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Specific Enhancement of SK Channel Activity Selectively Potentiates the Afterhyperpolarizing Current IAHP and Modulates the Firing Properties of Hippocampal Pyramidal Neurons

2005; Elsevier BV; Volume: 280; Issue: 50 Linguagem: Inglês

10.1074/jbc.m509610200

ISSN

1083-351X

Autores

Paola Pedarzani, James E. McCutcheon, Gregor Rogge, Bo Jensen, Palle Christophersen, Charlotte Hougaard, Dorte Strøbæk, Martin Stocker,

Tópico(s)

Cardiac electrophysiology and arrhythmias

Resumo

SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability. SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability. In hippocampal pyramidal neurons voltage-independent, Ca2+-activated K+ channels are responsible for the generation of two distinct afterhyperpolarizing currents, IAHP 5The abbreviations used are: IAHPapamin-sensitive afterhyperpolarizing currentAHPafterhyperpolarizationsIAHPslow afterhyperpolarizing currentsAHPslow AHPmAHPmedium AHPDCEBIO5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one1-EBIO1-ethyl-2-benzimidazolinoneNS3096,7-dichloro-1H-indole-2,3-dione-3-oximecAMPcyclic AMP8CPT-cAMP8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphateSK channelsmall conductance Ca2+-activated K+ channelIK channelintermediate conductance Ca2+-activated K+ channelHEKhuman embryonic kidney.5The abbreviations used are: IAHPapamin-sensitive afterhyperpolarizing currentAHPafterhyperpolarizationsIAHPslow afterhyperpolarizing currentsAHPslow AHPmAHPmedium AHPDCEBIO5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one1-EBIO1-ethyl-2-benzimidazolinoneNS3096,7-dichloro-1H-indole-2,3-dione-3-oximecAMPcyclic AMP8CPT-cAMP8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphateSK channelsmall conductance Ca2+-activated K+ channelIK channelintermediate conductance Ca2+-activated K+ channelHEKhuman embryonic kidney. and sIAHP (1Sah P. Faber L.E.S. Prog. Neurobiol. 2002; 66: 345-353Crossref PubMed Scopus (407) Google Scholar, 2Vogalis F. Storm J.F. Lancaster B. Eur. J. Neurosci. 2003; 18: 3155-3166Crossref PubMed Scopus (102) Google Scholar, 3Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar, 4Stocker M. Nat. Rev. Neurosci. 2004; 5: 758-770Crossref PubMed Scopus (410) Google Scholar). IAHP is characterized by a time constant of decay of ∼100 ms and by its sensitivity to the bee venom toxin, apamin, and to the scorpion toxins, scyllatoxin and tamapin (5Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (331) Google Scholar, 6Pedarzani 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, 7Sailer C.A. Hu H. Kaufmann W.A. Trieb M. Schwarzer C. Storm J.F. Knaus H.G. J. Neurosci. 2002; 22: 9698-9707Crossref PubMed Google Scholar). sIAHP is characterized by a slower time course (in the range of seconds), by its lack of sensitivity to apamin or any other classical K+channel blocker, and by its modulation by several neurotransmitters (1Sah P. Faber L.E.S. Prog. Neurobiol. 2002; 66: 345-353Crossref PubMed Scopus (407) Google Scholar, 2Vogalis F. Storm J.F. Lancaster B. Eur. J. Neurosci. 2003; 18: 3155-3166Crossref PubMed Scopus (102) Google Scholar, 3Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar, 8Lancaster B. Adams P.R. J. Neurophysiol. 1986; 55: 1268-1282Crossref PubMed Scopus (415) Google Scholar). Based on their kinetic and pharmacological features and on the results obtained from genetically manipulated mice, SK channels mediate IAHP, whereas the molecular correlate for sIAHP is still unknown (2Vogalis F. Storm J.F. Lancaster B. Eur. J. Neurosci. 2003; 18: 3155-3166Crossref PubMed Scopus (102) Google Scholar, 3Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar, 4Stocker M. Nat. Rev. Neurosci. 2004; 5: 758-770Crossref PubMed Scopus (410) Google Scholar, 9Bond C.T. Herson P.S. Strassmaier T. Hammond R. Stackman R.W. Maylie J. Adelman J.P. J. Neurosci. 2004; 24: 5301-5306Crossref PubMed Scopus (224) Google Scholar, 10Villalobos C. Shakkottai V.G. Chandy K.G. Michelhaugh S.K. Andrade R. J. Neurosci. 2004; 24: 3537-3542Crossref PubMed Scopus (95) Google Scholar).In addition to the use of selective blockers, an important contribution to the elucidation of the physiological role of SK and IK channels has arisen from the use of a small organic compound that enhances channel activity, the benzimidazolinone 1-EBIO (11Devor D.C. Singh A.K. Frizzell R.A. Bridges R.J. Am. J. Physiol. 1996; 271 (-L784): L775PubMed Google Scholar, 12Jensen B.S. Strobaek D. Christophersen P. Jorgensen T.D. Hansen C. Silahtaroglu A. Olesen S.P. Ahring P.K. Am. J. Physiol. 1998; 275 (-C856): C848Crossref PubMed Google Scholar, 13Syme C.A. Gerlach A.C. Singh A.K. Devor D.C. Am. J. Physiol. 2000; 278 (-C581): C570Crossref PubMed Google Scholar, 14Pedarzani P. Mosbacher J. Rivard A. Cingolani L.A. Oliver D. Stocker M. Adelman J.P. Fakler B. J. Biol. Chem. 2001; 276: 9762-9769Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Cao Y. Dreixler J.C. Roizen J.D. Roberts M.T. Houamed K.M. J. Pharmacol. Exp. Ther. 2001; 296: 683-689PubMed Google Scholar). 1-EBIO enhances the activity of SK channels in the presence of the physiological activator, intracellular Ca2+, by increasing the apparent sensitivity of SK channels to Ca2+ (14Pedarzani P. Mosbacher J. Rivard A. Cingolani L.A. Oliver D. Stocker M. Adelman J.P. Fakler B. J. Biol. Chem. 2001; 276: 9762-9769Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). As a consequence, 1-EBIO increases the amplitude of SK-mediated AHP currents and their duration in a variety of neurons, leading to profound changes in neuronal activity and firing patterns (14Pedarzani P. Mosbacher J. Rivard A. Cingolani L.A. Oliver D. Stocker M. Adelman J.P. Fakler B. J. Biol. Chem. 2001; 276: 9762-9769Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 16Wolfart J. Neuhoff H. Franz O. Roeper J. J. Neurosci. 2001; 21: 3443-3456Crossref PubMed Google Scholar, 17Cingolani L.A. Gymnopoulos M. Boccaccio A. Stocker M. Pedarzani P. J. Neurosci. 2002; 22: 4456-4467Crossref PubMed Google Scholar, 18Hallworth N.E. Wilson C.J. Bevan M.D. J. Neurosci. 2003; 23: 7525-7542Crossref PubMed Google Scholar). Although 1-EBIO has been a useful tool to elucidate the function of SK channels in their native context, it has some important limitations. First, it affects not only the SK channels but also the as yet unidentified Ca2+-dependent K+ channels underlying sIAHP (14Pedarzani P. Mosbacher J. Rivard A. Cingolani L.A. Oliver D. Stocker M. Adelman J.P. Fakler B. J. Biol. Chem. 2001; 276: 9762-9769Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Additionally, prolonged applications of 1-EBIO have been shown to lead to a decrease in Ca2+ currents in hippocampal neurons (14Pedarzani P. Mosbacher J. Rivard A. Cingolani L.A. Oliver D. Stocker M. Adelman J.P. Fakler B. J. Biol. Chem. 2001; 276: 9762-9769Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Finally, and most importantly, 1-EBIO displays a relatively low potency (EC50 on SK channels ∼700 μm) (14Pedarzani P. Mosbacher J. Rivard A. Cingolani L.A. Oliver D. Stocker M. Adelman J.P. Fakler B. J. Biol. Chem. 2001; 276: 9762-9769Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These limitations of 1-EBIO have prompted the development of novel, more potent SK channel enhancers. DCE-BIO, a dichlorinated analogue of 1-EBIO, has been reported to enhance the activity of intermediate conductance Ca2+-activated K+ channels (IK channels) (19Singh S. Syme C.A. Singh A.K. Devor D.C. Bridges R.J. J. Pharmacol. Exp. Ther. 2001; 296: 600-611PubMed Google Scholar). Moreover, recently 6,7-dichloro-1H-indole-2,3-dione-3-oxime (NS309) (20Strøbaek D. Teuber L. Jørgensen T.D. Ahring P.K. Kaer K. Hansen R.S. Olesen S.P. Christophersen P. Skaaning-Jensen B. Biochim. Biophys. Acta. 2004; 1665: 1-5Crossref PubMed Scopus (151) Google Scholar) has been described as a more potent enhancer of the activity of recombinant SK and IK channels.In the present study, we provide the first characterization of the effect of DCEBIO on recombinant SK channels and a quantification of the potency differences between DCEBIO, NS309, and 1-EBIO on recombinant SK2 channels, the predominant SK channel subtype in hippocampus. We have furthermore investigated the actions of DCEBIO and NS309 on the native SK channels mediating IAHP, on the distinct Ca2+-activated K+ current sIAHP, and on the firing behavior of CA1 pyramidal neurons in hippocampal slices.MATERIALS AND METHODSElectrophysiology on Recombinant hSK2 and Kv7.2/7.3 Channels— HEK293 cells stably expressing human SK2 (20Strøbaek D. Teuber L. Jørgensen T.D. Ahring P.K. Kaer K. Hansen R.S. Olesen S.P. Christophersen P. Skaaning-Jensen B. Biochim. Biophys. Acta. 2004; 1665: 1-5Crossref PubMed Scopus (151) Google Scholar) or co-expressing Kv7.2 and Kv7.3 channel subunits (21Schrøder R.L. Jespersen T. Christophersen P. Strøbaek D. Jensen B.S. Olesen S.P. Neuropharmacology. 2001; 40: 888-898Crossref PubMed Scopus (115) Google Scholar) were plated on coverslips 12-24 h prior to the experiments. For each experiment, a coverslip was placed in a 15-μl perfusion chamber (flow rate ∼1 ml/min). All experiments were performed at room temperature (20-22 °C) using borosilicate pipettes (resistance 2-3 megohms) controlled by a micromanipulator (Patch-Man, Eppendorf, Germany). The extracellular solution contained (in mm): 140 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, adjusted to pH 7.4 with KOH. The osmolarity of the extracellular solution was 285 mosm. For inside-out experiments on the hSK2 currents, three bath (intracellular) solutions were used and denoted as 0.01, 0.2, and 10 to indicated their calculated free [Ca2+] in μm. Free concentrations of Ca2+ and Mg2+ were calculated using EqCal software (Biosoft, United Kingdom) and verified by a Ca2+-selective electrode (World Precision Instruments). The solutions containing 0.01 and 0.2 μm free [Ca2+] included (in mm): 1 free Mg2+, 10 EGTA, 10 HEPES, and 123 KCl or 110 KCl, respectively. The solution containing 10 μm free [Ca2+] included (in mm): 1 free Mg2+, 1 EGTA, 9 NTA, 10 HEPES, and 120 KCl. Adjustment of pH with KOH resulted in a final [K+] of 154 mm. The osmolarity of intracellular solutions was ∼280 mosm. Kv7.2/7.3 currents were measured in whole-cell experiments with an intracellular solution consisting of (in mm): 120 KCl, 5.374 CaCl2, 1.75 MgCl2, 10 HEPES, 4 Na-ATP, 0.4 GTP (pH 7.2 with KOH). In hSK2 experiments, linear voltage ramps (-80 to +80 mV, 200 ms duration) were applied every 5 s from a holding potential of 0 mV. The Kv7.2/7.3 channels were activated by a 1-s step to -30 mV (close to the voltage of half-maximal activation), and the deactivation was followed for 1 s at -60 mV (close to the activation threshold) before stepping to the holding potential of -90 mV. The protocol was applied every 5 or 10 s.Electrophysiology on Brain Slices—350 μm thick transverse hippocampal slices were prepared from Sprague-Dawley rats (20-23 days old) with a vibratome (VT 1000S, Leica, Germany) and subsequently incubated in a humidified interface chamber at room temperature for ≥1 h. Tight seal whole-cell voltage clamp recordings were obtained from 58 CA1 pyramidal neurons using the "blind method" (22Blanton M.G. Lo Turco J.J. Kriegstein A.R. J. Neurosci. Methods. 1989; 30: 203-210Crossref PubMed Scopus (820) Google Scholar). Patch electrodes (4-6 megohms) were filled with an intracellular solution containing (in mm): 135 potassium gluconate, 10 KCl, 10 HEPES, 2 Na2-ATP, 0.4 Na3-GTP, 1 MgCl2 (osmolarity 280-300 mosm, pH 7.2-7.3, with KOH). 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (8CPT-cAMP; 50 μm) was included to suppress sIAHP when the apamin-sensitive IAHP was measured in isolation. Recordings were performed in a submerged recording chamber with a constant flow of artificial cerebrospinal fluid (2 ml/min) at room temperature. Artificial cerebrospinal fluid contained (in mm): 125 NaCl, 1.25 KCl, 2.5 CaCl2, 1.5 MgCl2, 1.25 KH2PO4, 25 NaHCO3, 16 d-glucose, and was bubbled with carbogen (95% O2/5% CO2).All neurons included in this study had a resting membrane potential below -55 mV (-63 ± 1 mV) and an input resistance of 195 ± 10 megohms. Neurons were voltage-clamped at -50 mV, and 100-ms-long depolarizing pulses to +10 mV were delivered every 30 s to elicit robust and reliable Ca2+ action currents in the presence of 0.5 μm tetrodotoxin and 1 mm tetraethylammonium, leading to the activation of the Ca2+-dependent IAHP and sIAHP (see Refs. 5Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (331) Google Scholar, 23Pedarzani P. Storm J.F. Neuron. 1993; 11: 1023-1035Abstract Full Text PDF PubMed Scopus (238) Google Scholar, and 24Zhang L. Pennefather P. Velumian A.A. Tymianski M. Charlton M. Carlen P.L. J. Neurophysiol. 1995; 74: 2225-2241Crossref PubMed Scopus (73) Google Scholar). The amplitude of IAHP and sIAHP were estimated 50-80 ms and 1 s after the end of the depolarizing pulse, respectively. In current clamp recordings, tetrodotoxin and tetraethylammonium were omitted, and action potentials were elicited by current injections from the resting membrane potential. Only cells with a stable resting potential throughout the current clamp protocols (±1 mV) were included in the analysis of the current clamp data. Series resistance (range 15-25 megohms) was monitored at regular intervals throughout the recording. All recordings included in this study presented minimal variations (≤10%) of the series resistance and of the amplitude and duration of the Ca2+ action current, well within the limits needed to maintain a stable amplitude of IAHP and sIAHP under control conditions. Data are reported without corrections for liquid junction potentials.Data Acquisition and 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 G4 or Power PC. Analysis was made using the Pulse and Pulsefit (HEKA, Lambrecht, Germany), Igor Pro (Wave Metrics), SigmaPlot (SPSS, Inc.), InStat (Graphpad), and Excel (Microsoft) software. Values are presented as mean ± S.E. For statistical analysis, the Student's t test was used, and differences were considered statistically significant when p < 0.05. Concentration-response relationships were fitted to the Hill equation I/Imax = [E]n/([E]n + (EC50)n) to obtain EC50 values and Hill-coefficients (n). [E] is the concentration of the enhancer.Pharmacology on Cells and Brain Slices—Drugs were applied in the bath solution. NS309, DCEBIO, 1-EBIO, and retigabine were dissolved in Me2SO as 500-1000-fold concentrated stock solutions and stored at -20 °C, diluted prior to use, and bath-applied in the perfusing artificial cerebrospinal fluid. All controls were performed in Me2SO at the same final concentration as during NS309, DCEBIO, or 1-EBIO application (0.005-0.3%). NS309 was synthesized at NeuroSearch A/S 6Jensen, B. S., Jørgensen, T. D., Ahring, P. K., Christophersen, P., Strøbaek, D., Teuber, L., and Olesen, S. P. (1999) NeuroSearch A/S, assignee, International Patent Application WO 00/33834.; DCEBIO was from Tocris (Bristol, UK) or synthesized at NeuroSearch A/S; 1-EBIO was from Sigma-Aldrich; retigabine (N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) was synthesized at NeuroSearch; tetraethylammonium, potassium gluconate, Na2-ATP, Na3-GTP, 8CPT-cAMP, and dimethyl sulfoxide (Me2SO) were obtained from Sigma; tetrodotoxin was from Alomone Laboratories (Jerusalem, Israel); noradrenaline and d-tubocurarine were from RBI (Natick, New Jersey); apamin was from Latoxan (Rosans, France); all other salts and chemicals were obtained from Merck or Sigma.RESULTSDCEBIO and NS309, SK Channel Enhancers More Potent than 1-EBIO—DCEBIO and NS309 have been shown to modulate recombinant IK and SK channels, respectively, at lower concentrations than required for 1-EBIO. To compare their relative potency on recombinant SK channels, we have first characterized the effect of DCEBIO on SK2 channels, in view of the essential role played by the SK2 (KCa2.2) subunits in mediating IAHP in hippocampal pyramidal neurons (5Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (331) Google Scholar, 7Sailer C.A. Hu H. Kaufmann W.A. Trieb M. Schwarzer C. Storm J.F. Knaus H.G. J. Neurosci. 2002; 22: 9698-9707Crossref PubMed Google Scholar, 9Bond C.T. Herson P.S. Strassmaier T. Hammond R. Stackman R.W. Maylie J. Adelman J.P. J. Neurosci. 2004; 24: 5301-5306Crossref PubMed Scopus (224) Google Scholar). We then compared the effect of DCEBIO to that of NS309 and 1-EBIO on SK2 channels. The three enhancers augment SK channel activity by increasing the apparent sensitivity to Ca2+, and concentration-response experiments were therefore performed in the inside-out configuration, which allows full control of the free [Ca2+]. Activation curves yielded an EC50 of 0.42 μm for Ca2+ (n = 8) (data not shown) with maximal activation obtained at 10 μm free Ca2+. The concentration-response curves for the three enhancers were performed at 200 nm free Ca2+, which activated 5% (0.048 ± 0.009) of the maximal SK current. Fig. 1A shows the control currents (Ctrl) as well as the currents obtained in the presence of the enhancers upon application of voltage ramps from -80 to +80 mV. For all compounds, potentiation was concentration-dependent but not voltage-dependent. The traces shown in Fig. 1A were obtained from experiments similar to the one shown in Fig. 1B, where the current at -75 mV is depicted as a function of time. The inside-out patch was exposed first to 0.01 μm Ca2+ to determine the background current level and subsequently to 10 μm Ca2+ to define the maximal current, which was used to normalize the currents. The concentration-response for DCEBIO was then determined at the subthreshold Ca2+ concentration of 200 nm, with control of maximal and background currents at the end of the experiment (Fig. 1B). The currents measured at the steady-state level of activation were plotted as a function of the DCEBIO concentration as illustrated in Fig. 1C, together with the values obtained for 1-EBIO and NS309 in similar experiments. Fig. 1C underscores the higher potency on SK2-mediated currents of DCEBIO (EC50 = 27 μm; n = 1.4) and even more remarkably of NS309 (EC50 = 0.62 μm; n = 1.4) when compared with 1-EBIO (EC50 = 453 μm; n = 1.6). Furthermore, all three compounds were found to have an efficacy of 100% with respect to saturating [Ca2+].Kv7 channels underlie IM, a current contributing to the generation of the mAHP in hippocampal neurons (26Storm J.F. J. Physiol. (Lond.). 1989; 409: 171-190Crossref Scopus (265) Google Scholar, 27Roche J.P. Westenbroek R. Sorom A.J. Hille B. Mackie K. Shapiro M.S. Br. J. Pharmacol. 2002; 137: 1173-1186Crossref PubMed Scopus (70) Google Scholar, 28Shah M.M. Mistry M. Marsh S.J. Brown D.A. Delmas P. J. Physiol. (Lond.). 2002; 544: 29-37Crossref Scopus (169) Google Scholar). We have therefore tested the SK channel enhancers on recombinant Kv7.2/7.3 channels. Fig. 1, D-F, illustrates the lack of effect of DCEBIO (100 μm), NS309 (10 μm), and 1-EBIO (1 mm) on these channels at the highest concentrations used for recordings in brain slices in this study. Fig. 1E shows the Kv7.2/7.3 currents activated by a 1-s-long step to -30 mV before and after the addition of the SK enhancers, as well as the reference Kv7 activator retigabine (3 μm). The time course in Fig. 1F depicts the current at the end of the step to -30 mV (open circles) and at the end of the step to -60 mV (open boxes). The bar diagram in Fig. 1D summarizes the effects from 5-6 experiments and shows that neither DCEBIO nor NS309 nor 1-EBIO significantly affect the Kv7.2/7.3 current, even at the highest concentrations tested.DC-EBIO and NS309 Selectively Increase IAHP but Not sIAHP in Hippocampal Pyramidal Neurons—When tested on CA1 pyramidal neurons in acute hippocampal slices, DCEBIO potentiated the SK-mediated Ca2+-activated K+ current IAHP without affecting the sIAHP at all concentrations tested (10 and 50 μm; n = 4) (data not shown) (100 μm; n = 7) (Fig. 2). In particular, 100 μm DCEBIO increased the amplitude of the SK-mediated IAHP by 195 ± 40% and prolonged its duration by 319 ± 42%. sIAHP co-exists with IAHP in CA1 pyramidal neurons but is mediated by channels clearly distinct from the SK channels underlying IAHP and of as yet unknown molecular identity (2Vogalis F. Storm J.F. Lancaster B. Eur. J. Neurosci. 2003; 18: 3155-3166Crossref PubMed Scopus (102) Google Scholar, 3Stocker M. Hirzel K. D'Hoedt D. Pedarzani P. Toxicon. 2004; 43: 933-949Crossref PubMed Scopus (70) Google Scholar, 4Stocker M. Nat. Rev. Neurosci. 2004; 5: 758-770Crossref PubMed Scopus (410) Google Scholar, 5Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (331) Google Scholar,9Bond C.T. Herson P.S. Strassmaier T. Hammond R. Stackman R.W. Maylie J. Adelman J.P. J. Neurosci. 2004; 24: 5301-5306Crossref PubMed Scopus (224) Google Scholar). Neither the sIAHP amplitude (118 ± 10%) nor its time constant of decay (112 ± 5%) were significantly increased by DCEBIO (Fig. 2). The remarkable potentiation of IAHP led to an increase of the charge transfer, estimated as the integral of the two AHP currents (IAHP + sIAHP), by 154 ± 16% (n = 7) (Fig. 2C). The specificity of the DCEBIO effect on neuronal SK channels was confirmed by the full block of the enhanced IAHP upon application of the SK channel blocker d-tubocurarine (curare, 100 μm) (Fig. 2A). sIAHP was instead identified by its suppression by noradrenaline (1 μm) (Fig. 2A), known to inhibit this current by activating the cAMP/protein kinase A pathway in hippocampal neurons (23Pedarzani P. Storm J.F. Neuron. 1993; 11: 1023-1035Abstract Full Text PDF PubMed Scopus (238) Google Scholar, 29Nicoll R.A. Science. 1988; 241: 545-551Crossref PubMed Scopus (448) Google Scholar). These results demonstrate that DCEBIO is a more potent enhancer of both recombinant and neuronal SK currents when compared with 1-EBIO.FIGURE 2Effect of DCEBIO on neuronal AHP currents. A,IAHP and sIAHP were measured in CA1 pyramidal neurons in response to depolarizing pulses activating Ca2+ influx through voltage-gated Ca2+ channels. IAHP was enhanced by application of DCEBIO (100 μm). The enhanced IAHP was blocked by the SK channel blocker d-tubocurarine (curare, 100 μm), whereas the remaining, unaffected sIAHP was inhibited by noradrenaline (1 μm). B, superimposition of IAHP and sIAHP traces, before (Control, gray) and after (DCEBIO, black) application of 100 μm DCEBIO, emphasizing the effect of this compound on the amplitude and time course of deactivation of IAHP. C, bar diagram summarizing the effect of 100 μm DCEBIO on the IAHP and sIAHP peak amplitudes (ampl.) and total integral of the two AHP currents in seven cells. * indicates statistical significance (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The rest of our study focuses on NS309, which displays an enhanced potency on recombinant SK2 channels compared with both 1-EBIO and DCEBIO (Fig. 1C). A crucial question is how effective and selective this compound is on SK channels in their native, neuronal environment. When tested on IAHP and sIAHP in CA1 pyramidal neurons in hippocampal slices, 10 μm NS309 induced a marked increase of IAHP amplitude with respect to the control currents recorded prior to application of the compound (Fig. 3A). IAHP was measured in isolation, upon inhibition of sIAHP by the cAMP analogue 8CPT-cAMP (50 μm). The relative increase in amplitude of the apamin-sensitive IAHP was 182 ± 22% (n = 8) (Fig. 3, A and C). NS309 had an even more prominent effect on the time constant of deactivation of IAHP, which was slowed by ∼6-fold, changing from 119 ± 19 ms to 654 ± 77 ms after NS309 application (n = 8). As a consequence, the charge transfer of IAHP measured as the integral of the current was increased by almost 10-fold (949 ± 165%; n = 8) (Fig. 3, A-C). By comparison, the application of the same concentration of DCEBIO (10 μm) resulted in an increase of the IAHP amplitude by 158 ± 20% and in an increase of its decay time constant by 149 ± 20% (n = 4) (data not shown). NS309 was applied for several minutes to yield a maximal and stable potentiation of IAHP, as illustrated by the time course of action of this drug (Fig. 3B). The augmentation of IAHP by NS309 was only scarcely reversible, even after prolonged wash out periods (data not shown). The application of NS309 (10 μm) did not affect the input resistance of the neurons (n = 18).FIGURE 3NS309 enhances the apamin-sensitive IAHP in CA1 pyramidal neurons. A and D,IAHP measured in isolation in the presence of 50 μm 8CPT-cAMP (left panels in A and D) is potentiated by the application of 10 μm NS309 (A) and even by 1 μm NS309 (D). The potentiated IAHP is fully blocked by 200 μm d-tubocurarine (A; Curare)or25 nm apamin (D). The right panels in A and D show a superimposition of scaled IAHP traces before (Control, black) and after (NS309, gray) application of 10 μm (A) and 1 μm NS309 (D), emphasizing the effect of this compound on the time course of deactivation of IAHP. B and E, time course of action of NS309 (10 μm in B; 1 μm in E) on the IAHP shown as charge transfer and estimated from the integral of each current trace. IAHP was elicited every 30 s. NS309 was applied until the potentiated IAHP reached a stable amplitude (ampl.) and area. The application of NS309 was followed by d-tubocurarine (d-TC; 200 μm) in B and apamin (25 nm) in E, which completely blocked the enhanced IAHP. In B, points were omitted after IAHP reached steady-state level to allow for testing of the access resistance. The diagrams in B and E are from the same representative cells shown in A and D. C and F, bar diagrams summarizing the effects of 10 μm NS309 (C) and 1 μm NS309 (F) on the IAHP peak amplitude and charge transfer in eight (C) and three cells (F).View Large Image Figure ViewerDownload Hi-res image Download (PPT)At a lower concentration (1 μm), NS309 had qualitatively similar but slightly less pronounced effects on both amplitude and charge transfer of IAHP. Thus, IAHP amplitude was increased to 139 ± 10% (n = 3) (Fig. 3, D and F). Similar to what was previously observed at higher concentrations, 1 μm NS309 slowed the deactivation of IAHP by ∼4-fold, increasing its time constant of decay (τ) from 86 ± 9 ms to 361 ± 39 ms (n = 3). As a consequence, the IAHP charge transfer increased by ∼5-fold (500 ± 18%; n = 3) (Fig. 3, D-F). Also at 1 μm, the action of NS309 developed slowly (Fig. 3E). The effect of NS309 can be entirely ascribed to an enhancement in the activity

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