Large-conductance Ca2+-activated K+ (BKCa) channels are widely distributed and play key roles in various cell functions. We previously reported the chemical synthesis of several benzofuroindole compounds that act as potent openers of BKCa channels. In this study, we investigated the mechanism of channel potentiation by one of the compounds, 7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (TBIC), using electrophysiological means. This chemical highly activated cloned BKCa channels from extracellular side independent of β subunits and regardless of the presence of intracellular Ca2+. The EC50 and Hill coefficient for rat BKCa channel α subunit, rSlo, were estimated as 8.9 ± 1.5 μM and 0.9, respectively. TBIC shifted the conductance-voltage curve of rSlo channels to more hyperpolarized potentials without altering its voltage dependence. Single-channel recording revealed that TBIC increased the open probability of the channel in a dose-dependent manner without any changes in single-channel conductance. Strong potentiation by TBIC was also observed for native BKCa channels from rat hippocampus pyramidal neurons. Thus, TBIC and the related benzofuroindole compounds can be useful tools to unravel the mechanism of this novel allosteric activation of BKCa channels.
Large-conductance Ca2+-activated K+ channels (BKCa or maxi-K channels) are a family of potassium-selective ion channels activated in response to membrane depolarization and are modulated by intracellular concentration of Ca2+ (for reviews, see Toro et al., 1998; Weiger et al., 2002). These channels are widely expressed in many different types of both excitable and nonexcitable cells. They have significant physiological roles in regulation of frequency of firing, action potential afterhyperpolarization, and neurotransmitter release (Vergara et al., 1998; Kaczorowski and Garcia, 1999). Their activities play a pivotal role in the negative feedback control of intracellular Ca2+ concentration and protect neuronal cells from excess Ca2+ influx through the voltage-dependent Ca2+ channels during pathophysiological environments (Lawson, 2000). In hyperactive neuronal cells, activation of BKCa channels is thought to be required for restoring resting membrane potential by down-regulating the activity of voltage-dependent Na+ and Ca2+ channels (Brenner et al., 2000; Jaggar et al., 2000). In addition, activation of BKCa channel significantly contributes to action potential repolarization and afterhyperpolarization during excitation-contraction coupling in smooth muscle cells (Ohi et al., 2001).
For these reasons, BKCa channel openers may be effective in protecting neuronal cells from damage during or after ischemic stroke and in down-regulating hyperactivity in smooth muscle cells (for review, see Shieh et al., 2000). BKCa channels are composed of two different subunits: the poreforming α subunit and the auxiliary β subunits. Although channels formed only by four α subunits can be functional, β subunits alter the biophysical and pharmacological properties of homomeric channels, including Ca2+ and voltage sensitivity, and gating kinetics (Valverde et al., 1999; Wallner et al., 1999; Xia et al., 1999; Qian et al., 2002; Ha et al., 2004). Several compounds have been developed and reported to be BKCa channel openers (e.g., dehydrosoyasaponin-I, maxik-diol, NS-1619, BMS-204352, 17β-estradiol, ethylbromide tamoxifen, pimaric acid, and epoxyeicosatrienoic acids) (Vergara et al., 1998; Valverde et al., 1999; Coghlan et al., 2001; Dick et al., 2002; Imaizumi et al., 2002). Although some synthetic activators, such as NS-1619 and BMS-204352, act on the α subunit, other openers of BKCa channels, including dehydrosoyasaponin-I and 17-β-estradiol, require β subunit for their action (Giangiacomo et al., 1998; Valverde et al., 1999). Several activators derived from natural products such as dehydrosoyasaponin-I are impermeable to the cell membrane and act only on intracellular side of BKCa channels (Kaczorowski and Garcia, 1999).
Benzofuroindole analogs were shown to relax smooth muscles of bladder possibly via the activation of BKCa channels (Butera et al., 2001). In our previous study, we reported the chemical synthesis of new benzofuroindole derivatives and the screening for their efficacy on cloned BKCa channels expressed in Xenopus laevis oocytes (Gormemis et al., 2005). One of the initial compounds, referred to as “compound 8,” highly up-regulated the activity of BKCa channels. In the present study, we further investigated this compound, 7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (TBIC) (Fig. 1, inset), to reveal its mechanism of action with respect to channel activation. We found that TBIC activated BKCa channel in a dose-dependent manner at micromolar concentration from extracellular side and its activation was independent of β subunits. TBIC shifted the conductance-voltage relationship of the channel to more hyperpolarized potentials without altering voltage dependence. In addition, single-channel analysis showed that the compound increased the open probability (Po) of the channel by altering gating kinetics without affecting its single-channel conductance.
Materials and Methods
Functional Expression of Cloned BKCa Channel Subunits in X. laevis Oocytes. The cDNAs of rat BKCa channel α subunit (rSlo), human β1 subunit (hβ1), and rat β4 subunit (rβ4) were subcloned into pGH vector for expression in X. laevis oocytes. The sequence information of rSlo, hβ1, and rβ4 used in this study are listed with GenBank under the accession numbers AF135265 (Ha et al., 2000), NM004137 (Meera et al., 1996), and AY028605 (Ha et al., 2004), respectively. Each cDNA was subcloned into pGH expression vector containing the 5′- and 3′-untranslated regions of X. laevis β-globin gene, because it is known to enhance the protein expression of certain mammalian messages in X. laevis oocytes (Liman et al., 1992). cRNA of each construct was prepared in vitro as described in previous studies (Ha et al., 2000, 2004). Plasmid DNA was purified (midi-prep columns; QIAGEN, Valencia, CA) and digested with a restriction enzyme, NotI. RNA was synthesized from linearized plasmid DNA using T7 RNA polymerase in the presence of a cap analog, m7G(5′)ppp(5′)G, and NTPs. Oocytes of stages V to VI were surgically removed from the ovarian lobes of anesthetized female X. laevis frogs (Xenopus I, Dexter, MI) and transferred into Ca2+-free OR medium (86 mM NaCl, 1.5 mM KCl, 2 mM MgCl2, 10 mM HEPES, and 50 μg/ml gentamicin at pH 7.6). The follicular cell layer was removed by incubating oocytes in Ca2+-free OR medium containing 3 mg/ml collagenases (Worthington Biochemicals, Freehold, NJ) for 2 h. The oocytes were then washed extensively with and kept in ND-96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and 50 μg/ml gentamicin at pH 7.6) at 18°C. Each oocyte was injected with 50 nl containing approximately 1 ng of cRNA for single-channel and 50 ng for macroscopic current recordings, respectively, using a microdispenser (Drummond Scientific, Broomall, PA). Injected oocytes were incubated at 18°C for 3 to 5 days in sterile ND-96 medium. Immediately before patch-clamp experiments, the vitelline membrane was removed manually with fine forceps.
Primary Culture of Pyramidal Neurons in Rat Hippocampus. Primary culture of rat hippocampal pyramidal neuron has been described previously (Abdel-Hamid and Baimbridge, 1997). Sprague-Dawley rats were anesthetized and decapitated at embryonic day 18. The hippocampus was surgically dissected and isolated from fetal brain and minced in Ca2+, Mg2+-free Hanks' balanced salt solution (Invitrogen, Carlsbad, CA). The tissue was digested with 0.1% trypsin-EDTA to the medium and then stopped by adding the same volume of fetal bovine serum. Cell dissociation was accomplished by gentle mechanical agitation. After removing cell debris by centrifugation for 2 min, the cell pellet was resuspended in minimal essential medium (Invitrogen) with 10% of a 1:1 mixture of heat-inactivated horse and fetal bovine serum. The dissociated cells were plated on 35-mm tissue culture dishes (Falcon; BD Biosciences Discovery Labware, Franklin Lakes, NJ) coated with poly-l-lysine (Sigma-Aldrich, St. Louis, MO). The cells were incubated at 37°C in a humidified, 5% CO2 incubator. The medium was renewed after 24 h and half-exchanged twice a week with feeding media. The feeding media contained apo-transferrin (200 μg/ml; Sigma-Aldrich), insulin (1 μg/ml; Sigma-Aldrich), sodium selenite (30 nM; Sigma-Aldrich), putrescine (100 μM; Sigma-Aldrich), progesterone (20 μM; Sigma-Aldrich), 5% equine serum (Hyclone Laboratories, Logan, UT), and minimal essential medium (Invitrogen). The cultured neurons were used for electrophysiological recording during 12 to 18 days in culture.
Electrophysiological Recordings and Data Analysis. All single-channel and macroscopic current recordings were performed using gigaohm-seal patch-clamp method in either excised inside-out or outside-out configuration (Hamil et al., 1981). Patch pipettes were fabricated from borosilicate glass (WPI, Sarasota, FL) and fire-polished to the resistance of 2 to 5 MΩ for macroscopic patches and 5 to 8 MΩ for single-channel recording, respectively. For single-channel recordings, patch pipettes were coated with beeswax to reduce electrical noise. The channel currents were amplified using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 1 or 2 kHz using a four-pole Bessel filter, and digitized at a rate of 10 or 20 points/ms using a Digidata 1200A (Axon Instruments). No series resistance compensation was used and linear leak currents were subtracted from macroscopic currents.
In recording rat hippocampal neurons, pyramidal cells were distinguished from mixed population including glia by morphological features. Before electrophysiological recording, the culture medium was washed multiple times with Na+-saline solution containing 140 mM NaCl, 4 mM KCl, 0.24 mM KH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM d-glucose, and 10 mM HEPES, pH 7.4. Cells were then rinsed in symmetrical 124 mM K+ solution for recordings. Patch recordings were made from the soma of cultured pyramidal neurons in the outside-out patch configuration at room temperature. From a total of 76 patches in neuron cells, 22 recordings showed single-channel level activities of BKCa channels
Single rSlo or native BKCa channels were readily activated at high concentration of intracellular Ca2+ and by briefly delivered membrane potentials to 100 mV. For single-channel analysis, transitions between closed and open states were determined by setting the threshold at half of the unitary current amplitude. To determine the single-channel conductance of expressed channels, mean amplitudes of channel currents were obtained from histograms fitted with Gaussian distributions, and the mean currents were plotted against transmembrane voltages. Slope-conductance values were obtained from linear regression.
Macroscopic currents of expressed channels were activated by voltage-clamp pulses delivered from a holding potential of -50 mV for [Ca2+]i at 0 μM, -100 mV at 1 μM, and -150 mV at 5 μM, respectively, to membrane potentials ranging usually from -150 to 200 mV in 10- or 20-mV increments. Solutions for both single- and macroscopic channel recordings contained gluconates as a nonpermeant anion to prevent the activation of endogenous calcium-activated chloride channels. The intracellular and extracellular solutions contained the following components unless specified otherwise: 120 mM potassium gluconate, 10 mM HEPES, 4 mM KCl, and 5 mM EGTA, pH 7.2. To provide required free [Ca2+]i, the appropriate amount of total Ca2+ to add to the intracellular solution was calculated using the program MaxChelator (Patton et al., 2004; http://www.standford.edu/~cpatton/maxc.html). To compare the channel characteristics accurately, an identical set of intracellular solutions was used throughout the entire experiments. Commercial software packages, such as Clampex 8.0 or 8.1 (Axon Instruments) and Origin 6.1 (OriginLab Corp., Northampton, MA), were used for the acquisition and the analysis of both single-channel and macroscopic recording data.
Reagents. The chemical synthesis of TBIC was described in a previous study where the compound was referred to as “compound 8” (Gormemis et al., 2005). TBIC was dissolved in dimethyl sulfoxide (Sigma-Aldrich) in 500 mM stock solutions and stored at -20°C until use. All reagents, buffered in bath solution to pH 7.2, were applied directly to membrane patches by gravity perfusion with 10 volumes of recording chamber at a flow rate of 1 to 2 ml/min.
Statistical Analysis. All data were presented as means ± S.E.M., where n indicates the number of independent experiments. For each data set, the statistical significance of the difference was tested using analysis of variance for independent observations. In all cases, P < 0.05 was considered significant. Each macroscopic current trace represents an average of three records in succession.
Effects of TBIC, a Benzofuroindole, on Macroscopic Currents of a Cloned BKCa Channel Expressed in X. laevis Oocytes. To understand the potentiation mechanism of TBIC on BKCa channels, we first characterized its effects on macroscopic currents of rat BKCa channel α subunit (rSlo) expressed in X. laevis oocytes. As shown in Fig. 1, the time-dependent effects of TBIC were monitored from a membrane patch containing hundreds of rSlo channels by applying 50-ms step-pulses to 50 mV every second. Small rSlo currents were initially evoked by the voltage pulses (Fig. 1a), because the intracellular (pipette) solution contained only 0.5 μM Ca2+. When 20 μM TBIC was applied on to the extracellular side of membrane patch, a large increase in the rSlo currents was observed. The full potentiation was achieved in two phases: the initial fast-activation occurring within 10 s (Fig. 1b) and the following slower phase over a few minutes (Fig. 1c). Upon cessation of TBIC application, the channel activity rapidly decreased within in 10 s (Fig. 1d). In some cases, the channel activity did not return fully to the pretreatment level, and a slight increase in channel activity remained (Fig. 1, a and d). However, the fast onset and offset of TBIC effects in cell-free recording condition suggest strongly that the compound interacts directly with the channel from extracellular side and enhances its activity.
Concentration Dependence of TBIC on Macroscopic rSlo Channels and Effects of β Subunits. We then determined the concentration dependence of TBIC on macroscopic rSlo currents. As increasing concentrations of TBIC were applied to the extracellular side of membrane patches, the activation rate as well as the level of steady-state current was increased in a concentration-dependent manner (Fig. 2A). To compare the effects of TBIC measured at specific concentrations, we normalized the ionic currents in the presence of a given concentration of TBIC (I) with the current in the absence of TBIC (I0). The relative -fold increase (I/I0) was plotted against the concentration of TBIC and fitted with a Hill equation (Fig. 2A, right). Although we were not able to obtain a concentration of TBIC higher than 300 μM because of its solubility in water, we noticed that TBIC-induced current increases reached plateau levels at around 100 μM (Fig. 2A). The half-effective concentration (EC50) and Hill coefficient (n) were obtained by fitting individual titration data to Hill equation (Fig. 2 legend), and the statistical means and standard errors were calculated using the values obtained from more than three independent experiments. The EC50 and n of extracellular TBIC for rSlo channels were determined as 8.9 ± 1.5 μM and 0.9 ± 0.1, respectively (Fig. 2A, •; n = 5). We also measured the effects of TBIC using inside-out patch configuration to determine whether this compound also affects channel activity from the intracellular side. The intracellular Ca2+ concentration was fixed at 2 μM to activate rSlo channels, and different concentrations of TBIC were added to the intracellular side of the membrane. Although intracellular TBIC also increased rSlo currents with a similar apparent affinity, EC50 of 12.7 ± 5.8 μM, its -fold increase was much smaller than that obtained from extracellular side (Fig. 2A, ▪; n = 3).
The functional characteristics of BKCa channels are altered by auxiliary β subunits, and the efficacy of some activators and inhibitors is greatly influenced by coassembly of β subunits. Thus, we asked whether the potentiating effects of TBIC are affected by coexpression of β subunits. We expressed rSlo together with either human β1 or rat β4 subunit in X. laevis oocytes and measured the channel currents in the presence of different concentrations of extracellular TBIC. We usually used 12-fold molar excess of the hβ1 and rβ4 transcripts to ensure the sufficient coassembly of β subunits with rSlo subunit. The activities of both rSlo/hβ1 (Fig. 2B; n = 4) and rSlo/rβ4 (Fig. 2C; n = 4) were increased by micromolar concentration of the compound in a concentration-dependent manner. EC50 values of extracellular TBIC were determined as 10.0 ± 1.5 μM for rSlo/hβ1 heteromeric channels (Fig. 2B, ▴) and 4.5 ± 0.7 μM for rSlo/rβ4 heteromeric channels (Fig. 2C, ♦) with Hill coefficients of 0.7 ± 0.1 and 1.1 ± 0.2, respectively, indicating that minor but statistically significant differences in both the apparent affinity and the cooperativity of TBIC were produced by the coexpression of different β subunits. However, these results indicate that TBIC can potentiate BKCa channel without the coassembly of β subunits and argue that the receptor site of TBIC locates within the main subunit of BKCa channel, the Slo protein.
Efficacy of Extracellular TBIC in Different Concentrations of Intracellular Ca2+. Because the activity of BKCa channels is modulated by intracellular Ca2+, we wondered how intracellular Ca2+ interplay affects the action of TBIC from the extracellular side and whether TBIC can activate channel in the absence of intracellular Ca2+.
We tested the effects of extracellular TBIC at two different concentrations in the absence of intracellular Ca2+ (Fig. 3A). To keep [Ca2+]i in the subnanomolar range, 5 mM EGTA was supplemented into the intracellular pipette solution. Even in the absence of intracellular Ca2+, the activation of rSlo channels was observed at extreme positive voltages, greater 100 mV (top row, control). The application of 30 μM extracellular TBIC greatly potentiated the channel activity, and large outward currents were measured (top row, 30 μM [TBIC]o). As illustrated in the current-voltage (I-V) relationship (top row, right), TBIC shifted the threshold voltage of channel activation to less positive voltages, and the channel currents were observed at voltages as low as 60 mV in 30 μM TBIC (○). In the presence of 100 μM TBIC, I-V relationship of rSlo channel was further shifted, and the currents were activated near 20 mV (▵). Robust outward currents were consistently recorded at the membrane voltages greater than 20 mV. In the presence of 1 μM [Ca2+]i,30 μM TBIC also shifted the I-V relationship to less positive voltages, and large tail currents evoked by rSlo were observed (middle row, 30 μM [TBIC]o). Because the channels were activated near -60 mV in the presence of 100 μM TBIC, we were able to detect inward currents at negative test-voltages (middle row, 100 μM [TBIC]o; right, half-filled triangles). Inward currents were even more dramatic when [Ca2+]i was increased to 5 μM (Fig. 3A, bottom row). With the treatment of 30 μM TBIC, the activation of rSlo channel became evident even at -100 mV, and the peak inward K+ currents was observed at around -40 mV (•). The most impressive effect of TBIC was seen at 100 μM in the presence of 5 μM [Ca2+]i. rSlo channels were activated robustly from -130 mV, and resulted in large inward currents that peaked near -70 mV (bottom row, 100 μM [TBIC]o; right, ▴). It is intriguing that the linear I-V relationship, a characteristic of the BKCa channel, could be appreciated by measuring steady-state currents instead of instantaneous tail currents.
The effects of TBIC on macroscopic rSlo channel are summarized in Fig. 3, B and C. Sets of conductance-voltage (G-V) relationships and their half-activation voltages (V½) were shown for four different concentrations of TBIC—0 μM (squares), 10 μM (circles), 30 μM (triangles), and 100 μM (inverted triangles)—in the presence of three different concentrations of [Ca2+]i: 0 μM (open), 1 μM (half-filled), and 5 μM (filled). The significant effects of TBIC on G-V relationship were observed at the concentrations higher than 1 μM, and the further increase resulted in steady shifts in G-V curves toward the negative direction. Although the addition of TBIC up to 100 μM shifted the G-V curve -134 mV, from 210 ± 7.5 to 76 ± 4.0 mV, in the absence of Ca2+, a smaller shift of approximately -83 mV was observed by identical concentration of TBIC at 5 μM [Ca2+]i. Despite the large shifts in their positions, no significant change was detected in the steepness of G-V curves, the measure of voltage dependence in channel activation, within a set of identical [Ca2+]i. These results indicate that BKCa channel can be activated by TBIC in the absence of intracellular Ca2+ and that the potentiation is because of the shift of its voltage-activation profile to a more negative range without affecting its voltage sensitivity.
Effects of Extracellular TBIC on Single-Channel Currents of rSlo. To obtain further insight into the mechanism of action, the effects of TBIC were investigated at the single-channel level. For each outside-out patch, we depolarized the membrane voltage to more than 80 mV to significantly activate rSlo channels to count the number of channels in the membrane. Only those patches containing a single rSlo channel were used for subsequent experiments. Single-channel recordings were performed at various durations to obtain accurate values of steady-state kinetic constants: 5 to 8 min at hyperpolarized voltages and 0.5 to 2 min at depolarized voltages. Representative traces of a single rSlo channel in the absence and the presence of 20 μM TBIC were shown in Fig. 4A. The channel currents were recorded at 2 μM [Ca2+]i at the specified membrane voltages. The opening of the channel was highly dependent on the membrane voltages as expected. The gating behavior, however, was dramatically altered by the application of 20 μM TBIC to the extracellular side (Fig. 4A). Although the rSlo channel rarely opens in control solution at -25 mV, the addition of 20 μM TBIC to the extracellular side made the channel open readily. In addition, Po was greatly increased at 25 mV by TBIC treatment. To examine the effects of TBIC on single-channel conductance, we measured the unitary current amplitudes of rSlo at various membrane voltages in the absence and presence of TBIC, and the single-channel I-V relationships were plotted (Fig. 4B). Single-channel conductances were estimated as 246.1 ± 5.4 pS in control and 247.7 ± 4.2 pS in TBIC, respectively, indicating that the drug did not affect the single-channel conductance of rSlo. We then analyzed the effects of TBIC on single-channel Po of the channel. Under control conditions, the increase in Po was highly dependent on membrane voltage in the range of -75 and 25 mV, and Po values were well fitted by a Boltzmann function (Fig. 4C, open symbols). The voltage required for half-maximum activation, V½, was determined as 38.1 ± 3.1 mV. In the presence of 20 μM TBIC, the Po versus voltage curve shifted in a parallel manner to the negative direction by 33 mV and V½ was estimated as 4.8 ±3.4 mV. It is worth noting that the slopes of voltage activation curve remained un-changed, 0.031 for control currents and 0.035 for TBIC-potentiated currents, respectively. These results are in a good agreement with the previous findings in macroscopic rSlo currents, where the G-V curve was also shifted in parallel by TBIC (Fig. 3B) further indicating that the potentiation of BKCa channel activity by TBIC is the direct result of the Po increase.
Effects of TBIC on Single BKCa Channels of Cultured Hippocampus Pyramidal Neuron. Because BKCa channels in brain neurons are known to express as a heterogenous population because of extensive RNA splicing, coassembly with β4 subunit, and post-translational modifications, we wondered whether the activity of native neuronal BKCa channels could also be potentiated by TBIC. We thus performed outside-out patch recording on pyramidal neurons of rat hippocampus. Although in most instances more than one BKCa channels was observed in single patches, we were able to determine unambiguously the number of channels using brief depolarizing pulses. Representative current traces, obtained from a membrane patch containing four neuronal BKCa channels, were shown in Fig. 5. Similar to the single-channel recordings of rSlo channel expressed in X. laevis oocytes (Fig. 4A), neuronal BKCa channel showed high single-channel conductances and steep voltage dependence. Although extracellular treatment of 20 μM TBIC highly increased the Po of native BKCa channels at all membrane voltages tested, the effects were more dramatic for negative voltage ranges. Although channel openings were observed only rarely in control solution containing 2 μM [Ca2+]i at -25 mV, the opening of all four BKCa channels were frequently observed in the presence of 20 μM TBIC. The time-dependent effects of TBIC on neuronal BKCa channels were compared at two different voltages, 25 and -25 mV (Fig. 5, B and C). The Po increase induced by TBIC treatment was almost instantaneous and readily reversible. Thus, we can conclude that this compound highly potentiates native BKCa channels from rat hippocampal neurons as well as the cloned rSlo channel.
In the present study, we characterized the effects of a benzofuroindole derivative, TBIC, on BKCa channels. This compound highly activates both native and cloned BKCa channels in a dose-dependent manner from the extracellular side of the membrane at low micromolar concentrations. TBIC potentiates the channel activity by shifting its Po-voltage relationship to more negative voltages without affecting the single-channel conductance or the voltage sensitivity. However, the action of TBIC is noncooperative, and thus the dose-response curve of TBIC is best fitted with Hill coefficient of 1.
TBIC is a derivative of benzofuroindole with a carboxylic acid and a trifluoromethyl moiety at the position 1 and 7, respectively (Fig. 1, inset). In our previous study, we showed that a negative charge at the position 1 and a strong electron-withdrawing group at the position 7 are critical for the activity of TBIC (Gormemis et al., 2005). Because TBIC acts from extracellular side of the membrane and does not require β subunit for its action, we assume that the binding site of this negatively charged compound is in the extracellular region of the main subunit, Slo protein. The binding sites have not been identified for the previously known BKCa channel activators, especially those targeting the α subunit, such as NS-1619 and BMS-204352 (for reviews, see Starrett et al., 1996; Coghlan et al., 2001). Therefore, it remains unclear whether benzofuroindoles act on the identical site for their action. Thus, it is our desire to localize the receptor site for benzofuroindoles and to understand the molecular mechanism of this novel modulation. Slo protein, the α subunit of BKCa channel, has seven membrane-spanning regions (S0-S6) with an amino terminus of approximately 8 kDa and three loops facing the extracellular side of the membrane. The β1 subunit confers its potentiating effect by interacting with the extracellular N terminus and the first transmembrane helix, S0. Therefore, it is conceivable that the TBIC may interact with the same region and activates the channel activity (Meera et al., 1997; Cox and Aldrich, 2000; Dick et al., 2001). It remains to be seen whether the deletion of the N-terminal region removes the effect of TBIC. There has been much progress in predicting optimum binding site of a given compound based on detailed structure-activity relationship (Dick et al., 2001). The structure-activity profiles of benzofuroindole derivatives, reported in the previous studies (Butera et al., 2001; Gormemis et al., 2005), can be used to predict the potential sites for TBIC on BKCa channel α subunit.
TBIC can activate native neuronal BKCa channels as well as the heterologously expressed Slo channels. At the single-channel level, the compound never failed to potentiate the BKCa channels in excised membrane of cultured neuron. We noticed that the potentiating effects of TBIC on neuronal BKCa channel might be even greater than those on cloned BKCa channel, although we were not able to quantify this precisely. This variability might be the result of the splicing variants of Slo or post-translational modification of BKCa channels in specific neurons. In a previous report, benzofuroindole derivatives were described to activate BKCa channels of smooth muscles only in rat bladder but not in arteries (Butera et al., 2001). Because the efficacy of TBIC was not significantly affected by different β subunits (Fig. 2, B and C), we wonder whether differential splicing of the Slo message affects the efficacy of TBIC on BKCa channels in different tissues. Thus, it will be important to assess the effects of TBIC in BKCa channels in different tissues and to correlate the efficacy with the nature of BKCa channels in the future.
In conclusion, our results provide the mechanistic details of benzofuroindole action on BKCa channel as a potentiator. Because TBIC and related compounds can activate BKCa channels so effectively, they can be used as experimental probes for a new allosteric site important for channel activation and be served as lead compounds for developing synthetic activators of BKCa channel for pharmaceutical purposes.
We are grateful to the members of Laboratory of Molecular Neurobiology (Gwangju Institute of Science and Technology) for valuable comments and timely help throughout the work and to M. Walden (Brandeis University. Waltham, MA) for critical reading of the manuscript.
This research was supported by grant R01-2002-000-00354-0 from Korea Science and Technology Foundation and grant 21C Frontier, 03K2201-00320 from the Ministry of Science and Technology of Korea (to C.-S.P.) and grant Korea Health 21 R&D Project, 0405-NS01-0704-0001-13 from the Ministry of Health and Welfare (to Y.-C.K.).
ABBREVIATIONS: BKCa, large-conductance Ca2+-activated K+ channel; TBIC, 7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid; rSlo, rat BKCa channel α subunit; hβ1, human β1 subunit of BKCa channel; rβ4, rat β4 subunit of BKCa channel; I-V, current-voltage; G-V, conductance-voltage; NS-1619, 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one; BMS-204352, (3S)-3-(5-chloro-2-methoxyphenyl)-3-fluoro-6-(trifluoromethyl)-1,3-dihydro-2H-indol-2-one.
↵1 Current affiliation: Department of Pharmacology and Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas.
- Received June 28, 2005.
- Accepted December 6, 2005.
- The American Society for Pharmacology and Experimental Therapeutics