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• W2038251790 abstract "In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK α-subunits and calmodulin. The channels are activated by Ca2+ binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca2+ from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca2+ concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg2+ in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system. In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK α-subunits and calmodulin. The channels are activated by Ca2+ binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca2+ from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca2+ concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg2+ in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system. action potential afterhyperpolarization small conductance Ca2+-activated potassium 1-ethyl-2-benzimidazolinone medium AHP calmodulin 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate CaM binding domain In many central nervous system neurons, action potentials (APs)1 are followed by a prolonged afterhyperpolarization (AHP) of the membrane potential that controls excitability and firing pattern (1Sah P. Trends Neurosci. 1996; 4: 150-154Abstract Full Text PDF Scopus (802) Google Scholar, 2Storm J.F. Prog. Brain Res. 1990; 83: 161-187Crossref PubMed Scopus (642) Google Scholar). Two classes of AHPs and the underlying Ca2+-activated currents may be distinguished based on their time course and pharmacological properties. IAHP underlies part of the medium AHP (mAHP) following single or repetitive APs, is sensitive to the bee venom toxin apamin, and exhibits fast rise and decay (time constants ≈100 ms). sIAHP mediates the slow AHP that follows trains of APs, is apamin-insensitive, has much slower kinetics (range of seconds), and is modulated by several neurotransmitters (1Sah P. Trends Neurosci. 1996; 4: 150-154Abstract Full Text PDF Scopus (802) Google Scholar). The mAHP controls the tonic firing frequency of neurons, whereas the slow AHP is responsible for spike frequency adaptation, a prominent reduction in the firing frequency in the late phase of responses to prolonged depolarizing stimuli (1Sah P. Trends Neurosci. 1996; 4: 150-154Abstract Full Text PDF Scopus (802) Google Scholar). The Ca2+-activated currents underlying different phases of the AHP have been ascribed to the activation of small conductance Ca2+-activated potassium (SK) channels (1Sah P. Trends Neurosci. 1996; 4: 150-154Abstract Full Text PDF Scopus (802) Google Scholar, 3Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates Inc., Sunderland, MA1992Google Scholar). SK channels are potassium-selective and voltage-independent and are activated by an increase in intracellular Ca2+([Ca2+]i) such as occurs during an AP. Three highly homologous mammalian SK channels (SK1, SK2, and SK3) have been cloned (4Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (783) Google Scholar), as well as a related channel with an intermediate conductance (IK1; Refs. 5Ishii 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 (506) Google Scholar, 6Joiner W.J. Wang L.-Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (312) Google Scholar, 7Logsdon 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). Whereas IK channels are predominantly found in epithelial and blood cells (8Brugnara 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, 9Jensen B.S. Strobaek D. Christophersen P. Jorgensen T.D. Hansen C. Silahtaroglu A. Olesen S.P. Ahring P.K. Am. J. Physiol. 1998; 275: C848-C856Crossref PubMed Google Scholar, 10Jensen B.S. Odum N. Jorgensen N.K. Christophersen P. Olesen S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10917-10921Crossref PubMed Scopus (97) Google Scholar, 11Mahaut-Smith M.P. J. Physiol. ( Lond. ). 1995; 484: 15-24Crossref PubMed Scopus (39) Google Scholar), SK channels are widely expressed in the central nervous system (4Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (783) Google Scholar) with high levels of expression in the regions presenting prominent AHP currents such as neocortex (SK1 and SK2), monoaminergic neurons (SK3), and CA1–3 layers in the hippocampus (SK1 and SK2) (12Stocker M. Pedarzani P. Mol. Cell. Neurosci. 2000; 15: 476-493Crossref PubMed Scopus (307) Google Scholar). As for their native counterparts, these channels are gated by [Ca2+]iin the submicromolar range independent of the transmembrane voltage. At steady state, [Ca2+]i of 0.3 - 0.5 μm leads to half-maximal activation of the channels, whereas saturation is observed with [Ca2+]i of around 10 μm (13Fanger 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 (255) Google Scholar, 14Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 15Xia 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 (718) Google Scholar). Analysis of Ca2+ gating showed that SK and IK channels use calmodulin (CaM) constitutively associated with the C terminus of the SK/IK α-subunit as a high-affinity Ca2+ sensor (13Fanger 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 (255) Google Scholar, 15Xia 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 (718) Google Scholar). Activation of the channels occurs by Ca2+ binding to at least one of the N-terminal EF hands of CaM in each of the four SK α-subunits, which, by the subsequent conformational changes in CaMs and the SK C termini, leads to channel opening. Channel deactivation is the reverse process and occurs upon dissociation of Ca2+from CaM (14Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 15Xia 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 (718) Google Scholar). When modeled, Ca2+ gating of SK channels is adequately described by a multiple-state model analogous to that used for voltage-dependent gating inShaker K+ channels (14Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 16Zagotta W.N. Hoshi T. Dittman J. Aldrich R.W. J. Gen. Physiol. 1994; 103: 279-313Crossref PubMed Scopus (272) Google Scholar, 17Zagotta W.N. Hoshi T. Aldrich R.W. J. Gen. Physiol. 1994; 103: 321-362Crossref PubMed Scopus (435) Google Scholar). In this model, the rate-limiting transitions between closed stated are controlled by Ca2+ and strongly depend on [Ca2+]i, the open-state transition occurs rapidly and is independent of [Ca2+]i (14Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 18Hirschberg B. Maylie J. Adelman J.P. Marrion N.V. J. Gen. Physiol. 1998; 111: 565-581Crossref PubMed Scopus (149) Google Scholar, 19Hirschberg B. Maylie J. Adelman J.P. Marrion N.V. Biophys. J. 1999; 77: 1905-1913Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Recently, it was shown that the compound 1-ethyl-2-benzimidazolinone (EBIO) activates IK channels in colonic epithelia as well as in transfected cultured cells when applied extracellularly under physiological conditions (10Jensen B.S. Odum N. Jorgensen N.K. Christophersen P. Olesen S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10917-10921Crossref PubMed Scopus (97) Google Scholar, 20Devor D.C. Singh A.K. Frizzell R.A. Bridges R.J. Am. J. Physiol. 1996; 271: L775-L784PubMed Google Scholar, 21Pedersen K.A. Schroder R.L. Skaaning-Jensen B. Strobaek D. Olesen S.P. Christophersen P. Biochim. Biophys. Acta. 1999; 1420: 231-240Crossref PubMed Scopus (68) Google Scholar, 22Warth R. Hamm K. Bleich M. Kunzelmann K. von Hahn T. Schreiber R. Ullrich E. Mengel M. Trautmann N. Kindle P. Schwab A. Greger R. Pfluegers Arch. 1999; 438: 437-444Crossref PubMed Scopus (115) Google Scholar). Pedersen et al. (21Pedersen K.A. Schroder R.L. Skaaning-Jensen B. Strobaek D. Olesen S.P. Christophersen P. Biochim. Biophys. Acta. 1999; 1420: 231-240Crossref PubMed Scopus (68) Google Scholar) showed an EBIO-induced shift in the Ca2+concentration-response relationship of IK1 currents such that [Ca2+]i of about 30 nm was enough to elicit robust channel activity. The molecular mechanism behind this shift in Ca2+ sensitivity remained unclear. Here we investigated the molecular mechanism underlying the EBIO-mediated activation of cloned SK channels at subthreshold Ca2+ concentrations and tested the effects of EBIO on the excitability and firing pattern of hippocampal neurons and cortical neuronal networks. The results suggest that EBIO affects the interaction between channel α subunits and CaM and that modulating SK channel activity is an effective mechanism for tuning excitability in central neurons. In vitro mRNA synthesis and oocyte injections were performed as described previously (23Fakler B. Brandle U. Glowatzki E. Weidemann S. Zenner H.P. Ruppersberg J.P. Cell. 1995; 80: 149-154Abstract Full Text PDF PubMed Scopus (314) Google Scholar). Giant patch recordings were made 3–7 days after injection. Pipettes made from thick-walled borosilicate glass had resistances of ≈0.3 megohms when filled with 120 mm KOH, 10 mm HEPES, and 0.5 mm CaCl2 (pH adjusted to 7.2 with methanesulfonic acid) or with 115 mm NaOH, 5 mmKOH, 10 mm HEPES, and 0.5 mm CaCl2(pH adjusted to 7.2 with methanesulfonic acid). Inside-out patches were superfused with an intracellular solution containing 119 mmKOH, 1 mm KCl, 10 mm HEPES, and 1 mm EGTA (pH adjusted to 7.2 with methanesulfonic acid); all chemicals used were of the highest grade; Millipore water was treated with Chelex100 (Bio-Rad, Hercules, CA) before solution preparation. The amount of CaCl2 required to yield the concentrations indicated was calculated as described by Fabiato (24Fabiato A. Methods Enzymol. 1988; 157: 378-417Crossref PubMed Scopus (973) Google Scholar) and added to the EGTA solution under pH meter control; thereafter, pH was readjusted to 7.2 with KOH. EBIO (Sigma (St. Louis, MO) and Tocris Cookson, Bristol, UK) was added to the intracellular solution to yield the final concentrations indicated. Rapid exchange of Ca2+ was achieved using a piezo-driven application system (25Oliver D. Klocker N. Schuck J. Baukrowitz T. Ruppersberg J.P. Fakler B. Neuron. 2000; 26: 595-601Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar); the time constant of solution exchange with this system was 0.5 ms. Data analysis and fitting were performed with IgorPro (WaveMetrics, Lake Oswego, OR) on a Macintosh PowerPC; for the Ca2+ concentration responses in symmetrical and asymmetrical K+, currents recorded at −80 mV and −120 mV were used, respectively. All data are presented as mean ± S.D. of n experiments. Transverse hippocampal slices (300 μm thick) were prepared from Wistar rats (23–28 days old) with a vibratome (VT 1000S Leica) and subsequently incubated in a humidified interface chamber at room temperature for ≥1 h. Tight-seal whole-cell voltage-clamp recordings were obtained from 28 CA1 pyramidal neurons using the “blind method” (55Blanton M.G. Lo Turco J.J. Kriegstein A.R. J. Neurosci. Methods. 1989; 30: 203-210Crossref PubMed Scopus (819) Google Scholar). Patch electrodes (4–7 megohms) were filled with an intracellular solution containing 135 mmpotassium gluconate, 10 mm KCl, 10 mm HEPES, 2 mm Na2-ATP, 0.4 mmNa3-GTP, and 1 mm MgCl2(osmolarity, 280–300 mosmol; pH 7.2–7.3 with KOH). 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (8CPT-cAMP; 50 μm) was included to measure the apamin-sensitive IAHP in isolation. All neurons included in this study had a resting membrane potential below −55 mV (−61 ± 1 mV;n = 19) and an input resistance of 215 ± 5 megaohms (n = 19). Recordings were performed in a submerged recording chamber with a constant flow of artificial cerebrospinal fluid (2 ml/min) at room temperature. Drugs were applied in the bath solution. Artificial cerebrospinal fluid contained 125 mm NaCl, 1.25 mm KCl, 2.5 mmCaCl2, 1.5 mm MgCl2, 1.25 mm KH2PO4, 25 mmNaHCO3, and 16 mm d-glucose and was bubbled with carbogen (95% O2/5% CO2). EBIO was dissolved in Me2SO and stored at −20 °C as a 0.4m stock solution, diluted before use, and bath-applied in the perfusing artificial cerebrospinal fluid. All controls were performed in Me2SO at the same final concentration used during EBIO application (0.25%), and no significant effects of Me2SO were detected. Neurons were voltage-clamped at −50 mV, and 100-ms-long depolarizing pulses to +10 mV were delivered every 30 s. These pulses led to unclamped Ca2+ action currents sufficient to activate IAHP and sIAHP. Series resistance was monitored at regular intervals throughout the recording, and only recordings with stable series resistances ≤25 megohms were included in this study. No series resistance compensation and no corrections for liquid junction potentials were made. Only cells with a stable resting potential throughout the current-clamp protocols (±1 mV) were included in the analysis. Data were acquired using a patch-clamp EPC9 amplifier (HEKA, Lambrecht, Germany), filtered at 0.25–1 kHz, sampled at 1–4 kHz, and stored on a Macintosh PowerPC. Analysis was performed using the programs Pulse and Pulsefit (HEKA), Igor Pro 3.01 (Wave Metrics), and Excel (Microsoft). Values are presented as mean ± S.E. For statistical analysis, the Student's t test was used, and differences were considered statistically significant ifp ≤ 0.05. Tetraethylammonium, potassium gluconate, Na2-ATP, Na3-GTP, 8CPT-cAMP, and Me2SO were obtained from Sigma; tetrodotoxin was obtained from Alomone Laboratories (Jerusalem, Israel); noradrenaline was obtained from RBI (Natick, MA); apamin was obtained from Latoxan (Rosans, France); samples of EBIO were obtained from Sigma and from Tocris Cookson; and all other salts and chemicals were obtained from Merck. Primary cultures of cortical neurons were prepared from embryonic day 17 Harlan Sprague-Dawley rats as described by Wang and Gruenstein (26Wang X. Gruenstein E.I. Brain Res. 1997; 767: 239-249Crossref PubMed Scopus (75) Google Scholar). Cells were incubated at 37 °C in 5% CO2 for 7–10 days. About 15 min before the experiments, the culture medium was removed, and cells were loaded with 2 μm fluo-4 (Molecular Probes) in Hanks' balanced salt solution (Life Technologies, Inc.) supplemented with 10 mm HEPES (pH adjusted to 7.4). After loading, cells were washed twice in Hanks' balanced salt solution + 2 mmCa2+ without Mg2+ and then transferred into a fluorescence plate reader I fluorescence image plate reader (Molecular Devices). Fluo-4 fluorescence from ≈5000 cells (on an area of ≈10) was measured at a sampling rate of 0.5 Hz, drug application (20 μl of a 5-fold concentrated stock in 80 μl of Hanks' balanced salt solution + 2 mm Ca2+without Mg2+) was performed within 1 s simultaneously in 96 wells during measurement. Fluorescence oscillations were analyzed by determining (i) the number of peaks above threshold and (ii) the area under curve (AUC) after baseline subtraction during intervals before and after drug application. The effect of EBIO on cloned SK channels was investigated in giant inside-out patches excised from Xenopus oocytes expressing either hSK1 (SK1) or rSK2 (SK2) channels. Fig. 1 illustrates the response of SK2 channels to cytoplasmic application of EBIO at various [Ca2+]i; SK currents were recorded under symmetrical K+ conditions at a membrane potential of −80 mV (intermittently stepped to 50 mV for 50 ms every 1 s). EBIO induced robust activation of SK2 channels at Ca2+concentrations as low as 50 and 100 nm, which, in the absence of the benzimidazolinone, did not elicit any SK currents (Fig.1 A). In the absence of Ca2+, however, EBIO failed to activate SK currents, similar to reports on IK channels (21Pedersen K.A. Schroder R.L. Skaaning-Jensen B. Strobaek D. Olesen S.P. Christophersen P. Biochim. Biophys. Acta. 1999; 1420: 231-240Crossref PubMed Scopus (68) Google Scholar). Moreover, EBIO did not increase the amplitude of SK currents when applied at a saturating [Ca2+]i of 10 μm (Fig. 1 A). The results suggest that EBIO shifts the apparent Ca2+sensitivity of SK channels. This was quantified in Ca2+concentration-response relationships determined for SK1 and SK2 currents in the absence and presence of EBIO under symmetrical and physiological K+ conditions. As shown in Fig. 1,B and C, EBIO induced a leftward shift of the concentration-response curve by about 7-fold for either SK subtype, with [Ca2+]i required for half-maximal activation (EC50) of SK1 and SK2 channels of 81.6 ± 31.4 nm (n = 6) and 69.4 ± 3.2 nm (n = 5), respectively. This shift in the Ca2+ concentration-response curve was independent of the extracellular K+ concentration (120 or 5 mm; Fig. 1, B and C). Additionally, the steepness of the concentration-response curves obtained in the presence of EBIO appeared slightly increased with respect to the controls (Hill coefficients were 4.0 ± 1.0 and 5.6 ± 0.5 for SK2 channels in the absence and presence of EBIO; the respective values for SK1 were 4.4 ± 1.0 and 6.6 ± 1.1). The observations that EBIO was ineffective either in the absence of [Ca2+]i or with saturating [Ca2+]i suggested that the compound may operate by interacting with the gating apparatus of SK channels rather than working as a classical channel opener requiring Ca2+ as a “cofactor.” Therefore, the kinetics of channel gating were examined in “fast application” experiments where activation and deactivation kinetics of Ca2+ gating can be monitored separately (15Xia 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 (718) Google Scholar,25Oliver D. Klocker N. Schuck J. Baukrowitz T. Ruppersberg J.P. Fakler B. Neuron. 2000; 26: 595-601Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Fig. 2 A shows SK2 currents measured in response to fast application and removal of a saturating [Ca2+]i of 10 μm to inside-out patches in the absence (gray trace) and presence of EBIO. Whereas the time course of channel activation was not affected by EBIO at this [Ca2+]i, the current decay upon removal of Ca2+ was markedly slowed down by the benzimidazolinone (Fig. 2 A, top panel). When fitted with monoexponentials, the respective time constants for activation (τon) and deactivation (τoff) were 4.4 ± 0.5 ms (n = 8) and 294.2 ± 21.9 ms in the presence of EBIO, whereas control values were 4.4 ± 1.1 ms (n= 7) and 39.4 ± 4.1 ms. EBIO prolonged channel deactivation by about 7.5-fold (Fig. 2 B), and the increase of τoff was only observed when EBIO was present after removal of Ca2+; washout of EBIO together with Ca2+ resulted in a current decay identical to that recorded in controls (Fig. 2 A, middle panel; τoff = 40.2 ± 5.8 ms; n = 4). To test whether EBIO may also affect channel activation, fast applications were done with [Ca2+]i of 0.5 μm, a value close to the EC50 for SK channels (Fig. 1, B and C). Under these conditions, application of EBIO changed both τon and τoff; τoff increased by about 6.8-fold, whereas τon decreased by a factor of 1.5 (Fig. 2 A, bottom panel and Fig. 2 B). Thus, the predominant effect of EBIO was on channel deactivation, whereas the activation kinetics were only slightly affected. The EBIO-induced increase of τoff was independent of channel activation (Fig. 2 C). In the presence of EBIO, either 50 or 100 nm Ca2+ activated currents that decayed with essentially the same τoff of about 300 ms (283.2 ± 7.6 and 303.9 ± 13.7 at 50 and 100 nm, respectively). In contrast, channel activation strongly depended on [Ca2+]i; τon exhibited values of 608.3 ± 49.3 and 83.6 ± 7.3 ms (n= 8) for 50 and 100 nm Ca2+, respectively (Fig.2 C). These results suggested that EBIO modulates the interaction between the SK channel α-subunit and Ca2+-bound CaM (Ca2+-CaM). This was further tested in experiments probing the influence of the SK α-subunit and CaM on the EBIO effect. In a first set of experiments, EBIO was tested on SK2 channels coexpressed with either wild-type CaM or mutant CaMs in which Ca2+ binding was largely impaired in both N-terminal EF hands (Mut CaM1, 2 and Mut CaM1–4, (14Keen J.E. Khawaled R. Farrens D.L. Neelands T. Rivard A. Bond C.T. Janowsky A. Fakler B. Adelman J.P. Maylie J. J. Neurosci. 1999; 19: 8830-8838Crossref PubMed Google Scholar, 15Xia 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 (718) Google Scholar)). Fig. 3 A shows that whereas EBIO effectively activated SK channels at [Ca2+]iof 50 nm when coexpressed with wild-type CaM, little or no current induction was observed for SK channel coexpression with Mut CaM1,2 or Mut CaM1–4 (data not shown). The small increase in current is most likely due to the fraction of SK2 channels coassembled with endogenous wild-type Xenopus CaM (15Xia 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 (718) Google Scholar); the amplitude of this current was 0.02 ± 0.01 (n = 7) of that obtained by coexpression with wild-type CaM. The second set of experiments probed the SK C terminus, the domain where CaM interacts with the channel α-subunit, as a candidate site for EBIO action (13Fanger 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 (255) Google Scholar, 15Xia 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 (718) Google Scholar). SK and IK channels share the basic Ca2+ gating mechanism mediated by CaM and exhibit similar Ca2+ concentration-response relations (13Fanger 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 (255) Google Scholar). However, the primary sequences of the C termini, including the CaM binding domains, show considerable variability (4Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (783) Google Scholar, 5Ishii 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 (506) Google Scholar, 6Joiner W.J. Wang L.-Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (312) Google Scholar, 7Logsdon 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). Therefore, concentration-response relations for EBIO-induced channel activation in the presence of a constant [Ca2+]i of 100 nm were determined for both channel types. As shown in Fig. 3 B, the concentration-response relationships for IK1 and SK2 differed in their EC50 by more than 20-fold with values of 28.4 and 654.2 μm for IK1 and SK2, respectively. Moreover, when the C terminus of IK1 was exchanged into the SK2 subunit (SK2-IK1C-term), the resulting chimeric channel showed an affinity for EBIO very similar to that obtained for IK1 (EC50 for SK2-IK1C-term was 22.3 μm; Fig. 3 B). Taken together with the effects on kinetics, the results shown are consistent with EBIO modulating the Ca2+ gating of SK channels by stabilizing the interaction between Ca2+-CaM and the SK α-subunit. In hippocampal pyramidal neurons, voltage-independent, Ca2+-activated K+ channels are responsible for the generation of two distinct phases of AHP, the mAHP and the slow AHP. According to their distribution and pharmacological properties, SK channels likely underlie at least part of the mAHP in hippocampal neurons (27Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (330) Google Scholar). Given the observed effects of EBIO on the Ca2+ gating of cloned SK channels, the compound was tested on the Ca2+-activated K+ currents underlying mAHP (IAHP) and slow AHP (sIAHP) in CA1 pyramidal neurons in acute hippocampal slices. In whole-cell voltage-clamp experiments, currents were elicited by a standard protocol (27Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (330) Google Scholar) in the presence of tetrodotoxin (0.5 μm) and tetraethylammonium (1 mm) to block Na+ channels and Ca2+- and voltage-dependent K+ (BK) channels, respectively. As shown in Fig.4 A, EBIO induced a marked increase of the IAHP amplitude with respect to the controls recorded under steady-state conditions before application of the compound. The relative increase of the IAHP was 3.5 ± 0.4 (n = 5) (Fig. 4 D). Moreover, the enhanced IAHP was fully suppressed by apamin applied to the neurons in the presence of the benzimidazolinone (Fig.4 B). EBIO also increased the apamin-insensitive sIAHP. When applied together with 50 nm apamin to block IAHP (27Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (330) Google Scholar), EBIO induced an increase of the remaining sIAHP by a factor of 1.5 ± 0.1 with respect to the control (Fig. 4, C and D; n = 4). The EBIO-enhanced current was fully inhibited by noradrenaline at 1 μm (data not shown), identifying it as the sIAHP (28Nicoll R.A. Science. 1988; 241: 545-551Crossref PubMed Scopus (446) Google Scholar, 29Pedarzani P. Storm J.F. Neuron. 1993; 11: 1023-1035Abstract Full Text PDF PubMed Scopus (236) Google Scholar). The effect of EBIO on the time course of IAHP was examined in isolation by inhibiting sIAHP by including the cAMP analogue 8CPT-cAMP in the patch pipette (27Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (330) Google Scholar, 29Pedarzani P. Storm J.F. Neuron. 1993; 11: 1023-1035Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 30Madison D.V. Nicoll R.A. J. Physiol. ( Lond. ). 1986; 372: 221-244Crossref PubMed Scopus (331) Google Scholar). Consistent with the effect of EBIO on activation and deactivation kinetics of cloned SK channels (Fig. 2), the time constant of decay of the IAHP(τ decay) was increased by the benzimidazolinone. The values for τ decay were 90.5 ± 7.5 and 214.5 ± 9.8 ms (n = 6) in the absence and presence of EBIO, respectively (Fig. 4, E and F). In contrast, the rise time (time-to-peak) of the current remained esse" @default.
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• W2038251790 title "Control of Electrical Activity in Central Neurons by Modulating the Gating of Small Conductance Ca2+-activated K+ Channels" @default.
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