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• W2108774434 abstract "Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail ofhSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1.3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed withhIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open. Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail ofhSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1.3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed withhIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open. Ca2+-activated K+ large conductance KCa IKCa intermediate conductance KCa carboxyl-terminal tail calmodulin charybdotoxin rat basophilic leukemia trifluoperazine wild-type glutathione S-transferase polyacrylamide gel electrophoresis Ca2+-mediated signaling events are central to the physiological activity of diverse cell types. Opening in response to changes in intracellular Ca2+([Ca2+]i), Ca2+-activated K+(KCa)1 channels play an important role in modulating the Ca2+ signaling cascade by regulating the membrane potential in both excitable and nonexcitable cells. Historically, these channels have been classified as large (BKCa), intermediate (IKCa), and small (SKCa) conductance channels based on their single-channel conductance in symmetrical K+ solutions (1Latorre R. Oberhauser A. Labarca P. Alvarez O. Annu. Rev. Physiol. 1989; 51: 385-399Crossref PubMed Scopus (636) Google Scholar). BKCa channels have a single channel conductance of 100–250 pS, are opened by elevated [Ca2+]i as well as by depolarization, and are blocked by the scorpion peptides charybdotoxin (ChTX) and iberiotoxin (2Kaczorowski G.J. Knaus H.G. Leonard R.J. McManus O.B. Garcia M.L. J. Bioenerg. Biomembr. 1996; 28: 255-267Crossref PubMed Scopus (264) Google Scholar). These channels are abundant in smooth muscle and in neurons and are also present in other cells (2Kaczorowski G.J. Knaus H.G. Leonard R.J. McManus O.B. Garcia M.L. J. Bioenerg. Biomembr. 1996; 28: 255-267Crossref PubMed Scopus (264) Google Scholar). BKCa channels are composed of an α- and a β-subunit. The α-subunit, encoded by the Slo gene (3Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-555Crossref PubMed Scopus (536) Google Scholar, 4Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Crossref PubMed Scopus (569) Google Scholar, 5Tseng-Crank J. Foster C.D. Krause J.D. Mertz R. Godinot N. DiChiara T.J. Reinhart P.H. Neuron. 1994; 13: 1315-1330Abstract Full Text PDF PubMed Scopus (383) Google Scholar), is a seven-transmembrane region protein with an extracellular N terminus (6Meera P. Wallner M. Song M. Toro L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14066-14071Crossref PubMed Scopus (251) Google Scholar). The β-subunit is a two-transmembrane region protein that, when associated with the channel, enhances the Ca2+ sensing and toxin binding properties of the channel (7Wallner M. Meera P. Ottolia M. Kaczorowski G.J. Latorre R. Garcia M.L. Stefani E. Toro L. Recept. Channels. 1995; 3: 185-199PubMed Google Scholar, 8Hanner M. Schmalhofer W.A. Munujos P. Knaus H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2853-2858Crossref PubMed Scopus (82) Google Scholar). SKCa channels have unitary conductances of 4–14 pS; are highly sensitive to [Ca2+]i, with activation in the 200–500 nm range; and are voltage-independent (9Hille B. Ionic Channels of Excitable Membranes. 2nd ed. Sinauer, Sunderland, MA1993: 121-124Google Scholar, 10Sah P. Trends Neurosci. 1992; 19: 150-154Abstract Full Text PDF Scopus (805) Google Scholar). SKCa channels are highly expressed in the central nervous system, where they modulate the firing pattern of neurons via the generation of slow membrane after-hyperpolarizations (10Sah P. Trends Neurosci. 1992; 19: 150-154Abstract Full Text PDF Scopus (805) Google Scholar). SKCa channels have also been described in skeletal muscle (11Vergara C. Ramirez B.U. Exp. Neurol. 1997; 146: 282-285Crossref PubMed Scopus (11) Google Scholar) and in human Jurkat T-cells (12Grissmer S. Lewis R.S. Cahalan M.D. J. Gen. Physiol. 1992; 99: 63-84Crossref PubMed Scopus (117) Google Scholar). These channels are blocked by apamin, a peptide from bee venom, and by the scorpion peptide scyllatoxin (12Grissmer S. Lewis R.S. Cahalan M.D. J. Gen. Physiol. 1992; 99: 63-84Crossref PubMed Scopus (117) Google Scholar, 13Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (788) Google Scholar, 14Hanselmann C. Grissmer S. J. Physiol. 1996; 496: 627-637Crossref PubMed Scopus (35) Google Scholar). Three genes (SKCa1–3) within a novel subfamily encode SKCa channels (13Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (788) Google Scholar). SKCa1–3gene products bear 70–80% amino acid sequence identity to each other, and hydrophilicity analysis predicts that these proteins have six transmembrane helices with intracellular N and C termini (13Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (788) Google Scholar, 15Chandy K.G. Fantino E. Wittekindt O. Kalman K. Tong L.L. Ho T.H. Gutman G.A. Crocq M.A. Ganguli R. Nimgaonkar V. Morris-Rosendahl D. Gargus J.J. Mol. Psychiatry. 1998; 3: 32-37Crossref PubMed Scopus (183) Google Scholar). ThehSKCa3 gene has recently been implicated in schizophrenia (15Chandy K.G. Fantino E. Wittekindt O. Kalman K. Tong L.L. Ho T.H. Gutman G.A. Crocq M.A. Ganguli R. Nimgaonkar V. Morris-Rosendahl D. Gargus J.J. Mol. Psychiatry. 1998; 3: 32-37Crossref PubMed Scopus (183) Google Scholar, 16Bowen T. Guy C.A. Craddock N. Williams N. Spurlock G. Murphy K. Jones L. Cardno A. Gray M. Sanders R. McCarthy M. Chandy K.G. Fantino E. Kalman K. Gutman G.A. Gargus J.J. Williams J. McGuffin P. Owen M.J. O'Donovan M.C. Mol. Psychiatry. 1998; 3: 266-269Crossref PubMed Scopus (59) Google Scholar). IKCa channels, unlike SKCa channels, are predominantly expressed in peripheral tissues, including those of the hematopoietic system, colon, lung, placenta, and pancreas (17Gardos G. Biochim. Biophys. Acta. 1958; 30: 653-654Crossref PubMed Scopus (467) Google Scholar, 18Brugnara C. Armsby C.C. De Franceschi L. Crest M. Euclaire M.F. Alper S.L. J. Membr. Biol. 1995; 147: 71-82Crossref PubMed Scopus (45) Google Scholar, 19Mahaut-Smith M.P. J. Physiol. 1995; 484: 15-24Crossref PubMed Scopus (40) Google Scholar, 20Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (221) Google Scholar, 21Gallin E.K. Physiol. Rev. 1991; 71: 775-811Crossref PubMed Scopus (180) Google Scholar, 22Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 23Ghanshani S. Coleman M. Gustavsson P. Wu C.-L. Gutman G.A. Dahl N. Mohrenweiser H. Chandy K.G. Genomics. 1998; 51: 160-161Crossref PubMed Scopus (30) Google Scholar). These channels have intermediate single channel conductance values of 11–40 pS and can be pharmacologically distinguished from SKCa channels by their sensitivity to block by ChTX and clotrimazole and by their insensitivity to apamin (20Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (221) Google Scholar, 22Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Both SKCa and IKCa channels are voltage-independent and steeply sensitive to a rise in [Ca2+]i. At least one gene encoding an IKCa channel has been cloned from human and mouse tissues. Called IKCa1 (also calledKCa4, SK4, and KCNN4), this gene has been shown to encode the native IKCa channel in human T-lymphocytes (22Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 23Ghanshani S. Coleman M. Gustavsson P. Wu C.-L. Gutman G.A. Dahl N. Mohrenweiser H. Chandy K.G. Genomics. 1998; 51: 160-161Crossref PubMed Scopus (30) Google Scholar) and erythrocytes (24–27); some patients with Diamond-Blackfan anemia lack one allele of this gene (23Ghanshani S. Coleman M. Gustavsson P. Wu C.-L. Gutman G.A. Dahl N. Mohrenweiser H. Chandy K.G. Genomics. 1998; 51: 160-161Crossref PubMed Scopus (30) Google Scholar). hIKCa1 shares little sequence identity with theSlo proteins, and only about 40% identity with theSKCa1–3 gene products. Thus, hIKCa1 constitutes a distinct subfamily within the extended K+ channel supergene family. The Ca2+ sensor for BKCa channels resides in a negatively charged Ca2+ bowl domain in the C-tail of the α-subunit (28Wei A. Solaro C. Lingle C. Salkoff L. Neuron. 1994; 13: 671-681Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 29Schreiber M. Salkoff L. Biophys. J. 1997; 73: 1355-1363Abstract Full Text PDF PubMed Scopus (338) Google Scholar). The β-subunit also contributes to the gating of these proteins (7Wallner M. Meera P. Ottolia M. Kaczorowski G.J. Latorre R. Garcia M.L. Stefani E. Toro L. Recept. Channels. 1995; 3: 185-199PubMed Google Scholar). In marked contrast, the protein-coding regions ofSKCa1–3 and hIKCa1 do not contain any EF-hand or Ca2+ bowl motifs in their primary amino acid sequence, despite their exquisite Ca2+ sensitivity. This observation led us to speculate that the Ca2+ sensor for these channels either resides in a novel motif intrinsic to the channel or is provided by an accessory subunit that is tightly linked to channel activity. We investigated the latter possibility in a yeast two-hybrid system usinghIKCa1 as our prototype. The Ca2+-binding protein calmodulin (CAM) was identified as a strong interacting partner of the C-tail of hIKCa1. Recently, CAM was shown to confer Ca2+ sensitivity to SKCa channel subfamily members (30Xia X.-M. Fakler B. Rivard A. Wayman G. Johnson-Pais T. Keen J.E. Ishii T. Hirschberg B. Bond C.T. Lutsenko S. Maylie J. Adelman J.P. Nature. 1998; 395: 503-507Crossref PubMed Scopus (725) Google Scholar). Here, we report that CAM binds to and is required for Ca2+-dependent activation of hIKCa1. Biochemical studies demonstrate that both hIKCa1 andhSKCa3 are prebound tightly to CAM in a Ca2+-independent fashion. Finally, we show by expression and patch-clamp recording that four CAMs are required to mediate the Ca2+-dependent channel activity of thehIKCa1 tetramer. We have previously reported the cloning of hIKCa1 (22Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 23Ghanshani S. Coleman M. Gustavsson P. Wu C.-L. Gutman G.A. Dahl N. Mohrenweiser H. Chandy K.G. Genomics. 1998; 51: 160-161Crossref PubMed Scopus (30) Google Scholar), hSKCa3 (15Chandy K.G. Fantino E. Wittekindt O. Kalman K. Tong L.L. Ho T.H. Gutman G.A. Crocq M.A. Ganguli R. Nimgaonkar V. Morris-Rosendahl D. Gargus J.J. Mol. Psychiatry. 1998; 3: 32-37Crossref PubMed Scopus (183) Google Scholar), andmKv1.3 (31Grissmer S. Dethlefs B. Wasmuth J.J. Goldin A.L. Gutman G.A. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9411-9415Crossref PubMed Scopus (169) Google Scholar). Drosophila wild-type (WT) and mutant (B1234Q) CAMs with differing Ca2+ sensitivities have been reported previously (32Maune J.F. Klee C.B. Beckingham K. J. Biol. Chem. 1992; 267: 5286-5295Abstract Full Text PDF PubMed Google Scholar, 33Mukherjea P. Maune J.F. Beckingham K. Protein Science. 1996; 5: 468-477Crossref PubMed Scopus (37) Google Scholar). The B1234Q mutant has all four EF-hands mutated; glutamates 31, 67, 104, and 140 are replaced by glutamine (33Mukherjea P. Maune J.F. Beckingham K. Protein Science. 1996; 5: 468-477Crossref PubMed Scopus (37) Google Scholar). PAGA2 vector was a kind gift of Lutz Birnbaumer (University of California, Los Angeles, CA). This vector is a pGEM3-based version of the pAGA vector, both of which contain the 5′-untranslated region of alfalfa virus RNA 4 and a 92-base pair poly(A) tail to increase stability of message and for efficient in vitro translation. The segments of DNA encoding the C-terminal tails of hIKCa1(nucleotides 1252–1678; GenBankTM accession AF022797),hSKCa3 (nucleotides 1632–2193; GenBankTMaccession number AF031815) and mKv1.3 (nucleotides 1736–2112; GenBankTM accession number M30441) were subcloned into the PAGA2 vector using the polymerase chain reaction with engineered restriction sites. Both CAM clones were also subcloned into the PAGA2 vector. For co-precipitation and electrophysiology experiments (see below), the full-length hIKCa1(GenBankTM accession number AF033021) and mKv1.3coding regions were fused in-frame with a N-terminal His6 tag in the pcDNA3.1-His-C vector (Invitrogen, Carlsbad, CA). All clones were verified by sequencing. A 426-base pair fragment ofhIKCa1 coding for residues 286–427 in the cytoplasmic C-terminal tail of the channel was subcloned into the GAL4 DNA-binding vector (pAS2–1, CLONTECH, Palo Alto, CA) using polymerase chain reaction and engineered restriction sites. This construct was used as bait to screen an activated human leukocyte cDNA library (HL4021AB, CLONTECH). Screening procedures were performed according to the manufacturer's recommendations (CLONTECH PT3061-1). Several thousand putative positives were identified after first-round selection in growth medium; they were then subjected to the colony-liftlacZ assay. Positive blue colonies were sequenced using vector-specific primers. Two methods were used to test for CAM binding to the channel proteins. The initial deletion constructs ofhIKCa1 were generated by polymerase chain reaction as glutathione S-transferase (GST) fusions in the pGEX-6P-1 vector (Amersham Pharmacia Biotech), expressed in the Escherichia coli strain BL21-De3, and synthesis of the fusion proteins was induced with 0.1 mm isopropyl β-d-thiogalactoside in a liquid culture grown toA 600 of ∼1.0. After 2.5 h at 37 °C, cells were collected by centrifugation, resuspended in NETN lysis buffer (0.5% Nonidet P-40, 1 mm EDTA, 20 mmTris-HCl (pH 8.0), 100 mm NaCl; 1.0 ml per 20 ml of culture) containing protease inhibitor mixture (complete protease inhibitor mixture tablets, Boehringer Mannheim), and lysed by sonication. The lysate was cleared by centrifugation at 10,000 ×g for 10 min at 4 °C. GST fusion proteins in the supernatant were adsorbed for 30 min at room temperature to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) in NETN (1 volume of lysate:0.4 volume of 50% (v/v) slurry of Sepharose-GSH beads (Amersham Pharmacia Biotech) in NETN), which were then washed with binding buffer containing 1% (v/v) polyoxyethylene-9-lauryl ether, 100 mm NaCl, 20 mm Tris-HCl, pH 8.0. For experiments investigating Ca2+ dependence, the above buffer contained, in addition, 1 mm CaCl2 or 2 mm EDTA. Slurries (50% (v/v)) of the bound Sepharose-GSH beads (Sepharose-GSH:GST fusions) were then incubated for 30 min at room temperature in 50 μl of binding buffer containing [35S]methionine-labeled hCAM, synthesized by coupled transcription-translation (TnT, Promega, Madison, WI) as described (34Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (545) Google Scholar). The bound beads were washed three times with binding buffer and resuspended in 15 μl (three volumes) of 2× Laemmli's sample buffer. Proteins released from the beads by boiling in the presence of reducing reagent were analyzed by 4–20% gradient SDS-PAGE followed by autoradiography to detect retention of hCAM by the channel-GST fusion proteins. To ensure equivalent protein loading, gels were stained with colloidal blue (Novex, San Diego, CA) to visualize the major protein band in each lane prior to autoradiography. Binding of WT- and B1234Q-CAMs to the C-tail of hIKCa1 was also determined using the GST pull-down method as above. For all other experiments, channel constructs in the pAGA2 vector were radiolabeled with [35S]methionine during coupled transcription-translation using reagents from Promega. These constructs were incubated with CAM-Sepharose 4B beads (Amersham Pharmacia Biotech). Briefly, slurries of CAM beads (50% (v/v)) in binding buffer (as described above) were incubated with radiolabeled channel proteins that had been normalized for radioactive incorporation using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Equal cpms of each specific protein were added to 50 μl of binding buffer including either 2 mm EDTA or 1 mm Ca2+. Binding and washing conditions were the same as for the GST-Sepharose experiments above. Proteins released from the beads by boiling in the presence of reducing reagent were analyzed by 18% SDS-PAGE followed by autoradiography. mKv1.3 andhIKCa1 expression constructs in pcDNA-3.1His(C) were transfected into COS-7 cells using Fugene6 (Boehringer Manheim) according to the supplied protocol. About 40 h after transfection, 5 × 106 cells were lysed in 10 mm HEPES (pH 7.4), 40 mm KCl, 0.75 mm EDTA (free Ca2+ concentration, <1 nm), 1% Triton X-100, 10 mm β-mercaptoethanol, 0.25% deoxycholate, and protease inhibitors. After 20 min on ice, cells were Dounce-homogenized and centrifuged at 2900 × g for 15 min to remove insoluble material. The soluble lysate was transferred to a clean tube and mixed with an equal volume of 2× binding buffer (20 mmHEPES (pH 7.4), 200 mm KCl, 20% glycerol, 60 mm imidazole, 20 mm β-mercaptoethanol, and protease inhibitors). The diluted lysate containing the membrane fraction was incubated with Ni+-NTA resin (Qiagen, Valencia, CA) for ∼2 h at 4 °C in order to immobilize the His-tagged channel protein. After extensively washing the resin with wash buffer (10 mm HEPES (pH 7.4), 100 mm KCl, 10% glycerol, 0.25% Triton X-100, 30 mm imidazole, 0.2 mm EDTA, 10 mm β-mercaptoethanol, and protease inhibitors), the channel protein was eluted with elution buffer (same as wash buffer but containing 400 mmimidazole). Proteins from the elution fraction, as well as from the flow-through, were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. To determine whether CAM was preassociated with hIKCa1 or mKv1.3, a Western blot analysis was performed using an anti-CAM monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Rat basophilic leukemia (RBL) cells were maintained in a culture medium of Eagle's minimum essential medium (BIO-Whittaker, San Diego, CA) supplemented with 1 mml-glutamine (Sigma) and 10% heat-inactivated fetal calf serum (Summit Biotechnology, Fort Collins, CO) and grown in a humidified, 5% CO2 incubator at 37 °C. Cells were plated to grow nonconfluently on glass 1 day prior to use for cRNA injection and electrophysiological experiments. T-lymphocytes were isolated from human peripheral blood and activated with phytohemagglutinin (DIFCO, Detroit, MI) as described previously (20Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (221) Google Scholar). Prior to experimentation, cells were plated for 15 min on glass coverslips coated with poly-l-lysine (Sigma). For other experiments, we stably transfected the COS-7 cell line withhIKCa1; the biophysical properties of the hIKCa1channels in these cells are indistinguishable from those of IKCa channels in T-cells (data not shown). Plasmids containing the entire coding sequence of the hIKCa1 gene, WT-CAM, and B1234Q-CAM were linearized with NotI andin vitro transcribed with the T7 mMessage mMachine system (Ambion, Austin TX). Plasmids containing the mKv1.3 coding sequence were linearized with EcoRI and in vitrotranscribed with the Sp6 version of the same kit. The resulting cRNA was phenol/chloroform-purified and stored at −75 °C. RNA concentrations were determined to an accuracy of 25%, based on intensity of bands in agarose gel electrophoresis. The cRNA was diluted with fluorescein isothiocyanate-dextran (Sigma) (averageM r, 10,000; 0.1% in 100 mm KCl). RBL cells were injected with an Eppendorf (Hamburg, Germany) microinjection system (Micro-manipulator 5171 and Transjector 5246) using injection capillaries (Femtotips®, Eppendorf) filled with the cRNA/fluorescein isothiocyanate solution, as described previously (35Ikeda S.R. Soler F. Zuhlke R.D. Lewis D.L. Pfluegers Arch. Eur. J. Physiol. 1992; 422: 201-203Crossref PubMed Scopus (30) Google Scholar). Cells were visualized by fluorescence, and hIKCa1-specific currents were measured 4–8 h after injection. Cells measured in the whole cell configuration were normally bathed in normal Ringer solution containing 160 mm NaCl, 4.5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, 10 mm glucose; adjusted to pH 7.4 with NaOH, with an osmolarity of 290–320 mosm. In K+ Ringer solution, Na+ was replaced by K+. A simple syringe-driven perfusion system was used to exchange the bath solutions in the recording chamber. The internal pipette solution with 1 μm free Ca2+contained 145 mm K+ aspartate, 8.5 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, 10 mm K2 EGTA; adjusted to pH 7.2 with KOH, with an osmolarity of 290–310 mosm. EGTA was omitted in the high Ca2+ internal solution containing 1 mm CaCl2. Pipettes were pulled from glass capillaries, coated with Sylgard (Dow-Corning, Midland, MI), and fire-polished to resistances measured in the bath of 2–5 MΩ. Membrane currents were recorded with an EPC-9 patch-clamp amplifier (HEKA elektronik, Lambrecht, Germany) interfaced to a computer running acquisition and analysis software (Pulse and PulseFit; HEKA elektronik). Data were filtered at 1.5 kHz, and all voltages were corrected for a liquid junction potential offset of −13 mV for aspartate-based solutions. The holding potential in all experiments was −80 mV. For characterization of the hIKCa1 current, voltage ramp stimuli were used to assess channel activation by elevated [Ca2+]i. RBL cells express an endogenous inwardly rectifying K+ channel that did not interfere with KCa currents seen at depolarized potentials. Experiments were performed at room temperature (21–25 °C). The CAM antagonists W7, trifluoperazine (TFP), and calmidazolium were purchased from Calbiochem (La Jolla, CA). ChTX was obtained either from Peptides International, (Louisville, KY) or from BACHEM Biosciences (King of Prussia, PA). Clotrimazole was purchased from Sigma. All slope conductances reported in the text are written as mean ± S.E. (number of cells or experiments). We searched for accessory molecules that bind tohIKCa1 using the yeast two-hybrid system. We reasoned that if such a molecule is involved in Ca2+ sensing, it must be common to IKCa and SKCa channels, because their Ca2+ sensitivities and gating behavior are remarkably similar (10Sah P. Trends Neurosci. 1992; 19: 150-154Abstract Full Text PDF Scopus (805) Google Scholar, 13Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (788) Google Scholar, 20Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (221) Google Scholar, 22Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 24Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (510) Google Scholar). An amino acid alignment ofhIKCa1 with hSKCa1, rSKCa2, and hSKCa3revealed that except for the pore and the transmembrane regions, the proximal half of the cytoplasmic C-tail was the most highly conserved (Fig. 1); the C-tail of hIKCa1was therefore employed as the bait. We chose an activated human leukocyte cDNA library to screen for interaction partners becausehIKCa1 has been previously shown to be highly up-regulated in activated lymphocytes (20Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (221) Google Scholar, 22Logsdon N.J. Kang J. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). A primary screen using the triple nutrient selection (Trp+Leu+His+) resulted in the identification of several thousand positive clones. A subsequent subscreen of 500 colonies yielded nine clones that were positive for β-galactosidase activity. Seven of these clones encodedCAM. We next examined the ability of 35S-labeled in vitro translated hIKCa1-C-tail to bind CAM-Sepharose as assessed by running the product on an SDS-PAGE gel followed by autoradiography. As shown in Fig.2 A, a radiolabeled band of ∼28 kDa is visible (hIKCa1) consistent with the size of the hIKCa1 C-tail (Fig. 2 B), indicating thathIKCa1 and CAM interact. CAM also binds to the C-tail of the SKCa channel, hSKCa3 (Fig. 2 A, ∼22-kDa band (hSKCa3)), but not the C-tail of the voltage-gated K+ channel, mKv1.3 (Fig2 A, mKv1.3). As an additional specificity control, we performed GST pull-down experiments; CAM did not bind GST alone but interacted with the GST-hIKCa1 C-tail (Fig.3 A). Thus, CAM interacts specifically with members of the IKCa and SKCafamily.Figure 3Deletion analysis of hIKCa1C-tail. A, glutathione-Sepharose beads containing GST fusion constructs of either the entire C-tail or deletion fragments of hIKCa1 C-tail were incubated with 35S-labeled CAM in the presence of 1 mm Ca2+ (top panel) or in the presence of 2 mm EDTA and no added Ca2+ (bottom panel). Coomassie gels ensured equivalent loading of protein in all lanes (data not shown).B, deletion constructs 1–82, 1–72, and 1–62 were35S-labeled by transcription-translation and incubated with CAM-Sepharose beads in the presence of 1 mmCa2+ (left panel) or in buffers containing 2 mm EDTA and no added Ca2+ (right panel).View Large Image Figure ViewerDownload (PPT) Surprisingly, the C-tail fragments of hIKCa1 andhSKCa3 bound CAM efficiently in buffers containing 2 mm EDTA and no added Ca2+ (Fig. 2 A, hIKCa1 and mKv1.3, right panel). Deletion analysis revealed that the shorter 1–98 and 1–62 fragments of thehIKCa1 C-tail also bound CAM efficiently in Ca2+-free conditions (Fig. 3 A, bottom panel;Fig. 3 B, right panel). Four additional deletion fragments (45–98, 37–77, 1–72, and 1–82) bound CAM, but only in the presence of 1 mm Ca2+ (Fig. 3 A, top panel;Fig. 3 B, left panel), whereas two others (1–50 and 93–142) did not bind CAM at all (Fig. 3). Thus, CAM interacts with the C-tails of hIKCa1 and hSKCa3 in the absence of Ca2+, and this property resides in a domain within the first 62 residues of the hIKCa1 C-tail. The segment between residues 62 and 82 appears to mask the Ca2+-independent interaction of CAM with hIKCa1, because the 1–72 and 1–82 fragments bind CAM only in the presence of Ca2+, whereas residues 82–98 appear to reverse the negative effect of 62–82. Removal of as yet unidentified motifs between residues 1 and 37 appears to unmask a Ca2+-dependent interaction with CAM. Interestingly, the 1–98 segment of the hIKCa1-C-tail, which contains the Ca2+-independent and Ca2+-dependent modulatory domains, shares" @default.
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• W2108774434 date "1999-02-01" @default.
• W2108774434 modified "2023-10-17" @default.
• W2108774434 title "Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance KCa Channel,IKCa1" @default.
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• W2108774434 doi "https://doi.org/10.1074/jbc.274.9.5746" @default.
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