FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2113076389> ?p ?o ?g. }

• W2113076389 endingPage "24246" @default.
• W2113076389 startingPage "24234" @default.
• W2113076389 abstract "To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine- and ryanodine-sensitive Ca2+release channel in HEK293 cells. Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+conductance of 777 picosiemens in 250 mm KCl and a Ca2+ conductance of 137 picosiemens in 250 mmCaCl2 and displayed apCa2+/pK+ ratio of 6.3 and an open time constant of about 1.16 ms. The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nmand inactivated at about 10 mm. Ca2+ alone was able to activate the cloned RyR3 fully. Calmodulin activated the cloned RyR3 at low Ca2+ concentrations but inhibited the channel at high Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Cyclic ADP-ribose did not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maximal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3. To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine- and ryanodine-sensitive Ca2+release channel in HEK293 cells. Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+conductance of 777 picosiemens in 250 mm KCl and a Ca2+ conductance of 137 picosiemens in 250 mmCaCl2 and displayed apCa2+/pK+ ratio of 6.3 and an open time constant of about 1.16 ms. The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nmand inactivated at about 10 mm. Ca2+ alone was able to activate the cloned RyR3 fully. Calmodulin activated the cloned RyR3 at low Ca2+ concentrations but inhibited the channel at high Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Cyclic ADP-ribose did not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maximal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3. Ryanodine receptors are a family of intracellular Ca2+release channels that were originally identified in the sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; RyR, ryanodine receptor; GST, glutathione S-transferase; PBS, phosphate-buffered saline; cADPr, cyclic ADP-ribose; pS, picosiemens; CaM, calmodulin. of striated muscles. To date, three members of this family have been identified in mammalian tissues, namely the skeletal muscle (RyR1), the cardiac muscle (RyR2), and the brain (RyR3) ryanodine receptor. These proteins are the products of different genes and share 66–70% amino acid sequence identity (1McPherson P.S. Campbell K.P. J. Biol. Chem. 1993; 268: 13765-13768Abstract Full Text PDF PubMed Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 4Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar, 5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (364) Google Scholar). Earlier studies using RNA blot analysis revealed that the expression patterns of these isoforms were very different (6Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (353) Google Scholar). RyR1 was predominantly expressed in skeletal muscle, whereas RyR2 was mainly expressed in heart and brain. The expression of RyR3 was detected in smooth muscle tissues and certain regions of the brain. However, results of recent ribonuclease protection assay demonstrate that all three RyR isoforms are widely and differentially expressed (7Giannini G. Conti A. Mammarella S. Scrobogna M. Sorrentino V. J. Cell Biol. 1995; 128: 893-904Crossref PubMed Scopus (490) Google Scholar). These studies also indicate that most tissues express more than one RyR isoform. For example, skeletal muscles express both RyR1 and RyR3, although RyR3 is expressed at a much lower level than that of RyR1. The function and regulation of RyR1 and RyR2 have been extensively studied. They function as Ca2+ release channels of the sarcoplasmic reticulum and play an essential role in excitation-contraction coupling in striated muscles (1McPherson P.S. Campbell K.P. J. Biol. Chem. 1993; 268: 13765-13768Abstract Full Text PDF PubMed Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 4Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar). Both RyR1 and RyR2 have been purified and characterized. The single channel properties of RyR1 and RyR2 that have been incorporated into lipid bilayers are very similar. They both form a large conductance channel permeable to monovalent and divalent cations and can be activated by submicromolar Ca2+, ATP, and caffeine and inhibited by millimolar Ca2+, Mg2+, and ruthenium red. Ryanodine, a plant alkaloid, locks both channels in a subconductance state. However, differences in sensitivities to Ca2+activation, Ca2+ inactivation, and Mg2+inhibition have been reported (8Rousseau E. Smith J.S. Henderson J.S. Meissner G. Biophys. J. 1986; 50: 1009-1014Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 9Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 10Chu A. Fill M. Stefani E. Entman M.L. J. Membr. Biol. 1993; 135: 49-59Crossref PubMed Scopus (93) Google Scholar, 11Yamazawa T. Takeshima H. Sakurai T. Endo M. Iino M. EMBO J. 1996; 15: 6172-6177Crossref PubMed Scopus (52) Google Scholar). Differences in responses to cyclic ADP-ribose (12Meszaros L.G. Bak J. Chu A. Nature. 1993; 364: 76-79Crossref PubMed Scopus (318) Google Scholar, 13Morrissette J. Heisermann G. Cleary J. Ruoho A. Coronado R. FEBS Lett. 1993; 330: 270-274Crossref PubMed Scopus (50) Google Scholar), inorganic phosphate, and perchlorate (14Fruen B. Mickelson J.R. Roghair T.J. Cheng H.-L. Louis C.F. Am. J. Physiol. 1994; 266: C1729-C1735Crossref PubMed Google Scholar) have also been observed. On the other hand, little is known about the physiological role, regulation, and channel properties of the RyR3 isoform (5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (364) Google Scholar). Isolation and biochemical and biophysical characterization of RyR3 have been impeded by its low abundance and its coexpression with other RyR isoforms. Biochemical studies and single channel recordings of ryanodine-sensitive Ca2+ release channels isolated from brain and smooth muscle tissues have been reported (15Herrmann-Frank A. Darling E. Meissner G. Pfluegers Arch. 1991; 418: 353-359Crossref PubMed Scopus (115) Google Scholar, 16McPherson P.S. Kim Y.K. Valdivia H. Knudson C.M. Takekura H. Franzini-Armstrong C. Coronado R. Campbell K.P. Neuron. 1991; 7: 17-25Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 17Xu L. Lai F.A. Cohn A. Etter E. Guerrero A. Fay F.S. Meissner G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3294-3298Crossref PubMed Scopus (72) Google Scholar). It is, however, uncertain whether the observed properties correspond to those of the RyR3 isoform, since these tissues are known to express RyR3 as well as RyR2 and/or RyR1. Recently, Murayama and Ogawa (18Murayama R. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) were able to purify the rabbit brain RyR3 by using an RyR3-specific antibody. They demonstrated that the immunoprecipitated RyR3 was capable of binding [3H]ryanodine in a Ca2+-dependent and caffeine-sensitive manner (18Murayama R. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, channel properties of the purified RyR3 have not been characterized. Significant progress in understanding the function and regulation of RyR3 has recently been made through the generation of mice with a null mutation in the ryr1 gene (19Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar). Skeletal muscles isolated from these mutant mice lack RyR1 and RyR2 but contain RyR3, thus providing a valuable system to study RyR3. Analysis of SR Ca2+ release in these mutant muscles suggests that RyR3 is modulated by Ca2+, adenine nucleotide, caffeine, and ryanodine (20Takeshima H. Yamazawa T. Ikemoto T. Takekura H. Nishi M. Noda T. Iino M. EMBO J. 1995; 14: 2999-3006Crossref PubMed Scopus (128) Google Scholar). More recently, a mutant mouse lacking RyR3 has also been generated (21Takeshima H. Ikemoto T. Nishi M. Nishiyama N. Shimuta M. Sugitani Y. Kuno J. Saito I. Saito H. Endo M. Iino M. Noda T. J. Biol. Chem. 1996; 271: 19649-19652Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Ca2+ release studies have shown that SR Ca2+ release in the RyR1-deficient muscle (or RyR3 containing muscle) was much less sensitive to Ca2+activation than that in the RyR3-deficient muscle (or RyR1 containing muscle). Based on these data, it was suggested that RyR3 may function as a Ca2+-induced Ca2+ release channel with a high threshold in mammalian skeletal muscles (21Takeshima H. Ikemoto T. Nishi M. Nishiyama N. Shimuta M. Sugitani Y. Kuno J. Saito I. Saito H. Endo M. Iino M. Noda T. J. Biol. Chem. 1996; 271: 19649-19652Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). It remains, however, to be determined whether the intrinsic sensitivity of the RyR3 channel to Ca2+ activation differs from that of the RyR1 channel. To gain insights into the Ca2+ sensitivity, ligand gating properties, and single channel characteristics of RyR3, we have cloned the RyR3 cDNA from rabbit uterus and expressed the cDNA in HEK293 cells. The CHAPS-solubilized and sucrose gradient-purified cloned RyR3 was characterized by using single channel recordings in planar lipid bilayers. Our results demonstrate that the cloned RyR3 single channel is modulated by caffeine, ryanodine, and other physiological and pharmacological ligands. The sensitivity of the cloned RyR3 to Ca2+ activation was found to be similar to that of RyR1. However, the cloned RyR3 differs from RyR1 in the gating behavior, the extent of maximal activation by Ca2+, and the sensitivity to Ca2+ inactivation. Restriction endonucleases and DNA modifying enzymes were purchased from Boehringer Mannheim, Life Technologies Inc., and Pharmacia Biotech Inc. Ryanodine was obtained from Agri Systems International (Wind Gap, PA). Soybean phosphatidylcholine, brain phosphatidylethanolamine, and brain phosphatidylserine were from Avanti Polar Lipids. Calmodulin was obtained from Calbiochem. Horseradish peroxidase-conjugated anti-rabbit IgG antibody was purchased from Promega Biotech. Rhodamine-conjugated anti-rabbit IgG antibody was from Jackson ImmunoResearch Laboratories, Inc. Prestained protein standards were purchased from Bio-Rad. Cyclic ADP-ribose, CHAPS, and other chemicals were from Sigma. Total RNA from rabbit uterus, isolated by the method of Chomczynski and Sacchi (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63222) Google Scholar), was used to purify poly(A)+ RNA using the mRNA Purification Kit (Pharmacia) according to the manufacturer's instructions. First strand cDNA was prepared from mRNA (10 μg) using the SuperScript Preamplification System (Life Technologies Inc.) with random primers and was resuspended in 150 μl of H2O. Polymerase chain reactions (PCR) were carried out in a 50-μl solution containing 20 mm Tris-HCl, pH 8.8, 10 mm KCl, 10 mm(NH4)2SO4, 2.0 mmMgS04, 0.1% Triton X-100, 0.1 mg/ml bovine serum albumin, 50 ng of each DNA primer, 200 μm concentration of each dATP, dCTP, dGTP, and dTTP, 2.5 units of Pfu DNA polymerase, and 1.0–2.0 μl of first strand cDNA. The DNA was denatured for 3 min at 94 °C followed by 25–30 cycles of amplification. Each cycle consists of 45 s at 94 °C, 1 min at 46–53 °C, and 4 min at 72 °C. An additional extension for 5 min at 72 °C was performed after the final cycle. The annealing temperature and the number of cycles for each pair of primers were determined empirically. The entire 15-kilobase pair coding region of the RyR3 mRNA from rabbit uterus was amplified by PCR using 16 pairs of primers (TableI and Fig. 1). PCR primers were synthesized based on the reported brain RyR3 cDNA sequence (6Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (353) Google Scholar). A 5′-flanking BamHI restriction site is included in primers CP-1F, CP-1R, CP-2F, CP-2R, CP-3F, CP-3R, CP-5F, CP-5R, CP-7F, CP-7R, FP-6F, and FP-8F, whereas a 5′-flanking EcoRI site is included in primers CP-4F, CP-4R, CP-6F, CP-6R, and FP-6R. Primers CP-8F and CP-8R each contain a 5′-flanking XbaI site. CP-1F also contains a 5′-flanking NheI site downstream of theBamHI. Mutations that did not change amino acid sequence were introduced in overlapping PCR primers CP-1R and CP-2F, CP-2R and CP-3F, and CP-5R and CP-6F to generate three unique restriction sitesXhoI, SalI, and SmaI in the RyR3 cDNA.Table ISequences of PCR primersNameSequencePCR productCP-1F5′-ACTGGATCCAGCTAGCAGCCATGGCCGAAGGCGGAGA-3′PCR1CP-1R5′-ACTGGATCCTCGAGTCTGTCCAATTTACTGATAAGC-3′CP-2F5′-ACTGGATCCTCGAGTCTTCCTCAGGTATCCTGGA-3′PCR2aSP2–2R5′-TTCAGCCAGCCTGTCTC-3′SP2–2F5′-GCATCTCCTTCCGCATC-3′PCR2bCP-2R5′-ACTGGATCCATGTCAGGTCGACAGCCTGGCCT-3′CP-3F5′-ACTGGATCCAAGGCTGTCGACCTGACATTGAGCT-3′PCR3aSP3–2R5′-ACCGGGTTCTTCTCTTCAC-3′SP3–1F5′-GCAACGTGGACCTGGAG-3′PCR3bCP-3R5′-ACTGGATCCGCCAAACACAGAGGGATCGATGA-3′CP-4F5′-ACTGAATTCCCTCCTCATCGATCCCTCTGTGT-3′PCR4aSP4–2R5′-CTCCAGCAGGTAACTCAG-3′SP4–3F5′-ACCAGCATCCCAACCTC-3′PCR4bFP-6R5′-CAGGAATTCTCAGCCCATGTGCACAATCTCTTCT-3′FP-6F5′-CTGGGATCCAGGGTGCCATTAAAATCTCCG-3′PCR4cCP-4R5′-ACTGAATTCAGAATCGATCAGAGAGGTGTAGTGT-3′CP-5F5′-ACTGGATCCTGATCGATTCTACACTGCAGACAA-3′PCR5aSP5–2R5′-GAAGGCATCCAGCTCCAT-3′FP-8F5′-CTGGGATCCAGAGGACCAAAGAGGGTGAAGC-3′PCR5bCP-5R5′-ACTGGATCCCGGGGAGTTTTGGTGTTGAAGACT-3′CP-6F5′-ACTGAATTCCCGGGAAAGGTCTATTCTGGGGATG-3′PCR6aSP6–1R5′-CCGCTCCTGGTCCTGT-3′SP6–2F5′-ACCAACTCTTCCGCATG-3′PCR6bCP-6R5′-ACTGAATTCAGCCAAATCCTGTACTAGTTTCTCT-3′CP-7F5′-ACTGGATCCAAGAGAAACTAGTACAGGATTTGGCT-3′PCR7aSP7–1R5′-GTGAACTCATCATTCTGG-3′SP7–1F5′-GCCGAGATGGTCCTTCA-3′PCR7bSP7–4R5′-TCCATGGCCTTCTGGAATTC-3′SP7–4F5′-TTAACCAGCTCAGATAC-3′PCR7cCP-7R5′-ACTGGATCCTGCACTAGTTCAGTCGTGATGCCT-3′CP-8F5′-CAGTCTAGAAGGGGCAAAGAACATCAGAGTGACT-3′PCR8CP-8R5′-GTCTCTAGACCAGTGTCGTGCTGTAGCTTTCAC-3′FP-8F5′-CTGGGATCCAGAGGACCAAAGAGGGTGAAGC-3′FP-8FP-8R5′-CAGGAATTCTCATTCTCTGGAGAGCACAACGTTTG-3′FP-11F5′-GTCGGATCCAGGCAGCAGAGACGAAG-3′FP-11FF-11R5′-CAGGAATTCTCATCGCTCCTCTTCTTTGGCT-3′ Open table in a new tab These primers were used to generate 16 PCR fragments PCR1, PCR2a, PCR2b, PCR3a, PCR3b, PCR4a, PCR4b, PCR4c, PCR5a, PCR5b, PCR6a, PCR6b, PCR7a, PCR7b, PCR7c, and PCR8 (Table I and Fig. 1). These PCR fragments were subcloned into plasmid pBluescript. Individual clones of each PCR fragment were analyzed by restriction endonuclease digestion and DNA sequencing analysis by the Sanger dideoxy chain termination method (23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52743) Google Scholar) with α-35S-dATP (1000 Ci/mmol, Amersham Corp.) using the T7 Sequencing Kit from Pharmacia. All recombinant DNA manipulations were carried out using standard procedures (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar). Subfragments PCR2a and PCR2b, PCR3a and PCR3b, PCR4a, PCR4b and PCR4c, PCR5a and PCR5b, PCR6a and PCR6b, PCR7a, PCR7b, and PCR7c were joined together to form PCR2, PCR3, PCR4, PCR5, PCR6, PCR7, respectively (Fig. 1). PCR1 and PCR2, PCR3 and PCR5, and PCR6 and PCR8 were then connected together to yield PCR1,2, PCR3,5, and PCR6,8, respectively. These three fragments were then ligated together sequentially to form PCR1–3,5,6,8 (Fig. 1). PCR4 was ligated with PCR1–3,5,6,8 to form PCR1–6,8 which was subsequently subcloned into another plasmid pBlueBac4 (Invitrogen) to yield PCR1–6,8 (pBB4). The final gap was filled by ligating PCR7 with PCR1–6,8 to form pRyR3 (pBB4) (Fig. 1). Finally, the full-length RyR3 cDNA from pRyR3 (pBB4) (Fig. 1) was subcloned into the mammalian expression vector pcDNA3 (Invitrogen) to form pRyR3. Anti-FP8 and anti-FP11 antibodies were raised in rabbits against short sequences of the rabbit RyR3 in the form of GST fusion proteins. Oligodeoxynucleotides with a 5′-flanking BamHI restriction site for the forward primers (FP-8F and FP-11F) and 5′-flankingEcoRI restriction site and a stop codon for the reverse primers (FP-8R and FP-11R) were synthesized (Table I). Primers, FP-8F and FP-8R, were used to synthesize the DNA fragment encoding amino acids 2690–2734 of the rabbit RyR3 by PCR using PCR5b as DNA template. Primers, FP-11F and FP-11R, were used to synthesize the DNA fragment encoding amino acids 4328–4363 of RyR3 by PCR using PCR8 fragment as DNA template. PCR products were digested with BamHI andEcoRI, purified, and ligated into theBamHI/EcoRI sites in the polylinker in pGEX-3X to form pFP8 and pFP11. The sequences of pFP8 and pFP11 were confirmed by DNA sequencing. The expression and isolation of glutathioneS-transferase (GST) fusion protein FP8 and FP11 were carried out according to the standard protocols from Pharmacia. Rabbits were immunized subcutaneously with 200 μg of affinity purified FP8 or FP11 in complete Freund's adjuvant, and booster injections of the same amount in incomplete Freund's adjuvant were given at 17–19-day intervals. Antiserum was collected 2 weeks after each booster injection. GST protein and FP8 and FP11 fusion proteins were immobilized to separate Affi-Gel 15 columns (Bio-Rad) according to the standard protocol from Bio-Rad. Antiserum was first absorbed on the GST column. Absorbed antiserum was then loaded onto the FP8 or FP11 column, washed, and eluted with 0.1 m glycine, pH 2.5, and neutralized with 0.2 volume of 1 m Tris-HCl, pH 8.0. Affinity purified antibodies were dialyzed against phosphate-buffered saline (PBS) and concentrated in a Centricon-10 concentrator (Amicon). HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.1 mm minimum Eagle's medium nonessential amino acids, 4 mml-glutamine, 100 units of penicillin/ml, 100 mg of streptomycin/ml, 4.5 g of glucose/liter, and 10% fetal calf serum, at 37 °C under 5% CO2. DNA transfection was carried out using calcium phosphate (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cells were plated in 100-mm tissue culture dishes 20–22 h before transfection and were transfected with 8 μg of pRyR3 per dish. Control cells were treated in the same way either with no DNA or with only the expression vector DNA. Coverslips were placed in a 100-mm tissue culture dish. Cell culture and DNA transfection were then carried out as described above. Twenty-four hours after transfection, the coverslips were washed three times with PBS, fixed with 4% formaldehyde in PBS for 15 min, and washed once with PBS and three times with PBS containing 0.1% saponin 5 min each time. The coverslips were blocked with buffer A (2% skim milk powder, 0.1% saponin in PBS) for 30 min before washing and incubating with anti-FP11 antibody in buffer A for 1 to 2 h. The coverslips were washed with buffer A and incubated with rhodamine-conjugated anti-rabbit IgG in buffer A for 30–60 min. The coverslips were then washed, mounted in 95% glycerol, and analyzed with the Leica DMRB photomicroscope using a 63 × objective. Free cytosolic Ca2+ in transfected and non-transfected HEK293 cells was measured with the fluorescence Ca2+ indicator dye fluo 3 (26Minta A. Kao J.P.Y. Tsien R.Y. J. Biol. Chem. 1989; 264: 8171-8178Abstract Full Text PDF PubMed Google Scholar). Cells grown for 24 h after transfection were washed three times with KRH buffer without MgCl2 and CaCl2(KRH buffer: 125 mm NaCl, 5 mm KCl, 1.2 mm KH2PO4, 6 mmglucose, 1.2 mm MgCl2, 2 mmCaCl2, and 25 mm Hepes, pH 7.4) and incubated in the same buffer at 37 °C for 30–60 min. After being detached from culture dishes by pipetting, cells were collected by centrifugation at 2,500 rpm for 2 min in a Beckman TH-4 rotor. Cell pellets were washed twice with KRH buffer and loaded with 10 μm fluo 3 in KRH buffer plus 0.1 mg/ml bovine serum albumin and 250 μm sulfinpyasone at room temperature for 30 min, followed by washing with KRH buffer three times and resuspended in KRH buffer plus 0.1 mg/ml bovine serum albumin and 250 μm sulfinpyasone. Aliquot of 100–150 μl of fluo 3 loaded cells was added to 2 ml (final volume) KRH buffer in a cuvette. Fluorescence intensity of fluo 3 at 530 nm was measured in an SLM-Aminco series 2 luminescence spectrometer with 480 nm excitation at 25 °C (SLM Instruments, Urbana, IL). Heavy sarcoplasmic reticulum was isolated from bovine diaphragm or rabbit fast-twitch skeletal muscle according to the method described by Campbell and MacLennan (27Campbell K.P. MacLennan D.H. J. Biol. Chem. 1981; 256: 4626-4632Abstract Full Text PDF PubMed Google Scholar). Microsomal membranes were prepared from transfected and non-transfected HEK293 cells as described previously (28Chen S.R.W. Vaughan D.M. Coronado R. MacLennan D.H. Biochemistry. 1993; 32: 3743-3753Crossref PubMed Scopus (49) Google Scholar) with some modifications. Cells grown for 28 h after transfection on 100-mm tissue culture dishes were washed twice with 3 ml of PBS containing 5 mm EDTA and incubated in the same solution for 5 min at room temperature. Cells were detached from dishes by gentle pipetting and collected by centrifugation at 2,500 rpm for 2 min in a Beckman TH-4 rotor. Cell pellets were suspended in a solution containing 5 mm Tris-HCl, pH 7.0, 0.5 mm MgCl2, and a protease inhibitor mix (0.5 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 μg/ml aprotinin, and 2.5 mmdithiothreitol) and incubated on ice for 20–30 min. Cells were homogenized in a glass Dounce homogenizer for 50 strokes with pestle A. An equal volume of a solution containing 15 mm Tris-HCl, pH 7.0, 0.5 m sucrose, 0.3 m KCl, 100 μm CaCl2, and the protease inhibitor mix was added to the homogenate and homogenized for 35 strokes. The homogenate was centrifuged at 8,000 rpm for 20 min in a Beckman JA-20 rotor at 4 °C. The supernatant was centrifuged at 37,000 rpm in a Beckman Ti70 rotor for 60 min. The pellets were suspended in a buffer containing 25 mm Tris-Hepes, pH 7.4, 1 m NaCl, and the protease inhibitor mix and solubilized by 1.5% (final concentration) CHAPS in a final volume of 2.5 ml and a final membrane protein concentration of 2 mg/ml for 1 h on ice. The suspension was spun at 35,000 rpm in a Beckman Ti75 rotor for 1 h at 4 °C, and 2.5 ml of the supernatant was layered on top of a 10.5-ml (7.5–25%, w/w) linear sucrose gradient containing 25 mmTris-Hepes, pH 7.4, 0.3 m NaCl, 0.1 mmCaCl2, 0.25 mm phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, 5 mm dithiothreitol, 0.3% CHAPS, and 0.17% phosphatidylcholine. The tubes were centrifuged at 28,500 rpm in a Beckman SW-41 rotor for 17–18 h at 4 °C. Fractions of 0.7 ml each were collected and monitored for immunoreactivity by enzyme-linked immunosorbent assay as described below. The peak fractions of immunoreactivity were pooled, aliquoted, and stored at −80 °C. Aliquots of 50 μl of the sucrose density gradient fractions were added to microtiter wells containing 150 μl of 50 mm sodium carbonate buffer, pH 9.6. The microtiter plate was incubated at 4 °C for 18 h and then blocked with 5% skim milk powder in 50 mm sodium carbonate buffer, pH 9.6, for 1 h at room temperature. The plate was washed 8–10 times with cold tap water and incubated with anti-FP11 in PBS solution containing 5% milk powder and 0.1% saponin for 2–4 h, washed again, and allowed to react with alkaline phosphatase-conjugated anti-rabbit IgG for 1 h. The samples were again washed, and bound antibodies were quantified by the alkaline phosphatase reaction in diethanolamine buffer containing 10% diethanolamine, pH 9.8, 5 mm MgCl2, and one phosphatase substrate tablet (Sigma) per 5 ml of diethanolamine buffer. Microsomal membranes and sarcoplasmic reticulum membranes were denatured in Laemmli sample buffer at 100 °C for 2 min and separated in 5% SDS-polyacrylamide minigels (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207461) Google Scholar). The resolved proteins were transferred to nitrocellulose membranes at 20 V for 15–16 h at 4 °C in the presence of 0.01% SDS according to Towbinet al. (30Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44931) Google Scholar). The nitrocellulose membranes were blocked for 1 h with PBS plus 0.5% Tween 20 and 5% skim milk powder, incubated 2–4 h with anti-FP8 antibody in the same solution, and then washed three times for 15 min each with the same buffer. The bound antibodies were visualized by using horseradish peroxidase-conjugated anti-rabbit IgG and the enhanced chemiluminescence Western blotting analysis system from Amersham Corp. Single channel recordings were carried out using CHAPS-solubilized and sucrose density gradient-purified ryanodine receptors from either rabbit skeletal muscle heavy sarcoplasmic reticulum or from transfected HEK293 cells as described previously (28Chen S.R.W. Vaughan D.M. Coronado R. MacLennan D.H. Biochemistry. 1993; 32: 3743-3753Crossref PubMed Scopus (49) Google Scholar). Brain phosphatidylserine and brain phosphatidylethanolamine, dissolved in chloroform, were combined in a 3:5 ratio (w/w), dried under nitrogen gas, and suspended in 30 μl ofn-decane at a concentration of 35 mg of lipid/ml. Bilayers were formed across a 250-μm hole in a Delrin partition separating two chambers. The trans chamber (500 μl) was connected to the head stage input of an Axopatch 200A amplifier (Axon Instruments Inc.). The cis chamber (1.2 ml) was held at virtual ground. A symmetrical solution containing 250 mm KCl and 25 mm Hepes, pH 7.4, was used for recordings. A 12-μl aliquot of the sucrose density gradient-purified cloned RyR3 or a 4-μl aliquot of the sucrose density gradient-purified native rabbit skeletal muscle ryanodine receptor was added to the cischamber. Unless indicated otherwise, spontaneous channel activity was tested for sensitivity to EGTA and/or Ca2+, thereby providing information about Ca2+ sensitivity, orientation in the bilayer, and stability of the incorporated channel. Free Ca2+ concentrations were calculated using the computer program of Fabiato and Fabiato (31Fabiato A. Fabiato F. J. Physiol. ( Paris ). 1979; 75: 463-505PubMed Google Scholar). All subsequent additions were made to that chamber in which the addition of EGTA inhibited the activity of the incorporated channel. This chamber presumably corresponds to the cytoplasmic side of the Ca2+ release channel. Recordings were filtered at 1500 Hz using a low-pass Bessel filter (Frequency Devices, Haverhill, MA), digitized at 20 kHz, and recorded on optical disk cartridges. The data were analyzed using pClamp 6.0.3 software (Axon Instruments Inc.). To obtain the full-length RyR3 cDNA for expression and functional studies, we employed reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the entire coding sequence of the RyR3 cDNA from rabbit uterus. Sixteen overlapping cDNA fragments were obtained and sequenced (Fig. 1). The deduced amino acid sequence of the rabbit uterus RyR3 cDNA is identical to that of the rabbit brain RyR3 cDNA repo" @default.
• W2113076389 created "2016-06-24" @default.
• W2113076389 creator A5002765258 @default.
• W2113076389 creator A5021541475 @default.
• W2113076389 creator A5037817240 @default.
• W2113076389 creator A5063447635 @default.
• W2113076389 date "1997-09-01" @default.
• W2113076389 modified "2023-09-28" @default.
• W2113076389 title "Functional Characterization of the Recombinant Type 3 Ca2+ Release Channel (Ryanodine Receptor) Expressed in HEK293 Cells" @default.
• W2113076389 cites W1490746554 @default.
• W2113076389 cites W1498369873 @default.
• W2113076389 cites W1527086839 @default.
• W2113076389 cites W1565088140 @default.
• W2113076389 cites W1601936385 @default.
• W2113076389 cites W1641967096 @default.
• W2113076389 cites W1876995878 @default.
• W2113076389 cites W1914131776 @default.
• W2113076389 cites W1964443900 @default.
• W2113076389 cites W1965861043 @default.
• W2113076389 cites W1972823724 @default.
• W2113076389 cites W1974143212 @default.
• W2113076389 cites W1981752474 @default.
• W2113076389 cites W1984234875 @default.
• W2113076389 cites W1986897813 @default.
• W2113076389 cites W1989256274 @default.
• W2113076389 cites W1989286153 @default.
• W2113076389 cites W1990952447 @default.
• W2113076389 cites W1991753089 @default.
• W2113076389 cites W2004773321 @default.
• W2113076389 cites W2011809437 @default.
• W2113076389 cites W2012467728 @default.
• W2113076389 cites W2014405435 @default.
• W2113076389 cites W2014993252 @default.
• W2113076389 cites W2018417695 @default.
• W2113076389 cites W2019465508 @default.
• W2113076389 cites W2026571586 @default.
• W2113076389 cites W2033792131 @default.
• W2113076389 cites W2036790702 @default.
• W2113076389 cites W2037744654 @default.
• W2113076389 cites W2043767569 @default.
• W2113076389 cites W2051125453 @default.
• W2113076389 cites W2058179976 @default.
• W2113076389 cites W2060055554 @default.
• W2113076389 cites W2060571726 @default.
• W2113076389 cites W2063081959 @default.
• W2113076389 cites W206588626 @default.
• W2113076389 cites W2085326980 @default.
• W2113076389 cites W2087231244 @default.
• W2113076389 cites W2093144498 @default.
• W2113076389 cites W2093818160 @default.
• W2113076389 cites W2095201247 @default.
• W2113076389 cites W2096827547 @default.
• W2113076389 cites W2100837269 @default.
• W2113076389 cites W2101108802 @default.
• W2113076389 cites W2109172777 @default.
• W2113076389 cites W2113007237 @default.
• W2113076389 cites W2119956322 @default.
• W2113076389 cites W2128113643 @default.
• W2113076389 cites W2129646135 @default.
• W2113076389 cites W2138270253 @default.
• W2113076389 cites W2138450406 @default.
• W2113076389 cites W2141385342 @default.
• W2113076389 cites W2144389195 @default.
• W2113076389 cites W2172295889 @default.
• W2113076389 cites W2358927500 @default.
• W2113076389 cites W2394518603 @default.
• W2113076389 cites W2408350377 @default.
• W2113076389 cites W2411375269 @default.
• W2113076389 cites W2462739816 @default.
• W2113076389 cites W252963246 @default.
• W2113076389 cites W36028778 @default.
• W2113076389 cites W4294216491 @default.
• W2113076389 doi "https://doi.org/10.1074/jbc.272.39.24234" @default.
• W2113076389 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9305876" @default.
• W2113076389 hasPublicationYear "1997" @default.
• W2113076389 type Work @default.
• W2113076389 sameAs 2113076389 @default.
• W2113076389 citedByCount "150" @default.
• W2113076389 countsByYear W21130763892013 @default.
• W2113076389 countsByYear W21130763892014 @default.
• W2113076389 countsByYear W21130763892015 @default.
• W2113076389 countsByYear W21130763892016 @default.
• W2113076389 countsByYear W21130763892017 @default.
• W2113076389 countsByYear W21130763892018 @default.
• W2113076389 countsByYear W21130763892019 @default.
• W2113076389 countsByYear W21130763892020 @default.
• W2113076389 countsByYear W21130763892021 @default.
• W2113076389 countsByYear W21130763892022 @default.
• W2113076389 crossrefType "journal-article" @default.
• W2113076389 hasAuthorship W2113076389A5002765258 @default.
• W2113076389 hasAuthorship W2113076389A5021541475 @default.
• W2113076389 hasAuthorship W2113076389A5037817240 @default.
• W2113076389 hasAuthorship W2113076389A5063447635 @default.
• W2113076389 hasBestOaLocation W21130763891 @default.
• W2113076389 hasConcept C104317684 @default.
• W2113076389 hasConcept C113217602 @default.
• W2113076389 hasConcept C153911025 @default.
• W2113076389 hasConcept C170493617 @default.

• next