Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain

Abstract

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-κB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-κB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane. Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-κB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-κB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane. Na,K-ATPase is an energy transducing ion-pump expressed in all mammalian cells. Recent studies have revealed a new role for Na,K-ATPase as a signal transducer. Ouabain, an analogue of digitalis and a highly specific ligand of Na,K-ATPase, has been shown by several groups to trigger intracellular signaling pathways controlling cell proliferation, apoptosis, cell motility, heart contractility, and blood pressure (1Schoner W. Scheiner-Bobis G. Am. J. Physiol. Cell Physiol. 2007; 293: 509-536Crossref PubMed Scopus (394) Google Scholar, 2Xie Z. Cai T. Mol. Interv. 2003; 3: 157-168Crossref PubMed Scopus (352) Google Scholar, 3Li J. Zelenin S. Aperia A. Aizman O. J. Am. Soc. Nephrol. 2006; 17: 1848-1857Crossref PubMed Scopus (120) Google Scholar). Results from our laboratory have demonstrated that Na,K-ATPase and the inositol 1,4,5,-trisphosphate (IP3) 3The abbreviations used are: IP3, inositol 1,4,5,-trisphosphate; IP3R, IP3 receptor; PBS phosphate-buffered saline; Ank, ankyrin; siRNA, small interfering RNA; GST, glutathione S-transferase; aa, amino acids; FRAP, fluorescence recovery after photobleaching; WT, wild type. receptor (IP3R) can form a signaling microdomain that, in the presence of ouabain, triggers slow calcium oscillations and activates the NF-κB transcription factor (4Miyakawa-Naito A. Uhlen P. Lal M. Aizman O. Mikoshiba K. Brismar H. Zelenin S. Aperia A. J. Biol. Chem. 2003; 278: 50355-50361Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 5Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (255) Google Scholar). The interaction between Na,K-ATPase and IP3R is mediated by the N terminus tail of the catalytic α-subunit of Na,K-ATPase and the N-terminal portion 1-604 of the IP3R (6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Here we have addressed the question of whether a scaffolding protein may support the interaction between Na,K-ATPase and IP3R and, if so, what the functional role of this protein can be. Ankyrins belong to a ubiquitously expressed intracellular scaffolding protein family that includes Ank-B, Ank-G, and Ank-R (7Mohler P.J. Gramolini A.O. Bennett V. J. Cell Sci. 2002; 115: 1565-1566Crossref PubMed Google Scholar). Ankyrins associate with a diverse set of membrane, cytoskeletal, and cytoplasmic proteins and tether them into specialized membrane signaling domains. Both Ank-B and Ank-G have been reported to interact with Na,K-ATPase, but Ank-B is the only ankyrin isoform that has been reported to interact with IP3R (8Lencesova L. O'Neill A. Resneck W.G. Bloch R.J. Blaustein M.P. J. Biol. Chem. 2004; 279: 2885-2893Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 9Liu X.L. Miyakawa A. Aperia A. Krieger P. Neuroreport. 2007; 18: 597-600Crossref PubMed Scopus (28) Google Scholar, 10Mohler P.J. Davis J.Q. Bennett V. PLoS Biol. 2005; 3: e423Crossref PubMed Scopus (203) Google Scholar). Thus, Ank-B is a likely protein to be a partner of an Na,K-ATPase/IP3R signaling microdomain. Previous studies performed with a pulldown technique have suggested that ankyrin may bind to the second cytoplasmic domain of Na,K-ATPase (11Woroniecki R. Ferdinand J.R. Morrow J.S. Devarajan P. Am. J. Physiol. Renal Physiol. 2003; 284: 358-364Crossref Scopus (45) Google Scholar, 12Devarajan P. Scaramuzzino D.A. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2965-2969Crossref PubMed Scopus (134) Google Scholar) and the C terminus of the IP3R (13Bourguignon L.Y. Jin H. J. Biol. Chem. 1995; 270: 7257-7260Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Here we have examined whether Ank-B may bind directly to the N-terminal tail of the catalytic α-subunit of Na,K-ATPase and the N-terminal portion 1-604 of the IP3R, i.e. the sites for Na,K-ATPase/IP3R interaction. We found this to be the case. To test the functional role of Ank-B in ouabain/Na,K-ATPase/IP3R signaling, a cell line derived from fetal monkey kidneys, COS-7, was used. This cell line has previously been shown to respond to ouabain with slow calcium oscillations (4Miyakawa-Naito A. Uhlen P. Lal M. Aizman O. Mikoshiba K. Brismar H. Zelenin S. Aperia A. J. Biol. Chem. 2003; 278: 50355-50361Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). With use of small interfering RNA (siRNA) we specifically knocked down Ank-B and compared the calcium and NF-κB response to ouabain in wild type and Ank-B-silenced cells. Cells transfected with control siRNA were used as an additional control. The signaling response to ouabain was found to be altered in Ank-B-silenced cells. Determination of the plasma membrane expression of Na,K-ATPase and of its ion transporting function indicated that the effect of Ank-B silencing on Na,K-ATPase signaling was not due to internalization of the protein. Cell Culture and Transfection—COS-7 cells, a cell line derived from fetal monkey (Cercopithecus aethiops) kidney (ECACC no. 87021302) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mm l-glutamine. Plasmid DNA and synthetic siRNA duplexes were transfected using either Effectene or HiPerFect according to the manufacturer's protocol (Qiagen). siRNA optimal concentration was determined by titration to 10 nm. siRNAs were fluorescence-tagged with Label IT® Cy™3 labeling kit (Mirus) according to the manufacturer's protocol. Transfected cells were used for experiments 2 days after transfection unless otherwise stated. Measurement of Cell Viability and Proliferation—A WST-1 assay (Chemicon International) was used to measure cell viability in Ank-B siRNA and rat siRNA-transfected cells as described previously (3Li J. Zelenin S. Aperia A. Aizman O. J. Am. Soc. Nephrol. 2006; 17: 1848-1857Crossref PubMed Scopus (120) Google Scholar). Bromodeoxyuridine incorporation was used to determine cells proliferation. The cell proliferation kit (GE Healthcare) was used according to manufacturer's protocol. DNA Plasmid Constructs and siRNA—A full-length human Na,K-ATPase α1 subunit (hNa,K-ATPase α1) was constructed and cloned into pEGFP-C2 vector. This construct was modified to obtain cDNA that encodes hNa,K-ATPase α1 with truncation of first five N-terminal residues (MGKGV), which are cleaved during the posttranslational modification of the enzyme (14Pressley T.A. Allen J.C. Clarke C.H. Odebunmi T. Higham S.C. Am. J. Physiol. 1996; 271: C825-C832Crossref PubMed Google Scholar). The glutathione S-transferase (GST)-fused N-terminal fragment of Na,K-ATPase α1 (6-100 aa) and the fragment corresponding to the 1-604 residues of IP3R type 1 were generated as described previously (4Miyakawa-Naito A. Uhlen P. Lal M. Aizman O. Mikoshiba K. Brismar H. Zelenin S. Aperia A. J. Biol. Chem. 2003; 278: 50355-50361Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The monkey ankyrin B-targeted sequence is based on the sequence of Ank-B from Macaca mulatta, the closest available monkey Ank-B sequence in the data base (XM_001094169): AAC CTG GAC AAA GTC GTG GAA. For controls, we used siRNA generated by changing three nucleotides (bold) from targeted sequence AAC CTG TAC GAA CTC GTG GAA or siRNA based on rat Ank-B sequence (XM_227735): CGG GCA CAC TGT GGT GAA GAA. siRNA oligonucleotides were synthesized by Qiagen. RNA Isolation and Reverse Transcription-PCR—Total RNA was extracted from siRNA-transfected cells using Aurum Total RNA mini kit (Bio-Rad) according to the manufacturer's protocol. First-strand cDNA was synthesized from 2 μg of total RNA using Moloney murine leukemia virus reverse transcriptase and oligonucleotide (dT) primers (Promega). PCR primers were designed according to the sequence from monkey Ank-B (XM_001094169) (forward, 5′-CAC AAG GCT CCT GTT GAT GAT GTC AC-3′; reverse, 5′-GAA TGT TAG TGA CAT CTG GAG AGG C-3′), and PCR was carried with protocol. of and in fragment of N-terminal Na,K-ATPase fragment corresponding to residues 1-604 of IP3R, and were in were in the mm mm 2 mm 1 mm and The was for was with (GE Healthcare) for 1 and the were with mm of with proteins or were with of human Ank-B from in B mm mm 1 mm and for 1 The were with B 1 were with of for were to for or and the were α1 and control For the were Na,K-ATPase α1 Ank-B from and or with (GE were from unless otherwise stated. were with pm ouabain for 2 with phosphate-buffered on in mm mm 1 mm mm 1 mm and for protein concentration was by protein assay (Bio-Rad) using bovine serum as the were to an of total protein. The protein was with μg of α1 or for 2 were with of of protein with The the were with and proteins were with of and for in were in or by and to a membrane. were in in mm 10 mm and using the corresponding for 1 with the were with with (GE Healthcare) for 1 were with and the protein were by using (GE Cell cultured on were to a concentration in PBS for with with PBS and with PBS mm to the cells were in assay mm 1 mm mm with for 10 protein concentration was according to protein proteins were with of with The was as the and with an of cell by by Na,K-ATPase was determined by as described previously G. Aizman O. Aizman R. R. P. Aperia A. Am. J. Physiol. PubMed Google Scholar). was as the between in the of ouabain and in the presence of 1 mm ouabain of cells were with the in for 1 cells were with ouabain for of intracellular calcium was performed in the presence of ouabain using a on a 2 with a cells were and and fluorescence was with a an were for was used for cells, the were cells were as cells that of oscillations was performed using (6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). after is a used to the mobility of proteins L. Zelenin S. Malmersjo S. P. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). were carried using a with a line was used for both and cells were transfected with siRNA 1 after and with for protein to Na,K-ATPase the The cells were used for experiments 2 days after the of a plasma membrane was performed with for 10 and fluorescence recovery were with for 10 were by using and cell the and of was This was used for of fluorescence were for and the NF-κB to was used as an of NF-κB activation. wild type rat siRNA, and Ank-B siRNA-transfected cells were with 10 pm ouabain for The of NF-κB was determined using NF-κB kit the manufacturer's protocol. were from cells as described previously O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (255) Google Scholar) and to the with oligonucleotide NF-κB The NF-κB to oligonucleotide was using an the NF-κB to was and was with a from cells were used as a control. To binding an NF-κB wild type oligonucleotide and a NF-κB oligonucleotide were used. NF-κB was using as described previously O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (255) Google Scholar). are as the was performed in most of experiments using test the data from of Ank-B calcium response of responding and NF-κB were using of by the was to be Ank-B to the N-terminal of Na,K-ATPase and N-terminal 1-604 of the test if Ank-B may be with the Na,K-ATPase/IP3R signaling microdomain in cells, were Ank-B with the catalytic of Na,K-ATPase α1) and with IP3R. IP3R with Ank-B and Na,K-ATPase α1 Na,K-ATPase and IP3R interact the N-terminal tail of Na,K-ATPase α1 and the first amino of the IP3R N we if Ank-B may bind directly to the Na,K-ATPase α1 N terminus and the 1-604 portion of the IP3R. Ank-B was with Na,K-ATPase α1 or with IP3R 2 and show that Ank-B directly to both Ank-B to the N-terminal domain of of in binding of Ank-B and N-terminal domain (6-100 aa) of was used as a control. and Na,K-ATPase N-terminal The was three Ank-B to the IP3R. of in binding of Ank-B and IP3R. was used as a control. and IP3R. The was three of Ank-B for Na,K-ATPase/IP3R whether Ank-B may the function of the ouabain/Na,K-ATPase/IP3R signaling we used siRNA to down Ank-B. this was carried on cells from the siRNA duplexes were based on the sequence of Ank-B from M. mulatta, the closest available monkey Ank-B sequence in the data after siRNA transfection the expression of Ank-B not and protein were The protein in Ank-B siRNA-transfected cells between and that in and rat siRNA-transfected and wild type cells The Ank-B silencing had no effect on cell cell proliferation, or not Na,K-ATPase interaction was in Ank-B-silenced cells but not in the and rat siRNA-transfected cells were with rat and siRNA, we used rat siRNA as the control siRNA in the interaction with IP3R is attenuated in Ank-B cells. and using IP3R or Na,K-ATPase α1 that the of interaction between Na,K-ATPase α1 and IP3R was reduced in monkey Ank-B siRNA cells compared with rat and siRNA-transfected cells. The of IP3R and Na,K-ATPase were based on the the of the rat or siRNA-transfected cells was as of total cell The was three binding to Na,K-ATPase triggers highly regular calcium oscillations in cells as as in several cell The between Na,K-ATPase and IP3R is a for this effect to of the Na,K-ATPase/IP3R interaction with an was found to the number of cells responding with calcium oscillations (6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To the functional role of Ank-B in the interaction between Na,K-ATPase and IP3R, we compared the calcium response to ouabain in wild control siRNA, and monkey Ank-B siRNA-transfected cells. We used a concentration of pm This concentration not of and is in the of the ouabain concentration W. Scheiner-Bobis G. Am. J. 2007; PubMed Scopus Google Scholar). The number of responding cells was in monkey Ank-B siRNA-transfected cells in wild type cells and rat siRNA-transfected cells The calcium was in cells Ank-B had been as compared with wild type and rat siRNA-transfected cells and Ank-B NF-κB by is an effect of the ouabain/Na,K-ATPase/IP3R signaling (3Li J. Zelenin S. Aperia A. Aizman O. J. Am. Soc. Nephrol. 2006; 17: 1848-1857Crossref PubMed Scopus (120) Google Scholar, 5Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (255) Google Scholar, 6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To test whether Ank-B can the NF-κB response to ouabain, control and Ank-B-silenced cells were to a of ouabain or to a for The of NF-κB from the to the was either by and of NF-κB or with labeling of NF-κB in cells. In both wild type cells and control siRNA-transfected cells we a of the after ouabain In contrast, no in the distribution of NF-κB in Ank-B-silenced cells were and Ank-B the and of test whether silencing of Ank-B has an effect on the ion transporting function of Na,K-ATPase, we compared in Ank-B-silenced cells and in control cells. was in both groups in control siRNA and monkey Ank-B-silenced cells Cells were with ouabain The data were from three of total of total protein in a new To determine the distribution of the Na,K-ATPase, a assay was The of the Na,K-ATPase α1 that was in the plasma membrane in Ank-B-silenced cells and in control siRNA-transfected cells was and Na,K-ATPase in and Ank-B-silenced test whether the interaction between Na,K-ATPase and Ank-B has an effect on the mobility of Na,K-ATPase the plasma membrane, we the of Na,K-ATPase α1 subunit in cells. cells transfected with and Ank-B siRNA were in The recovery of Na,K-ATPase is in and recovery a membrane are shown in The was for control siRNA-transfected and Ank-B-silenced cells. the recovery the the of Na,K-ATPase can be L. Zelenin S. Malmersjo S. P. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar, A. 2003; PubMed Scopus Google Scholar). The was as the of recovery after In control siRNA-transfected cells the of Na,K-ATPase In Ank-B-silenced cells the was of Na,K-ATPase for We have previously demonstrated the of a signaling microdomain that Na,K-ATPase and IP3R and oscillations and of NF-κB. Here we show that Ank-B is an partner of this signaling microdomain. We show that Ank-B to the interaction between Na,K-ATPase and IP3R and that Ank-B is for the of the signaling function of the Na,K-ATPase/IP3R signaling from the Na,K-ATPase/IP3R is by the interaction between the N terminus of Na,K-ATPase α1 subunit and the N-terminal portion 1-604 of the IP3R (6Zhang S. Malmersjo S. Li J. Ando H. Aizman O. Uhlen P. Mikoshiba K. Aperia A. J. Biol. Chem. 2006; 281: 21954-21962Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Results from the show that of Na,K-ATPase α1 and IP3R bind directly to Ank-B. N novel binding sites for Ank-B. Z. Devarajan P. Morrow J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported in that ankyrin to in the second cytoplasmic domain of and Jin (13Bourguignon L.Y. Jin H. J. Biol. Chem. 1995; 270: 7257-7260Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) reported in that ankyrin to in the C terminus of the IP3R. This is to the IP3R The interaction between the N terminus of the IP3R and ankyrin was not The N terminus of the IP3R of functional the domain and the IP3 binding domain The of has been H. T. T. Mikoshiba K. M. Mol. 2005; 17: Full Text Full Text PDF PubMed Scopus Google Scholar). highly on the IP3 binding domain was found to be a for interaction with The binding of Ank-B to the N of Na,K-ATPase α1 and IP3R plays an role in Na,K-ATPase α1 and IP3R In Ank-B-silenced cells, the interaction between Na,K-ATPase α1 and IP3R was reduced as indicated by the The studies of the calcium and NF-κB to ouabain that Ank-B with both Na,K-ATPase and IP3R has for Na,K-ATPase/IP3R is a that can interact with The calcium in and Mol. Cell Biol. 2003; PubMed Scopus Google Scholar). the for of transcription and by of to the of cells, cells, and cells, respond to ouabain with slow calcium (4Miyakawa-Naito A. Uhlen P. Lal M. Aizman O. Mikoshiba K. Brismar H. Zelenin S. Aperia A. J. Biol. Chem. 2003; 278: 50355-50361Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 5Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (255) Google Scholar, 9Liu X.L. Miyakawa A. Aperia A. Krieger P. Neuroreport. 2007; 18: 597-600Crossref PubMed Scopus (28) Google Scholar). a highly regular oscillatory of calcium from intracellular plays a role for the of calcium oscillations S. M. K. H. S. S. Mikoshiba K. PubMed Scopus Google Scholar). calcium from intracellular is due to interaction between IP3R with the N terminus of Na,K-ATPase and is of IP3 (4Miyakawa-Naito A. Uhlen P. Lal M. Aizman O. Mikoshiba K. Brismar H. Zelenin S. Aperia A. J. Biol. Chem. 2003; 278: 50355-50361Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Results from the that Ank-B plays a role in the oscillatory a of ouabain, we a in the number of responding cells in the Ank-B-silenced as compared with the control in the response were oscillations is the most of all cell the cell can the of the oscillations Mol. Cell Biol. 2003; PubMed Scopus Google Scholar). The transcription factor NF-κB is by calcium oscillations K. PubMed Scopus Google Scholar). calcium oscillations NF-κB in a of cell (3Li J. Zelenin S. Aperia A. Aizman O. J. Am. Soc. Nephrol. 2006; 17: 1848-1857Crossref PubMed Scopus (120) Google Scholar, 5Aizman O. Uhlen P. Lal M. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13420-13424Crossref PubMed Scopus (255) Google Scholar, 9Liu X.L. Miyakawa A. Aperia A. Krieger P. Neuroreport. 2007; 18: 597-600Crossref PubMed Scopus (28) Google Scholar). not NF-κB in the Ank-B-silenced cells but in cells transfected with control Ank-B This the of the calcium oscillatory for the NF-κB is a In most cell of NF-κB has an effect M. H. A. 2004; PubMed Scopus Google Scholar, M. 2005; PubMed Scopus Google Scholar). We have shown that to doses of ouabain can cells from serum (3Li J. Zelenin S. Aperia A. Aizman O. J. Am. Soc. Nephrol. 2006; 17: 1848-1857Crossref PubMed Scopus (120) Google Scholar). The that Ank-B may an role for this effect to that Ank-B is for the interaction between Na,K-ATPase and IP3R, may a role in the of Na,K-ATPase to the plasma membrane. We found no of internalization of Na,K-ATPase in the Ank-B-silenced cells, and according to the was effect of Ank-B silencing on the Na,K-ATPase In the of Na,K-ATPase in the plasma membrane was by as from ouabain binding studies P.J. Gramolini A.O. S. K. H. Bennett V. 2003; PubMed Scopus Google Scholar). The Ank-B-silenced cells expressed of the Ank-B. This is likely to cell intracellular signaling pathways may be by the In a of and J. Cai T. Z. H. L. M. Mol. Biol. 2006; 17: PubMed Scopus Google Scholar, Z. Cai T. J. Z. Mol. Biol. 2005; PubMed Scopus Google Scholar, M. A. Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that Na,K-ATPase with and that ouabain binding to Na,K-ATPase to from a of effects of and of is of the IP3R and of from the intracellular Z. Cai T. J. Z. Mol. Biol. 2005; PubMed Scopus Google Scholar). The role of Ank-B for the between Na,K-ATPase and to be the of M. J. L. S. J. J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) the of of Na,K-ATPase in the plasma membrane, a and a suggested that the in of cells, function as the ouabain binding signaling transducer. In the studies we found that the of Na,K-ATPase was and the was of Ank-B had no effect on Na,K-ATPase from the studies be with the by that is the of Na,K-ATPase that with Ank-B and is the that and Na,K-ATPase in the plasma membrane. The role of Ank-B may be to the signaling of Na,K-ATPase and IP3R. on have shown that are heart and and and with in the Ank-B a of P.J. Gramolini A.O. S. K. H. Bennett V. 2003; PubMed Scopus Google Scholar, P.J. G. L. K. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). have been to of Ank-B of a number of ion of Ank-B in the of cells, is that the may be to of Na,K-ATPase ion to of Na,K-ATPase days S. M. Davis L. M. Bennett V. J. Cell Biol. PubMed Scopus (109) Google Scholar) and in the of the and of and of P. H. Bennett V. J. Cell Biol. PubMed Scopus Google Scholar). a role for ankyrin B in The from the have revealed a novel role for Ank-B as a of the signaling be an for studies to to which in the and may be due to of Na,K-ATPase/IP3R We and for and for of the