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• W1988049420 abstract "Small conductance calcium-activated potassium channels (SK, KCa) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1–S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity. Small conductance calcium-activated potassium channels (SK, KCa) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1–S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity. Ca2+-activated K+ channels (KCa) 3The abbreviations used are: KCa,Ca2+-activated potassium channel; CI, confidence interval; HEK293 cells, human embryonic kidney 293 cells; SK, small conductance Ca2+-activated potassium channel; rSK, rat SK1; TEA, tetraethylammonium; hSK1, human SK1. are activated by rises in intracellular Ca2+. The KCa potassium channel family includes at least three subfamilies, KCa1–3 (1Wei A.D. Gutman G.A. Aldrich R. Chandy K.G. Grissmer S. Wulff H. Pharmacol. Rev. 2005; 57: 463-472Crossref PubMed Scopus (305) Google Scholar). Channels containing the KCa1.1 α-subunit (BK channels) have large single channel conductance and are maximally activated by micromolar concentrations of intracellular free calcium and concurrent depolarization (2Hille B. Ion Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001: 143-147Google Scholar). Their kinetic and pharmacological properties are modified upon assembly with membrane standing β-subunits (3Orio P. Rojas P. Ferreira G. Latorre R. News Physiol. Sci. 2002; 17: 156-161PubMed Google Scholar). The KCa2 subfamily of small conductance Ca2+-activated K+ channels, also known as SK channels, has three closely related members SK1 (KCa2.1), SK2 (KCa2.2), and SK3 (KCa2.3), which are characterized by a small single channel conductance. The IK channel (KCa3.1) shows an intermediate single channel conductance. Both SK and IK channels are voltage-independent and activated by submicromolar concentrations of intracellular free Ca2+. The gating of SK and IK channels is induced upon Ca2+ binding to calmodulin, which is constitutively bound to each channel subunit. Ca2+ binding to calmodulin induces a conformational change, which leads to the opening of these channels (4Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 5Xia 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 (740) Google Scholar, 6Schumacher M.A. Rivard A.F. Bachinger H.P. Adelman J.P. Nature. 2001; 410: 1120-1124Crossref PubMed Scopus (501) Google Scholar). SK channels, predominantly of the SK2 type, have been identified in sensory systems such as the retina (7Klocker N. Oliver D. Ruppersberg J.P. Knaus H.G. Fakler B. Mol. Cell. Neurosci. 2001; 17: 514-520Crossref PubMed Scopus (23) Google Scholar) and the cochlear inner and outer hair cells (8Glowatzki E. Fuchs P.A. Science. 2000; 288: 2366-2368Crossref PubMed Scopus (242) Google Scholar, 9Oliver D. Klocker N. Schuck J. Baukrowitz T. Ruppersberg J.P. Fakler B. Neuron. 2000; 26: 595-601Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). SK channels have also been described in heart (10Schetz J.A. Anderson P.A. Cardiovasc. Res. 1995; 30: 755-762Crossref PubMed Scopus (7) Google Scholar, 11Xu Y. Tuteja D. Zhang Z. Xu D. Zhang Y. Rodriguez J. Nie L. Tuxson H.R. Young J.N. Glatter K.A. Vazquez A.E. Yamoah E.N. Chiamvimonvat N. J. Biol. Chem. 2003; 278: 49085-49094Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), liver (12Barfod E.T. Moore A.L. Lidofsky S.D. Am. J. Physiol. 2001; 280: C836-C842Crossref PubMed Google Scholar, 13Capiod T. Ogden D.C. J. Physiol. (Lond.). 1989; 409: 285-295Crossref Scopus (68) Google Scholar), skeletal muscle (14Hugues M. Schmid H. Romey G. Duval D. Frelin C. Lazdunski M. EMBO J. 1982; 1: 1039-1042Crossref PubMed Scopus (77) Google Scholar, 15Pribnow D. Johnson-Pais T. Bond C.T. Keen J. Johnson R.A. Janowsky A. Silvia C. Thayer M. Maylie J. Adelman J.P. Muscle Nerve. 1999; 22: 742-750Crossref PubMed Scopus (35) Google Scholar), and visceral smooth muscle (16Banks B.E. Brown C. Burgess G.M. Burnstock G. Claret M. Cocks T.M. Jenkinson D.H. Nature. 1979; 282: 415-417Crossref PubMed Scopus (284) Google Scholar), including the urinary bladder (17Herrera G.M. Nelson M.T. J. Physiol. (Lond.). 2002; 541: 483-492Crossref Scopus (107) Google Scholar). All three SK channels are expressed in the brain and show a differential distribution (18Stocker M. Pedarzani P. Mol. Cell. Neurosci. 2000; 15: 476-493Crossref PubMed Scopus (316) Google Scholar). The SK channel-mediated current (IAHP) has been studied in detail in various regions of the central nervous system (19Bond C.T. Maylie J. Adelman J.P. Curr. Opin. Neurobiol. 2005; 15: 305-311Crossref PubMed Scopus (142) Google Scholar, 20Sah P. Trends Neurosci. 1996; 19: 150-154Abstract Full Text PDF PubMed Scopus (808) Google Scholar, 21Stocker M. Nat. Rev. Neurosci. 2004; 5: 758-770Crossref PubMed Scopus (417) Google Scholar). The IAHP regulates membrane excitability, increases the precision of neuronal firing (22Schwindt P.C. Spain W.J. Foehring R.C. Stafstrom C.E. Chubb M.C. Crill W.E. J. Neurophysiol. 1988; 59: 424-449Crossref PubMed Scopus (286) Google Scholar, 23Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (336) Google Scholar, 24Pedarzani P. Kulik A. Muller M. Ballanyi K. Stocker M. J. Physiol. (Lond.). 2000; 527: 283-290Crossref Scopus (78) Google Scholar, 25Wolfart J. Neuhoff H. Franz O. Roeper J. J. Neurosci. 2001; 21: 3443-3456Crossref PubMed Google Scholar, 26Cingolani L.A. Gymnopoulos M. Boccaccio A. Stocker M. Pedarzani P. J. Neurosci. 2002; 22: 4456-4467Crossref PubMed Google Scholar, 27Edgerton J.R. Reinhart P.H. J. Physiol. (Lond.). 2003; 548: 53-69Crossref Scopus (195) Google Scholar, 28Hallworth N.E. Wilson C.J. Bevan M.D. J. Neurosci. 2003; 23: 7525-7542Crossref PubMed Google Scholar, 29Womack M.D. Khodakhah K. J. Neurosci. 2003; 23: 2600-2607Crossref PubMed Google Scholar, 30Bond C.T. Herson P.S. Strassmaier T. Hammond R. Stackman R. Maylie J. Adelman J.P. J. Neurosci. 2004; 24: 5301-5306Crossref PubMed Scopus (228) Google Scholar), and modulates synaptic plasticity by regulating excitatory synaptic transmission in the amygdala (31Faber E.S. Delaney A.J. Sah P. Nat. Neurosci. 2005; 8: 635-641Crossref PubMed Scopus (200) Google Scholar) and the hippocampus (32Ngo-Anh T.J. Bloodgood B.L. Lin M. Sabatini B.L. Maylie J. Adelman J.P. Nat. Neurosci. 2005; 8: 642-649Crossref PubMed Scopus (363) Google Scholar). Inhibition of SK channels facilitates hippocampal independent (33Fournier C. Kourrich S. Soumireu-Mourat B. Mourre C. Behav. Brain Res. 2001; 121: 81-93Crossref PubMed Scopus (43) Google Scholar, 34Messier C. Mourre C. Bontempi B. Sif J. Lazdunski M. Destrade C. Brain Res. 1991; 551: 322-326Crossref PubMed Scopus (110) Google Scholar) as well as dependent (35Stackman R.W. Hammond R.S. Linardatos E. Gerlach A. Maylie J. Adelman J.P. Tzounopoulos T. J. Neurosci. 2002; 22: 10163-10171Crossref PubMed Google Scholar) learning and improves memory performance (33Fournier C. Kourrich S. Soumireu-Mourat B. Mourre C. Behav. Brain Res. 2001; 121: 81-93Crossref PubMed Scopus (43) Google Scholar, 34Messier C. Mourre C. Bontempi B. Sif J. Lazdunski M. Destrade C. Brain Res. 1991; 551: 322-326Crossref PubMed Scopus (110) Google Scholar, 36Hammond R.S. Bond C.T. Strassmaier T. Ngo-Anh T.J. Adelman J.P. Maylie J. Stackman R.W. J. Neurosci. 2006; 26: 1844-1853Crossref PubMed Scopus (175) Google Scholar, 37Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar). The unequivocal, molecular correlation between native KCa currents with their corresponding channel subunit(s) was possible because of the availability of highly specific blockers, in particular peptide toxins, which selectively inhibit these channels (37Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar, 38Liegeois J.F. Mercier F. Graulich A. Graulich-Lorge F. Scuvee-Moreau J. Seutin V. Curr. Med. Chem. 2003; 10: 625-647Crossref PubMed Scopus (78) Google Scholar). The 18-amino acid bee venom toxin apamin has proven to be extremely valuable as a specific blocker for which the only known receptors are the SK channels. Besides apamin, the scorpion toxins scyllatoxin (39Shakkottai V.G. Regaya I. Wulff H. Fajloun Z. Tomita H. Fathallah M. Cahalan M.D. Gargus J.J. Sabatier J.M. Chandy K.G. J. Biol. Chem. 2001; 276: 43145-43151Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), P05 (39Shakkottai V.G. Regaya I. Wulff H. Fajloun Z. Tomita H. Fathallah M. Cahalan M.D. Gargus J.J. Sabatier J.M. Chandy K.G. J. Biol. Chem. 2001; 276: 43145-43151Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and tamapin (40Pedarzani P. D'Hoedt D. Doorty K.B. Wadsworth J.D. Joseph J.S. Jeyaseelan K. Kini R.M. Gadre S.V. Sapatnekar S.M. Stocker M. Strong P.N. J. Biol. Chem. 2002; 277: 46101-46109Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) have the ability to inhibit SK-mediated currents, whereas other scorpion toxins (maurotoxin, Pi1, P01, and Tsκ) compete with 125I-apamin binding (reviewed in Ref. 37Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar) but do not inhibit SK2 or SK3 channels (39Shakkottai V.G. Regaya I. Wulff H. Fajloun Z. Tomita H. Fathallah M. Cahalan M.D. Gargus J.J. Sabatier J.M. Chandy K.G. J. Biol. Chem. 2001; 276: 43145-43151Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The rat SK1 (rSK1) subunits, unlike the human SK1 (hSK1), do not form functional homomeric SK channels (41Benton D.C. Monaghan A.S. Hosseini R. Bahia P.K. Haylett D.G. Moss G.W. J. Physiol. (Lond.). 2003; 553: 13-19Crossref Scopus (62) Google Scholar, 42D'Hoedt D. Hirzel K. Pedarzani P. Stocker M. J. Biol. Chem. 2004; 279: 12088-12092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) but form heteromeric channels together with the SK2 subunit in heterologous expression systems (41Benton D.C. Monaghan A.S. Hosseini R. Bahia P.K. Haylett D.G. Moss G.W. J. Physiol. (Lond.). 2003; 553: 13-19Crossref Scopus (62) Google Scholar). The construction of chimeric rSK1 channels, including the amino- and/or carboxyl-terminal regions of rSK2 or hSK1, enabled the expression of homomeric rSK1-like channels displaying more than a 25-fold reduction in apamin sensitivity compared with hSK1 (42D'Hoedt D. Hirzel K. Pedarzani P. Stocker M. J. Biol. Chem. 2004; 279: 12088-12092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This finding was rather surprising, as hSK1 and rSK1 share an identical primary sequence within the pore region, where three amino acids have been identified as determinants of the apamin sensitivity (43Ishii T.M. Maylie J. Adelman J.P. J. Biol. Chem. 1997; 272: 23195-23200Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In this study, to test the hypothesis that amino acids outside the pore region contribute to apamin binding, chimera were generated to identify new regions in the SK channels involved in determining sensitivity to apamin. The introduction of point mutations and their analysis revealed the existence of an amino acid beyond the pore region that crucially influences SK channel sensitivity for apamin. This finding provides a new insight into the molecular determinants for the apamin receptor and extends our understanding of how the difference in sensitivity of the different SK channel subunits is generated. Furthermore, to our knowledge this is the first description of a toxin that inhibits potassium channels by interacting simultaneously with amino acids inside and outside the pore region. A detailed understanding of the structural basis of the differences in the pharmacological profile of SK channel subtypes is essential for the rational development of subunit-specific inhibitors. DNA Constructs—The coding regions of hSK1, rSK1, and rSK2 (GenBank™ accession numbers NM_002248, NM_019313, and NM_019314) were cloned into pcDNA3 or pcDNA5/FRT (40Pedarzani P. D'Hoedt D. Doorty K.B. Wadsworth J.D. Joseph J.S. Jeyaseelan K. Kini R.M. Gadre S.V. Sapatnekar S.M. Stocker M. Strong P.N. J. Biol. Chem. 2002; 277: 46101-46109Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 42D'Hoedt D. Hirzel K. Pedarzani P. Stocker M. J. Biol. Chem. 2004; 279: 12088-12092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Point mutations and chimera were generated by splicing of overlap extension (44Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar). The range of amino acids combined is given in plain numbers for residues corresponding to rSK1, boldface numbers for residues corresponding to hSK1, and underlined numbers for residues corresponding to rSK2. The chimera rSK1NSK2-CSK2 contains the transmembrane domains and the pore of rSK1 and the NH2 and COOH termini of rSK2. The generation of this chimera (1–121/89–372/406–580) has been described previously (42D'Hoedt D. Hirzel K. Pedarzani P. Stocker M. J. Biol. Chem. 2004; 279: 12088-12092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). L1-hSK1 (1–121/89–120/125–143/140–372/406–580) was generated by substituting the extracellular region between transmembrane segments S1 and S2 in rSK1NSK2-CSK2 for the corresponding region in hSK1. In L3-hSK1 (1–121/89–200/205–227/224–372/406–580) the extracellular region between transmembrane segments S3 and S4 of rSK1NSK2-CSK2 was exchanged for the corresponding region of hSK1. Finally, chimera hSK1-SK2-hSK1 (1–122/152–256/228–543) was created by exchanging the region from the end of transmembrane segment S1 until the beginning of transmembrane segment S4 of hSK1 for the corresponding region in rSK2. In the text, hSK1-QDN refers to point mutation hSK1-K310Q/E312D/H339N and hSK1-QDN+S refers to hSK1-K310Q/E312D/H339N+T216S. All modified sequences were verified by sequencing with the BigDye terminator cycle sequencing kit and an ABI3100 Avant Genetic-Analyzer (Applied Biosystems). Maintenance and Transient Transfection of HEK293 and Chinese Hamster Ovary Cells—Cell lines were cultured in a humidified atmosphere (5% CO2, 95% air) at 37 °C. HEK293 cells were grown in Dulbecco's modified Eagle's medium/F-12 with 2 mm l-glutamine. Chinese hamster ovary cell lines were grown in F-12 nutrient mixture (Ham) with GlutaMAX™ I. Both media were supplemented with 10% fetal calf serum and penicillin/streptomycin. For transient transfections cells were transfected using Lipofectamine 2000 (Invitrogen). 2 μg of plasmid DNA encoding one of the SK channel constructs was co-transfected with 0.5 μg of pEGFP-C2. Recordings were performed 1–3 days after transfection. Generation and Maintenance of Cell Lines Stably Expressing SK Channels—For SK channel constructs hSK1-T216S, hSK1, and hSK1(QDN+S), stable cell lines were generated. Constructs were cloned into the pcDNA5/FRT vector. HEK Flp-In cells (Invitrogen) were grown under the same conditions as HEK293 but in the presence of Zeocin (100 μg/ml). Cells were transfected using Lipofectamine 2000 or by CaPO4 precipitation. For Lipofectamine 2000 transfection, cells were plated into 60-mm culture dishes and co-transfected with a mixture of 0.7 μg of expression construct and 7.3 μg of pOG44 plasmid according to the manufacturer's protocol. For the CaPO4 precipitation method, cells were plated into 90-mm culture dishes and co-transfected with a mixture of 2 μg of expression construct and 23 μg of pOG44 plasmid. For polyclonal selection of cells, Zeocin was replaced by hygromycin B (75 μg/ml). Hygromycin-resistant cells were pooled and maintained in the presence of hygromycin B. Electrophysiology—Whole-cell patch clamp recordings of HEK cells transiently transfected or stably expressing SK channel constructs were performed. Transfected cells were grown on coverslips, placed in a recording chamber, and perfused at a flow rate of 1 ml/min. Currents were recorded using an EPC-10 amplifier (HEKA, Lambrecht, Germany). All recordings were performed at room temperature (22 °C). Data were acquired with Pulse/Pulsefit software (HEKA, Lambrecht, Germany). Pipettes were pulled from borosilicate glass with a horizontal patch electrode puller (Zeitz Instruments GmbH, Muenchen, Germany) and had a resistance of 1.8–3 megohms when filled with intracellular solution (see below). After gigaseal formation, the fast capacitative transients were automatically compensated. SK channels were activated by whole-cell dialysis with an intracellular solution containing (in mm) the following: 130 KCl, 10 HEPES, 10 EGTA, pH 7.2. MgCl2 and CaCl2 were added to obtain free magnesium and calcium concentrations of 1 mm and 1 μm, respectively, using EqCal (Biosoft, Cambridge, UK). Recordings were performed in a high potassium extracellular solution containing (in mm) the following: 144 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, 10 d-glucose, pH 7.4, with KOH, or a low potassium extracellular solution containing (in mm) the following: 4 KCl, 140 NaCl, 10 HEPES, 2 CaCl2, 1 MgCl2, 10 d-glucose, pH 7.4, with NaOH. The high potassium solution was used in most experiments, and inward currents were analyzed in order to avoid problems because of SK channel rectification and activation of endogenous voltage-gated K+ channels at positive potentials. In tetraethylammonium (TEA)-containing solutions, TEA-Cl replaced NaCl in the low potassium extracellular solution. Membrane currents were recorded upon application of 400-ms voltage ramps from –140 to +40 mV and repeated every 10 s. Given the large size of some currents, voltages were corrected off-line for the occurring voltage-clamp error. Cells that showed >20% residual current after application of 100 nm apamin were excluded from the analysis. Concentration-response curves were analyzed using a Langmuir-Hill equation of the type I/Imax = (1 – a0)/(1 + ([inhibitor]/IC50)h) + a0, where h corresponds to the Hill coefficient and IC50 to the concentration of inhibitor that produces a 50% inhibition. The value for curve minimum (a0) was not fixed. The data points for the concentration-response curves were obtained from different cells and therefore treated as independent replicates. The fraction of unblocked current (I/Imax) was calculated at –80 or –20 mV. Curve fitting was performed by using a least square fitting routine. All data were analyzed with Igor Pro 5.0 (Wave-Metrics, Lake Oswego, OR) or Prism 4.0 (GraphPad Software Inc., San Diego). Values are given as mean ± S.E. or 95% confidence interval (95% CI). Unpaired two-tailed Student's t test was used for statistical comparisons between groups. Amino Acids Outside the Pore Affect Apamin Sensitivity— The three members of the SK channel family, SK1, SK2, and SK3, can be distinguished by their sensitivity to apamin. Homomeric SK2 channels from rat (rSK2), mouse (mSK2), and human (hSK2) are the most apamin-sensitive, and homomeric hSK1 channels are the least sensitive channels of the KCa2 subfamily (21Stocker M. Nat. Rev. Neurosci. 2004; 5: 758-770Crossref PubMed Scopus (417) Google Scholar, 37Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar, 45Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (804) Google Scholar, 46Shah M. Haylett D.G. Br. J. Pharmacol. 2000; 129: 627-630Crossref PubMed Scopus (101) Google Scholar, 47Strobaek D. Jorgensen T.D. Christophersen P. Ahring P.K. Olesen S.P. Br. J. Pharmacol. 2000; 129: 991-999Crossref PubMed Scopus (162) Google Scholar). In heterologous expression systems, the rSK1 α-subunit does not form functional homomeric channels (41Benton D.C. Monaghan A.S. Hosseini R. Bahia P.K. Haylett D.G. Moss G.W. J. Physiol. (Lond.). 2003; 553: 13-19Crossref Scopus (62) Google Scholar, 42D'Hoedt D. Hirzel K. Pedarzani P. Stocker M. J. Biol. Chem. 2004; 279: 12088-12092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) but forms functional heteromeric channels with SK2 (41Benton D.C. Monaghan A.S. Hosseini R. Bahia P.K. Haylett D.G. Moss G.W. J. Physiol. (Lond.). 2003; 553: 13-19Crossref Scopus (62) Google Scholar). The chimeric rSK1 α-subunit (rSK1NSK2-CSK2), which contains the S1–S6 region of rSK1 with amino and carboxyl termini of rSK2, assembles into homomers (42D'Hoedt D. Hirzel K. Pedarzani P. Stocker M. J. Biol. Chem. 2004; 279: 12088-12092Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This chimera is ideally suited to obtain information on the pharmacological properties of the rSK1 subunit. The hSK1 channel and the rSK1NSK2-CSK2 chimera were transiently expressed in HEK293 cells. In solutions with nearly identical intra- and extracellular potassium concentrations, a calcium-activated potassium current was elicited by voltage ramps ranging from –140 to +40 mV in the presence of 1 μm intracellular free calcium. The observed current showed a reversal potential close to 0 mV and was shifted to hyperpolarized values upon lowering the extracellular potassium concentration to 4 mm (Fig. 1A). As expected the observed reversal potentials corresponded to values predicted by the Nernst equation for a potassium conductance. The primary sequence of the hSK1 α-subunit and the rSK1NSK2-CSK2 chimera is identical between transmembrane segments S5 and S6. This region includes the pore of potassium channels and has been ascribed the structural basis for the different apamin sensitivities of members of the KCa2 family (43Ishii T.M. Maylie J. Adelman J.P. J. Biol. Chem. 1997; 272: 23195-23200Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, the current generated by the rSK1NSK2-CSK2 chimera was not inhibited by 5 nm apamin (Fig. 1A), whereas 3 nm apamin inhibited hSK1 by nearly 50% (Fig. 1B; 42 ± 1%, n = 6). The identical pore regions of hSK1 and rSK1NSK2-CSK2 and the observed intriguing difference in apamin sensitivity (Fig. 1, A and B) strongly suggest the involvement of amino acids located outside the pore domain for apamin binding. The most obvious regions potentially harboring these amino acids are the hydrophilic extracellular loop L1, located between transmembrane segments S1 and S2, and L3, located between transmembrane segments S3 and S4. Two chimera, L1-hSK1 and L3-hSK1, were created to analyze these regions. L1-hSK1 was generated by substituting the extracellular loop between the transmembrane segments S1 and S2 in rSK1NSK2-CSK2 with the corresponding region of hSK1. In L3-hSK1 the extracellular loop between S3 and S4 (S3–S4 loop) of rSK1NSK2-CSK2 was exchanged for the corresponding region of hSK1. The chimera were transiently expressed in HEK293 cells, and currents were elicited as described above. Both chimera assembled into functional homomeric channels (Fig. 1, C and D). L1-hSK1 showed, like rSK1NSK2-CSK2, only a marginal inhibition upon application of 5 nm apamin (1 ± 1%, n = 4; Fig. 1C). In contrast, 5 nm apamin suppressed around 50% of the current generated by L3-hSK1 (44 ± 5%, n = 5; Fig. 1D), thereby demonstrating an apamin sensitivity similar to hSK1. These results show that the extracellular S3–S4 loop (L3) influences apamin sensitivity and is most likely sufficient to explain the observed difference in apamin sensitivity between hSK1 and rSK1. The Hydrophilic Extracellular Loop L3 between Transmembrane Segments S3 and S4 Influences Apamin Binding—Because amino acids located outside the pore influence the apamin sensitivity of the rSK1NSK2-CSK2 chimera (Fig. 1, A–D), we wondered whether it is a general feature of SK channels that regions outside the pore influence their sensitivity toward apamin. To address this question, we generated a chimera between hSK1 and SK2, which differ more than 100-fold in apamin sensitivity (46Shah M. Haylett D.G. Br. J. Pharmacol. 2000; 129: 627-630Crossref PubMed Scopus (101) Google Scholar, 47Strobaek D. Jorgensen T.D. Christophersen P. Ahring P.K. Olesen S.P. Br. J. Pharmacol. 2000; 129: 991-999Crossref PubMed Scopus (162) Google Scholar). In this chimera, hSK1-SK2-hSK1, the region from the end of the transmembrane segment S1 until the beginning of the transmembrane segment S4 of hSK1, was exchanged for the corresponding region of SK2 (Fig. 2A). If only amino acids in the pore region (between S5 and S6) are responsible for the apamin sensitivity of SK channels, the hSK1-SK2-hSK1 chimera should display an apamin sensitivity comparable with hSK1. If, however, amino acids outside the pore region influence the apamin sensitivity, hSK1-SK2-hSK1 should show an increased sensitivity when compared with hSK1. To test these predictions, hSK1-SK2-hSK1 was transiently expressed in HEK293 cells and calcium-activated potassium currents were elicited as described earlier (see also “Material and Methods”). Apamin at a concentration of 300 pm was applied and suppressed the current generated by the hSK1-SK2-hSK1 chimera by more than 50% (Fig. 2, A and D; 58 ± 3%, n = 5). The concentration of 300 pm apamin was used because it does not affect hSK1 (Fig. 2, B and D) but inhibits SK2 channels nearly completely (Fig. 2, C and D). The IC50 value obtained for the hSK1-SK2-hSK1 chimera (Fig. 2E) is 124 pm (95% CI, 100–156 pm). The clear inhibition of the hSK1-SK2-hSK1 current by low concentrations of apamin shows that the exchanged region contributes to the difference in apamin sensitivity between hSK1 and SK2, and thereby supports the hypothesis that amino acids located beyond the pore region influence the apamin sensitivity in SK channels in general. The observations made for the L1-hSK1 and L3-hSK1 constructs were therefore not an oddity only observed within the rSK1NSK2-CSK2 chimera. Next we were interested in identifying the amino acids located outside the pore region that influence the apamin sensitivity. Once more, we used chimeras of SK2 and hSK1, the most and the least apamin-sensitive SK channel subunits, respectively. Based on the observations made with the chimeric SK channels (Fig. 1D and Fig. 2A), we decided to focus on differences in the amino acid sequences of hSK1 and SK2 in the hydrophilic extracellular loop L3 (Fig. 3A). The alignment shows differences between the amino acid sequences of hSK1 and SK2 in eight positions. By introducing single or double point mutations, the amino acids differing at these eight positions in hSK1 were replaced by the corresponding amino acids found in SK2. Consequently, if any of these amino acids influence the apamin sensitivity in SK2, the corresponding hSK1 mutant should show a higher sensitivity for apamin when co" @default.
• W1988049420 created "2016-06-24" @default.
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• W1988049420 date "2007-02-01" @default.
• W1988049420 modified "2023-09-28" @default.
• W1988049420 title "An Amino Acid Outside the Pore Region Influences Apamin Sensitivity in Small Conductance Ca2+-activated K+ Channels" @default.
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