 was expressed in the oocytes. An apparent desensitizing current observed during a constant GABA application was determined to be secondary to an E(Cl) shift. Detailed kinetic analyses and parameter estimation for a five-state kinetic model revealed that the channel is best described by a set of rate constants with a notably faster GABA unbinding K(off) rate compared to the parameters proposed for the same subunit expressed in the oocytes. The same subunit expressed in hippocampal neurons showed activation and deactivation kinetics identical to the current characterized in HEK293 cells. The kinetic properties of rho1 subunit expressed in a nonoocyte model system may be better described quantitatively by the rate constants presented here.     

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.     

Endothelin ET(A) and ET(B) receptors in postnatal intestine

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with (125)I-labeled ET-1 in membranes from endothelium-denuded (E(-)) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive (125)I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E(+)) and E(-) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ET(A) receptor antagonist BQ-123, competitive (125)I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E(+) vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ET(B) receptor.     

Functional characteristics of TRPC4 channels expressed in HEK 293 cells

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by GTPγS was not desensitized. TPRC4 activation by GTPγS was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK _a of 7.3. Finally, TPRC4 activation by GTPγS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles. These authors contributed equally to this work.     

Properties of Xenopus Kv1.10 channels expressed in HEK293 cells

Voltage-gated K+ channels play important roles in shaping the characteristics of action potentials and electrical activity. In a previous study, we isolated cDNAs encoding several distinct K+ channel isoforms, including a novel isoform (XKv1.10) expressed in Xenopus laevis spinal cord neurons and myocytes. Here, we report the biophysical characterization of XKv1.10 expressed in transiently transfected HEK293 cells. Whole cell patch clamp recordings revealed a voltage-gated, rapidly activating and inactivating K+ current. Interestingly, the rate of inactivation of XKv1.10 channels showed apparent voltage dependence, with time constants between 77.7-213.3 ms. The predicted protein sequence of XKv1.10 does not appear to encode an N-terminal inactivating "ball and chain" domain, and instead these channels may inactivate via a C/P-type mechanism. Consistent with this, either increasing the external concentration of K+ or external application of tetraethylammonium caused a decrease in the rate of inactivation. Pharmacologically, XKv1.10 K+ channels were sensitive to 4-aminopyridine and tetraethylammonium with apparent IC50 values of 68.5 microM and 17.1 mM, respectively. When simulated action potentials were used as a voltage command, XKv1.10 was similar to XKv1.4 in that it carried more repolarizing current during the action potential than XKv1.2. However, while XKv1.4 was active during the interspike interval, XKv1.10 and XKv1.2 were not. Overall, the data suggest that XKv1.10 channels make a unique contribution to the developmental maturation of electrical signaling in Xenopus laevis.     

20-hydroxyeicosatetraenoic acid (20-HETE) activates mouse TRPC6 channels expressed in HEK293 cells

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 microM Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 microM) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 microM) showed outward rectification, as shown previously for these channels using 100 microM 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 microM. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels approximately 3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.     

Functional characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed in HEK293T cells

Mouse alpha4beta2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmalpha4beta2, was assessed using an aequorin-based luminescence method that measures agonist-evoked changes in intracellular calcium. Agonist-elicited changes in intracellular calcium were due primarily to direct entry of calcium through the alpha4beta2 channel, although release of calcium from intracellular stores contributed approximately 28% of the agonist-evoked response. Agonist pharmacologies were very similar between the mmalpha4beta2 cells and most cell lines that stably express human alpha4beta2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-beta-erythroidine (DHbetaE), the predominant alpha4beta2 nAchR expressed in the mmalpha4beta2 cells exhibits a pharmacology that most resembles the DHbetaE-sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmalpha4beta2 nAchR was found to be atypically sensitive to blockade by the presumed alpha7-selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 +/- 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine-stimulated dopamine release, a response that is mediated by beta2-subunit-containing nAchRs and not alpha7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for alpha7-containing nAchRs as previously thought.     

Modulation of cell surface GABA(B) receptors by desensitization, trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.     

Differential desensitization of Ca2+ mobilization and vasoconstriction by ET(A) receptors in the gerbil spiral modiolar artery

Endothelins are known to be among the most potent endogenous vasoconstrictors. Vasoconstriction of the spiral modiolar artery, which supplies the cochlea, may be implicated in hearing loss and tinnitus. The purpose of the present study was to determine whether the spiral modiolar artery responds to endothelin, whether a change in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) mediates the response and which endothelin receptors are present. The vascular diameter and [Ca²⁺]_i were measured simultaneously by videomicroscopy and microfluorometry in the isolated spiral modiolar artery from the gerbil. ET-1 induced a transient [Ca²⁺]_i increase and a strong and long-lasting vasoconstriction. The transient [Ca²⁺]_i increase underwent rapid desensitization, was independent of extracellular Ca²⁺ and inhibited by the IP₃-receptor blocker (75 μm) 2-aminoethoxydiphenyl borate (2-APB) and by depletion of Ca²⁺ stores with 10⁻⁶ m thapsigargin. In contrast, the vasoconstriction displayed no comparable desensitization. The initial vasoconstriction was independent of extracellular Ca²⁺ but maintenance of the constriction depended on the presence of extracellular Ca²⁺. The half-maximal concentration values (EC ₅₀) for the agonists ET-1, ET-3 and sarafotoxin S6c were 0.8 nm, >10 nm and >100 nm, respectively. Affinity constants for the antagonists BQ-123 and BQ-788 were 24 nm and 77 nm, respectively. These observations demonstrate that ET-1 mediates a vasoconstriction of the gerbil spiral modiolar artery via ET_A receptors and an IP₃ receptor-mediated release of Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. The marked difference in desensitization between Ca²⁺ mobilization and vasoconstriction suggests that Ca²⁺ mobilization is not solely responsible for the vasoconstriction and that other signaling mechanisms must be present. Received: 4 January 2001/Revised: 23 April 2001     

Ion permeation properties of a cloned human 5-HT3 receptor transiently expressed in HEK 293 cells

Summary Human 5-HT₃ receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na⁺ were Li⁺, 1.16; K⁺, 1.04; Rb⁺, 1.11; Cs⁺ 1.11; NMDG⁺, 0.04; Ca²⁺, 0.49, and Mg²⁺, 0.37. The permeability sequence of the alkali metal cations was Lⁱ⁺ > Rb⁺ = Cs⁺ > K⁺ > Na⁺. Increased external concentrations of Ca²⁺ or Mg²⁺ decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT₃ receptors measured by fluctuation analysis of whole-cell currents was 790 ± 100fS. Differences in the basic properties of 5-HT₃ receptors between species may explain interspecies differences in pharmacological properties.     

Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells

Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.     

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB-induced activation of procaspase-8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL-induced cell death. Our results suggest that Bcl-2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase-8 in UVB-irradiated keratinocytes.     

Fatty acid regulation of liver X receptors (LXR) and peroxisome proliferator-activated receptor alpha (PPARalpha ) in HEK293 cells

Fatty acids bind to and regulate the activity of peroxisome proliferator-activated (PPAR) and liver X receptors (LXR). However, the role lipid metabolism plays in the control of intracellular fatty acid ligands is poorly understood. We have identified two strains of HEK293 cells that display differences in fatty acid regulation of nuclear receptors. Using full-length and Gal4-LBD chimeric receptors in functional assays, 20:4,n6 induced PPARalpha activity approximately 2.2-fold and suppressed LXRalpha activity by 80% (ED50 approximately 25-50 microm) in HEK293-E (early passage) cells but had no effect on PPARalpha or LXRalpha receptor activity in HEK293-L (late passage) cells. LXRbeta was insensitive to fatty acid regulation in both HEK293 strains. Metabolic labeling studies using [14C]20:4,n6 (at 100 microm) indicated that the uptake of 20:4,n6 and its assimilation into triacylglycerol, diacylglycerol, and polar lipids revealed no difference between the two strains. Such treatment increased total cellular 20:4,n6 ( approximately 11-fold) and its elongation product, 22:4,n6 ( approximately 3.6-fold), within 6 h. Non-esterified 20:4,n6 and 22:4,n6 represented 相似文献   

Purification and characterization of the creatine transporter expressed at high levels in HEK293 cells

The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (90% pure) by affinity chromatography on wheat germ agglutinin (WGA)-Sepharose and gel-filtration. The CreaT was shown to be an approximately 70 kDa glycoprotein by SDS-polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224 kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (95% purity) of the approximately 70kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises approximately 3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210 kDa. Circular dichroism showed a predominantly alpha-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts ( approximately 100 microg) that make it feasible to consider structural studies of a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter family.     

Selective regulation of endogenous G protein-coupled receptors by arrestins in HEK293 cells

Arrestins play an important role in regulating desensitization and trafficking of G protein-coupled receptors (GPCRs). However, limited insight into the specificity of arrestin-mediated regulation of GPCRs is currently available. Recently, we used an antisense strategy to reduce arrestin levels in HEK293 cells and characterize the role of arrestins on endogenous G(s)-coupled receptors (Mundell, S. J., Loudon, R. B., and Benovic, J. L. (1999) Biochemistry 38, 8723-8732). Here, we characterized GPCRs coupled to either G(q) (M(1) muscarinic acetylcholine receptor (M(1)AchR) and P2y(1) and P2y(2) purinergic receptors) or G(i) (somatostatin and AT1 angiotensin receptors) in wild type and arrestin antisense HEK293 cells. The agonist-specific desensitization of the M(1)Ach and somatostatin receptors was significantly attenuated in antisense-expressing cells, whereas desensitization of P2y(1) and P2y(2) purinergic and AT1 angiotensin receptors was unaffected by reduced arrestin levels. To further examine arrestin/GPCR specificity, we studied the effects of endogenous GPCR activation on the redistribution of arrestin-2 epitope tagged with the green fluorescent protein (arrestin-2-GFP). These studies revealed a receptor-specific movement of arrestin-2-GFP that mirrored the arrestin-receptor specificity observed in the antisense cells. Thus, agonist-induced activation of endogenous beta(2)-adrenergic, prostaglandin E(2), M(1)Ach, and somatostatin receptors induced arrestin-2-GFP redistribution to early endosomes, whereas P2y(1) and P2y(2) purinergic and AT1 angiotensin receptor activation did not. Thus, endogenous arrestins mediate the regulation of selective G(q)- and G(i)-coupled receptors in HEK293 cells.     

Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells

AimThe nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr¹⁰]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of ¹²⁵I-[Tyr¹⁴]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr¹⁰]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr¹⁰]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP–MOP receptors. We also examined if [Tyr¹⁰]N/OFQ(1-11) distinguished heteromeric NOP–MOP receptors from homomeric NOP receptors.Main methodsThe NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors.Key findingsIn vlPAG neurons, [Tyr¹⁰]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC₅₀: 8.98 μM) but equi-efficacious as N/OFQ. [Tyr¹⁰]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr¹⁰]N/OFQ(1-11) displayed similar concentration–response curves in decreasing cAMP accumulation.Significance[Tyr¹⁰]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP–MOP and homomeric NOP receptors.