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• W1982591971 abstract "Large conductance, calcium- and voltage-activated potassium (BK) channels control excitability in many tissues and are regulated by several protein kinases and phosphatases that remain associated with the channels in cell-free patches of membrane. Here, we report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase (PKA) to associate with the channel and regulate its activity. A synthetic polypeptide encompassing the central d position leucine residues in LZ1 blocks the regulation of recombinant mouse BK channels by endogenous PKA in HEK293 cells. In contrast, neither an alanine-substituted LZ1 peptide nor a peptide corresponding to another, more C-terminal putative leucine zipper, LZ2, had any effect on regulation of the channels by endogenous PKA. Mutagenesis of the central two LZ1 d position leucines to alanine in the BK channel also eliminated regulation by endogenous PKA in HEK293 cells without altering the channel sensitivity to activation by voltage or by exogenous purified PKA. Inclusion of the STREX splice insert in the BK channel protein, which switches channel regulation by PKA from stimulation to inhibition, did not alter the requirement for an intact LZ1. Although PKA does not bind directly to the channel proteinin vitro, mutation of LZ1 abolished co-immunoprecipitation of PKA and the respective BK channel splice variant from HEK293 cells. Furthermore, a 127-amino acid fusion protein encompassing the functional LZ1 domain co-immunoprecipitates a PKA-signaling complex from rat brain. Thus LZ1 is required for the association and regulation of mammalian BK channels by PKA, and other putative leucine zippers in the BK channel protein may provide anchoring for other regulatory enzyme complexes. Large conductance, calcium- and voltage-activated potassium (BK) channels control excitability in many tissues and are regulated by several protein kinases and phosphatases that remain associated with the channels in cell-free patches of membrane. Here, we report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase (PKA) to associate with the channel and regulate its activity. A synthetic polypeptide encompassing the central d position leucine residues in LZ1 blocks the regulation of recombinant mouse BK channels by endogenous PKA in HEK293 cells. In contrast, neither an alanine-substituted LZ1 peptide nor a peptide corresponding to another, more C-terminal putative leucine zipper, LZ2, had any effect on regulation of the channels by endogenous PKA. Mutagenesis of the central two LZ1 d position leucines to alanine in the BK channel also eliminated regulation by endogenous PKA in HEK293 cells without altering the channel sensitivity to activation by voltage or by exogenous purified PKA. Inclusion of the STREX splice insert in the BK channel protein, which switches channel regulation by PKA from stimulation to inhibition, did not alter the requirement for an intact LZ1. Although PKA does not bind directly to the channel proteinin vitro, mutation of LZ1 abolished co-immunoprecipitation of PKA and the respective BK channel splice variant from HEK293 cells. Furthermore, a 127-amino acid fusion protein encompassing the functional LZ1 domain co-immunoprecipitates a PKA-signaling complex from rat brain. Thus LZ1 is required for the association and regulation of mammalian BK channels by PKA, and other putative leucine zippers in the BK channel protein may provide anchoring for other regulatory enzyme complexes. cAMP-dependent protein kinase catalytic subunit of PKA leucine zipper protein kinase A-anchoring protein hemagglutinin immuno- precipitation Reversible protein phosphorylation represents a fundamental cellular regulatory mechanism to control the activity and function of plasma membrane ion channels (1Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Google Scholar). The co-ordination, specificity, and compartmentalization of ion channel regulation by reversible protein phosphorylation is facilitated by assembly with signaling complexes comprising cognate protein kinases and protein phosphatases. Assembly of ion channels with signaling complexes typically results from multiple protein-protein interactions mediated by distinct interaction domains (2Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Google Scholar) allowing signaling molecules to interact directly with an ion channel or indirectly as part of a higher order complex. Large conductance calcium- and voltage-activated potassium (BK) channels have been widely exploited as models of ion channel regulation by reversible protein phosphorylation; however, the molecular basis for kinase and phosphatase assembly with the BK channel is largely unknown (1Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Google Scholar, 3Wang J. Zhou Y. Wen H. Levitan I. J. Neurosci. 1999; 19: 1-7Google Scholar). BK channels play a central role in the regulation of cellular excitability because they are activated directly by both voltage and intracellular free calcium (4Vergara C. Latorre R. Marrion N. Adelman J. Curr. Opin. Neurobiol. 1998; 8: 321-329Google Scholar, 5Toro L. Wallner M. Meera P. Tanaka Y. News Physiol. Sci. 1998; 13: 112-117Google Scholar, 6Shipston M.J. Trends Cell Biol. 2001; 11: 353-358Google Scholar) and potently modulated by reversible protein phosphorylation (1Levitan I.B. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 3-22Google Scholar). For example, they provide a dynamic link between electrical and chemical signaling events in cells, are major determinants of vascular smooth muscle tone (5Toro L. Wallner M. Meera P. Tanaka Y. News Physiol. Sci. 1998; 13: 112-117Google Scholar, 7Brenner R. Perez G.J. Bonev A.D. Eckman D.M. Kosek J.C. Wiler S.W. Patterson A.J. Nelson M.T. Aldrich R.W. Nature. 2000; 407: 870-876Google Scholar), and regulate action potential duration and frequency as well as neurotransmitter and hormone release in neurons and endocrine cells (6Shipston M.J. Trends Cell Biol. 2001; 11: 353-358Google Scholar, 8Sah P. Trends Neurosci. 1996; 19: 150-154Google Scholar). A single gene (KCNMA1) encodes for the pore-forming α-subunits of BK channels in all mammalian tissues (9Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Google Scholar). Phenotypic variation in native BK channels results from extensive alternative exon splicing of the α-subunit (6Shipston M.J. Trends Cell Biol. 2001; 11: 353-358Google Scholar, 9Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Google Scholar, 10Xie J. McCobb D.P. Science. 1998; 280: 443-446Google Scholar) as well as through interaction with regulatory β-subunits and accessory proteins (11McManus O.B. Helms L.M.H. Pallanck L. Ganetzky B. Swanson R. Leonard R.J. Neuron. 1995; 14: 645-650Google Scholar, 12Schopperle W. Holmqvist M. Zhou Y. Wang J. Wang Z. Griffith L. Keselman I. Kusinitz F. Dagan D. Levitan I. Neuron. 1998; 20: 565-573Google Scholar, 13Xia X. Hirschberg B. Smolik S. Forte M. Adelman J. J. Neurosci. 1998; 18: 2360-2369Google Scholar). The BK channel α-subunit is a target for regulation by multiple protein kinases and protein phosphatases (3Wang J. Zhou Y. Wen H. Levitan I. J. Neurosci. 1999; 19: 1-7Google Scholar, 14Alioua A. Tanaka Y. Wallner M. Hofmann F. Ruth P. Meera P. Toro L. J. Biol. Chem. 1998; 273: 32950-32956Google Scholar, 15Reinhart P.H. Levitan I.B. J. Neurosci. 1995; 15: 4572-4579Google Scholar, 16Ling S.Z. Woronuk G. Sy L. Lev S. Braun A.P. J. Biol. Chem. 2000; 275: 30683-30689Google Scholar, 17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar, 18White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Google Scholar), and several protein kinases have been reported to co-immunoprecipitate with mammalian BK channels (3Wang J. Zhou Y. Wen H. Levitan I. J. Neurosci. 1999; 19: 1-7Google Scholar, 16Ling S.Z. Woronuk G. Sy L. Lev S. Braun A.P. J. Biol. Chem. 2000; 275: 30683-30689Google Scholar,17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). Although several consensus phosphorylation sites have been identified by mutagenesis within the intracellular C-terminal domain, BK channel α-subunits do not contain previously identified protein-protein interaction domains. Thus, the molecular basis for protein kinase or phosphatase targeting to mammalian BK channels is essentially unknown. In Drosophila, the catalytic subunit, but not the holoenzyme, of cAMP-dependent protein kinase (PKA)1 binds directly to the intracellular C terminus (3Wang J. Zhou Y. Wen H. Levitan I. J. Neurosci. 1999; 19: 1-7Google Scholar, 19Zhou Y. Wang J. Wen H. Kucherovsky O. Levitan I.B. J. Neurosci. 2002; 22: 3855-3863Google Scholar) of the channel. Although PKA co-immunoprecipitates with mammalian BK channels in a splice variant-independent manner, the mechanism of complex assembly is unknown (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). Recently a structural motif, the leucine zipper (LZ), originally described in classes of DNA-binding proteins (20Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Google Scholar), has been reported to play an important role in coordinating both the assembly of ion channels as well as their interaction with protein kinase and protein phosphatase signaling complexes (21Hulme J.T. Ahn M. Hauschka S.D. Scheuer T. Catterall W.A. J. Biol. Chem. 2002; 277: 4079-4087Google Scholar, 22Marx S.O. Reiken S. Hisamatsu Y. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Marks A.R. J. Cell Biol. 2001; 153: 699-708Google Scholar, 23Marx S.O. Kurokawa J. Reiken S. Motoike H. D'Armiento J. Marks A.R. Kass R.S. Science. 2002; 295: 496-499Google Scholar, 24Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Google Scholar). In several of these channels the catalytic subunit of PKA (PKAc) is targeted to the channel through a protein kinase A-anchoring protein (AKAP). The AKAP acts as an adapter protein by binding to both the LZ domain of the channel and the regulatory subunits of PKA. In this report we identify a putative LZ domain for BK channel assembly with a PKA signaling complex essential for the functional regulation of mammalian BK channels by PKA-dependent phosphorylation. The cloning and sub-cloning of the mouse BK channel splice variants ZERO and STREX into the mammalian expression vector pcDNA3 or pcDNA3.1+ (Invitrogen) have been described previously (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar, 25Shipston M.J. Duncan R.R. Clark A.G. Antoni F.A. Tian L.J. Mol. Endocrinol. 1999; 13: 1728-1737Google Scholar). A C-terminal hemagglutinin (HA) tag was introduced into each channel construct by replacing the normal stop codon with a sequence encoding the HA tag from a mouse BK channel construct kindly provided by Dr. Yi Zhou and Prof. Irwin B Levitan. The C terminus is thus identical to the mouse mbr5 (GenBankTM accession number GI:347144) sequence present in the original clones apart from the HA tag (REVEDECYPYDVPDYA*), where the italicized amino acids indicate HA-tag amino acids replacing the normal stop codon. Amino acid numbering in the subsequent text and figures is in accordance with the amino acid sequence of the mouse mbr5 clone (accession number: GI:347144; the start methionine being M1ELEH) for consistency. Alanine substitutions in the third and fourth LZ1 d position leucine residues (Fig. 1: amino acids Leu-530 and Leu-537) was performed by site-directed mutagenesis using a single mutagenic primer set with the QuikChange system according to the manufacturer (Stratagene, La Jolla, CA) to generate the HA-tagged LZ1 mutant channels ZEROL530A/L537A and STREXL530A/L537A. Thioredoxin fusion proteins were generated by PCR and subcloning fragments into the pBAD/Thio-TOPO vector (Invitrogen). Fusion proteins were constructed with an N-terminal His-patch (HP)-thioredoxin fusion and C-terminal V5 and hexahistidine epitopes to facilitate purification and immunoprecipitation. All soluble fusion proteins were induced and purified from BL21 RIL Escherichia coli using standard methods. All immunoprecipitations using thioredoxin fusion proteins were performed with the intact HP-thioredoxin fusion, because cleavage of the thioredoxin fusion resulted in proteins that were largely insoluble. The LZ1489–616 thioredoxin fusion protein was designed to span the LZ1 domain from the end of the predicted regulator of potassium conductance (RCK) domain βE strand (26Jiang Y.X. Lee A. Chen J.Y. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Google Scholar, 27Jiang Y.X. Pico A. Cadene M. Chait B.T. MacKinnon R. Neuron. 2001; 29: 593-601Google Scholar, 28Quirk J.C. Reinhart P.H. Neuron. 2001; 32: 13-23Google Scholar) starting at amino acid Gln-489 and extending to amino acid Ile-616 (Fig.5 a). LZ1489–616 thus does not include core RCK domain residues essential for tetramerization (28Quirk J.C. Reinhart P.H. Neuron. 2001; 32: 13-23Google Scholar). The ZERO580–984 and STREX580–984 thioredoxin fusion proteins were designed to span LZ2 and to include the mammalian STREX alternative site of splicing as well as the conserved PKA consensus site at S899 (Fig. 5 a). The starting amino acid in the respective construct was Val-580 and terminating in amino acid Ala-984. ZERO580–984 and STREX580–984 are, thus, identical apart from the addition of 59 amino acids of the mouse STREX (25Shipston M.J. Duncan R.R. Clark A.G. Antoni F.A. Tian L.J. Mol. Endocrinol. 1999; 13: 1728-1737Google Scholar) insert in STREX 580–984 (Fig. 5 a: amino acid numbering has been retained as for ZERO for consistency) and do not contain LZ1. All constructs were verified by DNA sequencing. Leucine zipper competing peptides were synthesized by Genemed Synthesis (South San Francisco, CA) and used at a final bath concentration of 25–80 μm. Concentrations of peptide <5 μm were largely ineffective (data not shown). The LZ1 peptide (D QSCLAQGLSTMLANLFS) corresponds to amino acids 523–539, spanning the 2nd, 3rd, and 4thd positions in the mouse 5-heptad repeat LZ1 motif (Fig.1 a) except that an N-terminal aspartate (D) residue was included to improve water solubility. The corresponding alanine substituted peptide (Ala-LZ1) was identical except that the 2nd, 3rd, and 4th LZ1 heptad repeat d residues were replaced by alanine (D ASCLAQGASTMLANAFS). The LZ2 peptide (DLRAVNINLCDMCVILS) corresponds to conserved residues 818–834 within a putative C-terminal 4-heptad repeat LZ motif in the mouse BK channel (Fig.1 a). HEK293 cells were subcultured essentially as previously described (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar, 25Shipston M.J. Duncan R.R. Clark A.G. Antoni F.A. Tian L.J. Mol. Endocrinol. 1999; 13: 1728-1737Google Scholar) except with one modification whereby cells were placed in serum-free (ITS, Invitrogen) medium 24 h before experiments. Briefly, cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. Cells were routinely passaged every 3–7 days using 0.25% trypsin in Hanks' buffered salt solution containing 0.1% EDTA. For immunoblotting studies cells were grown to 70–80% confluence in 75-cm2 flasks. For electrophysiological assays cells were plated on glass coverslips in 6-well cluster dishes. Twenty-four hours before the experiment cells were washed, and medium was replaced with Dulbecco's modified Eagle's medium containing ITS serum replacement (Invitrogen). For transient transfections of BK channels HEK293 cells were seeded onto glass coverslips in 6-well cluster dishes at a density to allow cells reaching 40–60% confluence after 24 h. Cells were then transfected with 1 μg of the respective cDNA using LipofectAMINE (Invitrogen) in Dulbecco's modified Eagle's medium essentially as described by the manufacturer. After 5 h medium was supplemented with 10% fetal calf serum, which was replaced after 24 h, and electrophysiological recordings made 24–72 h post-transfection. Stable cell lines were also created by selection and maintenance for Zeocin or Geneticin resistance using 0.2 mg ml−1 Zeocin (Invitrogen) or 0.8 mg ml−1Geneticin (Invitrogen) as appropriate. Immunoprecipitation (IP) of HA-tagged channels or PKA was performed using transient or stably expressing HEK293 cell lines essentially as previously described (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). Briefly, cells were solubilized in radioimmunoassay buffer containing 50 mm Tris-HCl, pH 7.5, 0.5 mmMgCl2, 0.2 m NaCl, 10 mmEDTA, 20 mm sodium pyrophosphate, 100 mm sodium fluoride, 1 mg/ml bovine serum albumin, 1% (v/v) Triton X-100, and protease inhibitors (Roche Molecular Biochemicals). Insoluble material was removed by centrifugation (10,000 × g, 15 min, 4 °C), and the lysate was pre-cleared by incubation with 20 μl of 50% (v/v) protein G-Sepharose with agitation for 1 h at 4 °C. PKA- or HA-tagged channels were immunoprecipitated from the cleared lysate with the respective antibody (prebound to 40 μl of 50% (v/v) protein G-Sepharose) for 4 h at 4 °C. IP antibodies used were mouse anti-HA monoclonal antibody (clone: 12CA5, Roche Diagnostics) for HA-tagged channels, a sheep anti-PKAc polyclonal antibody (ab365 (29Aspbury R. Fisher M. Rees H. Clegg R. Biochem. Biophys. Res. Commun. 1997; 238: 523-527Google Scholar), a generous gift from Dr Roger A Clegg), or a rabbit anti-PKAc polyclonal antibody (Santa Cruz Biotechnology Inc.). The immunoprecipitate was washed 5 times with 1 ml of radioimmunoassay buffer before SDS-PAGE analysis. For Western blot detection a rabbit anti-HA polyclonal (Y-11, Santa Cruz Biotechnology, Inc.) and the above sheep anti-PKAc polyclonal were used as described in figure legends (Figs. 4 and 5). IP of thioredoxin fusion proteins of the BK channel intracellular C terminus employed a mouse monoclonal anti-V5 antibody (Invitrogen). Detection was by enhanced chemiluminescence. All experiments were performed in the inside-out configuration of the patch clamp technique at room temperature (20–24 °C) using physiological potassium gradients essentially as described previously (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). The pipette solution (extracellular) contained 140 mm NaCl, 5 mm KCl, 0.1 mm CaCl2, 2 mm MgCl2, 20 mm glucose, 10 mm HEPES, pH 7.4. The bath solution (intracellular) contained 140 mm KCl, 5 mm NaCl, 2 mm MgCl2, 1 or 5 mm BAPTA, 30 mm glucose, 10 mm HEPES, 1 mm ATP, pH 7.3, with free calcium [Ca2+]i buffered to 0.2 μm unless indicated otherwise. For assays in which the catalytic subunit of PKA (PKAc, Promega, Madison, WI) was applied directly to patches, patches were exposed to the above intracellular bathing solution containing 0.1 mm dithiothreitol during control and PKAc application to exclude effects dues to dithiothreitol (30Erxleben C. Everhart A.L. Romeo C. Florance H. Bauer M.B. Alcorta D.A. Rossie S. Shipston M.J. Armstrong D.L. J. Biol. Chem. 2002; 277: 27045-27052Google Scholar) present in purified PKAc preparations. Data acquisition and voltage protocols were controlled by an Axopatch 200 A or B amplifier and pCLAMP6 software (Axon Instruments Inc., Foster City, CA). All recordings were sampled at 10 kHz and filtered at 2 kHz. After patch excision channel activity was allowed to stabilize for at least 10 min (typically 10–15 min after excision), and stability plot experiments demonstrated that BK channel activity was stable for >1 h under the recording conditions used (data not shown) in the absence of channel modulators. Application of cAMP or other reagents to the intracellular face of patches was by gravity-driven perfusion (10 volumes of the recording bath solution (bath volume, 0.5 ml) by gravity-driven perfusion at a flow rate of 1–2 ml/min) or direct application to the bath. Channel activity was determined during 30-s depolarizations to +40 mV. Single-channel open probability (P o) was derived either from single-channel analysis using pSTAT for patches with <4 channels or, in the case of patches with >4 channels, by an integration-over-baseline algorithm using Igor Pro 4.1 (WaveMetrics, Lake Oswego, OR). In the latter case N P o (number of functional channels × open probability of channel) values were determined as follows. All-point histograms were plotted to obtain the “offset,” i.e. leak current, as well as the single-channel current amplitude from the peak intervals. After subtraction of the offset from the traces these were integrated over 0.5–60-s segments. The integral divided by integration time and single-channel current amplitude gives NP o. To determine mean percent (%) change in channel activity after a treatment in patches with low to moderate levels of channel expression, mean P o or N × P o was averaged from several minutes of recording at +40 mV immediately before and 10 min after the respective drug treatment. Mean change in activity was expressed as a percentage (%) of the pretreatment control ±S.E. In the respective figure legends and text (see “Results”) a positive percentage (%) change in activity reflects activation, whereas a negative percentage (%) change reflects channel inhibition. The amino acid sequence of LZ motifs is typically characterized by a seven-residue (heptad) repeat (commonly denoted abcdefg, see Fig.1, a and b) with positions a and d in each heptad repeat occupied by hydrophobic residues (20Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Google Scholar). Leucine provides the most thermodynamically stable residue at position d (32Tripet B. Wagschal K. Lavigne P. Mant C.T. Hodges R.S. J. Mol. Biol. 2000; 300: 377-402Google Scholar). However, significant deviations in amino acid sequence from this “classical” leucine zipper motif may exist, for example, deletions or insertions of individual residues within the heptad repeat or the presence of polar residues at position a or d(22Marx S.O. Reiken S. Hisamatsu Y. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Marks A.R. J. Cell Biol. 2001; 153: 699-708Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Google Scholar, 32Tripet B. Wagschal K. Lavigne P. Mant C.T. Hodges R.S. J. Mol. Biol. 2000; 300: 377-402Google Scholar, 34Brown J.H. Cohen C. Parry D.A.D. Proteins. 1996; 26: 134-145Google Scholar). Allowing for such variability, manual inspection of the C-terminal amino acid sequence of mammalian BK channels revealed several putative LZ motifs. A C-terminal LZ motif (LZ1, Fig. 1, a andb, between residues 513 and 548 of the murine BK channel variant mbr5 (accession number GI:347144 (9Butler A. Tsunoda S. McCobb D.P. Wei A. Salkoff L. Science. 1993; 261: 221-224Google Scholar)) contains five heptad repeats downstream of residues that contribute to the proposed “fixed interface” of the BK channel regulator of K channel conductance (RCK, or tetramerization) domain (26Jiang Y.X. Lee A. Chen J.Y. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Google Scholar, 27Jiang Y.X. Pico A. Cadene M. Chait B.T. MacKinnon R. Neuron. 2001; 29: 593-601Google Scholar, 28Quirk J.C. Reinhart P.H. Neuron. 2001; 32: 13-23Google Scholar). Although the second dresidue of the five-heptad LZ1 repeat is glutamine (Q) and the fiftha residue is non-hydrophobic, the stability of “prototypical” LZ domains with a Gln (Q) residue at a singled position is, paradoxically, not significantly compromised compared with isoleucine or valine substitutions (32Tripet B. Wagschal K. Lavigne P. Mant C.T. Hodges R.S. J. Mol. Biol. 2000; 300: 377-402Google Scholar). Comparison of the amino acid sequence of mammalian BK channels with the structure of the RCK domain in calcium-activated potassium (MthK) channels fromMethanobacterium thermoautotrophicum suggests that LZ1 forms the αG helix and also contributes to an extended linker region between the αG helix and βG strand (26Jiang Y.X. Lee A. Chen J.Y. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Google Scholar, 27Jiang Y.X. Pico A. Cadene M. Chait B.T. MacKinnon R. Neuron. 2001; 29: 593-601Google Scholar, 28Quirk J.C. Reinhart P.H. Neuron. 2001; 32: 13-23Google Scholar). However, in MthK the extended linker is absent, and the mammalian LZ1 heptad repeats are not conserved in MthK or other prokaryotic RCK domains (Fig.1 a), suggesting LZ1 and the linker region play an additional role in mammalian BK channels. A second putative four-heptad repeat LZ domain (LZ2: residues 816–843, Fig. 1 a) is positioned between splice site 2 and the conserved PKA consensus site at serine residue Ser-899 (see Fig.1 a). At least two further three-heptad repeats may also be present in the C terminus. To investigate whether LZ1 or LZ2 plays a functional role in targeting PKA to BK channels we examined the regulation of two distinct murine BK channel splice variants, STREX and ZERO, that are differentially regulated by PKA-dependent phosphorylation when expressed in HEK293 cells (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). Two functional strategies were exploited, first, by designing competitive peptide inhibitors of LZ1 and LZ2 interactions in an attempt to disrupt PKA regulation of STREX and ZERO channels, respectively, and second, by investigating PKA-dependent regulation of STREX and ZERO channels in which candidate LZ1 motifs identified in the peptide inhibitor screen were disrupted by mutating the third and fourth d position leucine residues (Leu-530 and Leu-537) to alanine. Because the LZ1 domain is conserved in mammalian BK channels and distinct BK channel splice variants may be differentially regulated by PKA (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar), we addressed whether the LZ1 motif was required for regulation of distinct splice variants by endogenous PKA. To assay for regulation of BK channel splice variants by endogenous PKA we applied cAMP to the intracellular face of isolated inside-out patches from HEK293 cells to activate PKA closely associated with the channel as previously reported (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). The mouse ZERO variant of BK is activated by PKA closely associated with the channel and dependent upon a C-terminal serine residue (Ser-899) conserved in all mammalian BK channel splice variants (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar, 25Shipston M.J. Duncan R.R. Clark A.G. Antoni F.A. Tian L.J. Mol. Endocrinol. 1999; 13: 1728-1737Google Scholar). C-terminal HA-tagged ZERO channels were stimulated upon application of cAMP to the intracellular face of inside-out patches from HEK293 cells only in the presence of Mg-ATP (48.6 ± 8.0%, n = 8, Fig.2) in a similar fashion to untagged channels (17Tian L. Duncan R.R. Hammond S.L. Coghill L.C. Wen H. Rusinova R. Clark A.G. Levitan I.B. Shipston M.J. J. Biol. Chem. 2001; 276: 1717-1720Google Scholar). cAMP activation of ZERO channel activity was not observed in the presence of 25–80 μm LZ1 competing peptide (mean change in activity was −5.1 ± 14.7%,n = 8, Fig. 2). LZ1-competing peptide also blocked cAMP stimulation of the untagged channels (the mean change in activity was −1.9 ± 3.6%, n = 3). However, cAMP regulation of ZERO channels was unaffected by the alanine-substituted LZ1-competing peptide (the mean activation was 56.8 ± 14.6%,n = 4) or LZ2-competing peptide (the mean activation was 57.9 ± 8.9%, n = 4). Although the effect of the LZ1 peptide is specific, relatively high concentrations of peptide are required, LZ1 peptide concentrations <5 μm were largely ineffective (not shown). Thus, to confirm the requirement for a LZ1 domain in ZERO channel regulation by endogenous PKA, we mutated the third and fourth d position LZ1 leucine residues to alanine (ZEROL530A/L537A). This mutation had no significant effect on the half-maximal voltage required for channel activation (V 0.5) under the assay conditions;V 0.5 for the respective HA-tagged channels in the presence of 0.2 μm free calcium and Mg-ATP were 86 ± 7mV (n = 4) (ZERO) and 90 ± 10 mV (n = 3) (ZEROL530A/L537A). Importantly, ZEROL530A/L537A channels were unaffected by application of cAMP to their intracellular face (mean change in activity was 2.5 ± 5.6%, n = 6) (Fig. 2 b). To address whether mutation of the LZ1 motif prevents transduction of the effect of PKA-mediated phosphorylation of the mutant channel rather than preventing interaction of PKA with the channel, we applied the purified catalytic subunit of PKA (PKAc) to the intracellular face of patches containing ZEROL530A/L537A channels. Application of PKAc resulted in a 49.3 ±" @default.
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