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A novel extract SB-300 from the stem bark latex of Croton lechleri inhibits CFTR-mediated chloride secretion in human colonic epithelial cells

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A novel extract SB-300 from the stem bark latex of Croton lechleri inhibits CFTR-mediated chloride secretion in human colonic epithelial cells
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  Journal of Ethnopharmacology 93 (2004) 351–357 A novel extract SB-300 from the stem bark latex of   Croton lechleri  inhibitsCFTR-mediated chloride secretion in human colonic epithelial cells Horst Fischer a , Terry E. Machen b , Jonathan H. Widdicombe a , Thomas J.S. Carlson c , d ,Steven R. King c , John W.S. Chow c , Beate Illek  a , ∗ a Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA b  Department of Molecular and Cell Biology, 231 Life Science Addition, University of California Berkeley, Berkeley, CA 94720, USA c Shaman Pharmaceuticals, Inc., 213 East Grand Avenue, South San Francisco, CA 94080, USA d  Department of Integrative Biology, 1001 Valley Life Sciences, University of California Berkeley, Berkeley, CA 94720, USA Accepted 3 April 2004Available online 1 June 2004 Abstract An oligomeric proanthocyanidin (SP-303) extracted from the bark latex of the tree  Croton lechleri  (family Euphorbiaceae) is a potentinhibitor of cholera toxin-induced fluid accumulation and chloride secretion. The manufacturing process for SP-303 was optimized andsimplified to produce an increased yield of the herbal extract. The novel extract (named SB-300) contained on average 70 . 6 ± 7 . 2% SP-303 byweight (mean ± S . D . ;  n = 56 lots). Here, we describe the effectiveness of SB-300 on cAMP-regulated chloride secretion, which is mediatedby the cystic fibrosis transmembrane conductance regulator Cl − channel (CFTR) in human colonic T84 cells. Exposure of the apical surfaceto SB-300 blocked forskolin-stimulated Cl − secretion by 92 . 2 ± 3 . 0% with a half-maximal inhibition constant ( K  B ) of 4 . 8 ± 0 . 8  M. ForSP-303, stimulated Cl − currents were decreased by 98 . 0 ± 7 . 2% and  K  B  averaged 4 . 1 ± 1 . 3  M. There was no significant difference betweenthe blocking kinetics of SP-303 and SB-300. Forskolin-stimulated whole cell Cl − currents were effectively blocked by extracellular additionof SB-300 (63 ± 8 . 5%;  n  =  3) and to a similar extent by SP-303 (83  ±  0.6%;  n  =  2; at 50  M each). Both extracts inhibited a time-and voltage-independent Cl − conductance, which indicated the involvement of CFTR Cl − channels. We conclude that both SP-303 (used inProvir ® ) and SB-300 (used in NSF Normal Stool Formula TM ) are novel natural products that target the CFTR Cl − channel. SB-300 is a lowcost herbal extract and may present a complementary and alternative medicine approach for the treatment of fluid loss in watery diarrhea.© 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Croton lechleri ; Proanthocyanidin; Herbal extract; Dietary supplement; Diarrhea; Cystic fibrosis transmembrane conductance regulator chloridechannel; Chloride channel blocker 1. Introduction Secretory diarrhea and the associated massive loss of salt and water are a worldwide problem of enormous mag-nitude. It is the leading cause of death in infants in thedeveloping world (Giannella, 1981) and currently accounts for an estimated 2.5 million deaths in children under 5 yearsof age (Kosek et al., 2003). Childhood diarrhea is mostcommonly caused by rotavirus infections (World HealthOrganization, 1999) of both the epithelium and the entericnervous system that leads to secretion of both salt andwater (Lundgren et al., 2000). Coliform bacteria (such as  Escherichia coli ,  Bacillus cereus ,  Campylobacter fetus  and Vibrio parahemolyticus ) also cause watery diarrhea primar- ∗ Corresponding author. Tel.: + 1-510-450-7699; fax: + 1-510-450-7910.  E-mail address:  billek@chori.org (B. Illek). ily by release of bacterial enterotoxins. It has been shownthat both the heat-stable and the heat-labile  Escherichia coli enterotoxins increase intracellular concentrations of adeno-sine 3 ′ ,5 ′ -cyclic monophosphate or guanosine 3 ′ ,5 ′ -cyclicmonophosphate which upregulate protein kinase A (PKA)activity (Forte et al., 1992; Chao et al., 1994). PKA is the prime regulator of the cystic fibrosis transmembraneconductance regulator (CFTR) Cl − channel (Cheng et al.,1991; Berger et al., 1991).CFTR functions as an apical membrane Cl − channel inepithelia and modulates cAMP-dependent fluid secretion.CFTR is mutated and dysfunctional in the autosomal re-cessive inherited disease cystic fibrosis (CF). MessengerRNA encoding for CFTR and localization of CFTR proteinto the apical membrane have been detected in the duo-denum, jejunum, ileum and colon (Riordan et al., 1989;Crawford et al., 1991). The small intestine of CF patients 0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2004.04.005  352  H. Fischer et al./Journal of Ethnopharmacology 93 (2004) 351–357  exhibits decreased Cl − and fluid secretion that results inmeconium ileus in 10% of CF newborns and accumulationof mucus and intestinal obstruction in 20% of adult CFpatients (Grubb and Boucher, 1997). In contrast, excessive activation of the CFTR Cl channel in the intestinal epitheliacauses secretory diarrhea. It has been proposed that mu-tations in the CFTR gene and its resulting defective Cl − channel function presented an advantage for heterozygouscarriers during episodes of diarrhea, thereby promoting theretention and spread of CFTR mutations in the population(Rodman and Zamudio, 1991; Gabriel et al., 1994).Current antidiarrheal drugs largely rely on reductionof smooth muscle motility and contractility. Currently, nodrug treatments are available that specifically target andblock the CFTR chloride ion channel. However, the pri-mary cause of diarrhea is due to the cAMP-dependenthyper-activation of CFTR and drugs directed at this siteshould show greater effectiveness and selectivity. The redviscous bark latex of the tree  Croton lechleri  Muell.-Arg.(Euphorbiaceae) (“sangre de drago”) is known for its medic-inal properties in the treatment of diarrhea, inflammation,insect bites, viral infections and wounds (Jones, 2003). The sap of sangre de drago has been taken orally by indige-nous people of the Amazon basin of South America totreat different types of diarrhea, including cholera (Carlsonand King, 2000). Previously, a compound, termed SP-303,was isolated from the bark latex of   Croton lechleri  byUbillas et al. (1994), using a bioassay-guided fractionationand selection procedure. SP-303 is a purified, heteroge-neous proanthocyanidin oligomer. The basic monomersare mainly ( + )-gallocatechin and ( − )-galloepicatechin,and to a smaller amount ( + )-catechin and ( − )-epicatechin.The oligomer consists of linearly linked monomers (onaverage heptamers, ranging from penta- to 11-mers) of varying ratios (Ubillas et al., 1994). SP-303 was re-cently shown to inhibit cAMP-mediated Cl − and fluidsecretion by T84 and Caco-2 cells, as well as in intactloop studies in cholera toxin treated mice (Gabriel et al.,1999).The safety and efficacy of orally administered SP-303 forthe symptomatic treatment of diarrhea has been evaluated intravelers (DiCesare et al., 2002) and in patients with AIDS (Holodniy et al., 1999). In a double blind, randomized, placebo-controlled study among travelers to Jamaica andMexico, SP-303 shortened the duration of acute secretory di-arrhea by 21% without causing post-treatment constipation(DiCesare et al., 2002). An estimated 50–60% of patients with human immunodeficiency virus infection experiencediarrhea during their illness which is caused by medications,including protease inhibitors (Sherman and Fish, 2000). Ina double blind, randomized, placebo-controlled phase II hu-man clinical trial for treatment of HIV-associated diarrhea,SP-303 reduced stool weight and abnormal stool frequency(Holodniy et al., 1999). SP-303 was safe, well tolerated and had a significant impact on quality of life (Holodniy et al.,1999).The conventional isolation procedure of SP-303 from thetree bark resulted in a relatively low yield ( ∼ 2.5%) whichprompted us to optimize and, at the same time, simplify themanufacturing process. This resulted in the novel extracttermed SB-300, which is now commercially available asa dietary supplement (NSF/Normal Stool Formula TM ), de-veloped by Shaman Pharmaceuticals Inc. SB-300 containson average 70% SP-303 by weight. The remaining 30%of SB-300 contain, at least in part, proanthocyanidins thathave not been specifically characterized. The purpose of thepresent study was to compare the efficacy and potency of SB-300 and SP-303 on cAMP-regulated Cl − secretion inhuman intestinal T84 epithelial cells. 2. Methodology 2.1. Preparation of SB-300 SB-300 is derived from liquid bark latex of the  Crotonlechleri  tree. Basification of the liquid latex is achieved byaddition of NaOH to produce a neutral to slightly alkalinepH that results in a precipitate that is filtered out. The re-maining liquid filtrate goes through ultrafiltration to producea retentate that is mixed with CM sepharose resin and thenfiltered to yield a filtrate that is tray dried to produce thesolid SB-300. By HPLC analysis, SB-300 (Lot SL-1315)contained 65% purity of SP-303 by weight. SP-303 is anoligomeric proanthocyanidin compound in the polypheno-lic chemical class with a MW of approx. 2200Da (Ubillaset al., 1994). Polyphenol and Karl Fisher analyses showedthat the remaining 30% of the SB-300 contain other nonSP-303 polyphenolic compounds that have not been specif-ically characterized, as well as moisture. 2.2. Cell culture The human colonic epithelial cell line T84 (AmericanTypeCellCulturecollection,Manassas,Virginia)wasgrownin a 1:1 mix of Dulbeccos’s modified Eagle’s medium andHam’s Nutrient mixture F-12 (Life Technologies, GrandIsland, New York) supplemented with 10% fetal calf serum(Hyclone, Logan, Utah), 100U/ml penicillin, 100mg/mlstreptomycin and 4mM  l -glutamine. T84 cells were cul-tured at 37 ◦ C in a humidified atmosphere of 95% air and5% CO 2 . T84 cells were used at passages 65–68. For Uss-ing chamber studies, T84 cells were seeded on permeableand collagen-coated cell culture inserts with a 0.45  mpore size and 12mm diameter (Falcon, Becton Dickinson,Franklin Lakes, NJ) at a density of   ∼ 10 6 cells/cm 2 . Afterseeding, transepithelial resistance (  R T ) was monitored withan epithelial voltohm-meter (World Precision Instruments,Sarasota, FL) and monolayers with  R T  >  500  cm 2 wereused for Ussing chamber studies after 5–12 days in culture.For patch clamp experiments cells were seeded on glasscover slips and used after 24h.   H. Fischer et al./Journal of Ethnopharmacology 93 (2004) 351–357   353 2.3. Transepithelial Ussing chambers experiments T84 monolayers grown on cell culture inserts werecarefully cut from the plastic cup and mounted in Ussingchambers (World Precision Instruments, Saratoga, FL).Both chamber compartments were separately perfusedwith Krebs-Henseleit solutions and gassed with 95% O 2 and 5% CO 2  at pH 7.4. The exposed perfusing area was0.6cm 2 . Transepithelial voltage was clamped at 0mV us-ing a standard four-electrode voltage clamp (558C-voltageclamp, University of Iowa, Iowa City, IO). A pair of agarbridges (1M KCl) connected through half-cell electrodeswere used for measuring transepithelial potential. Ag/AgClpellets connected to agar bridges at the back of the half chambers were used for passing current. Positive currentswere defined as anion movement from serosa to mucosa.  R T  was monitored and calculated from current deflectionscaused by single voltage pulses of 2mV amplitude and 1sduration every 20s using Ohm’s law. Data were recordedto a computer through an analog-to-digital board (DataQInstruments, Akron, OH). Experiments were performed at37 ◦ C. T84 monolayers were bathed with a Cl − -containingsolution on the serosal side and a Cl − -free solution onthe mucosal side. This serosal-to-mucosal Cl − gradientwas used to increase the electrochemical driving forcefor Cl − secretion across the apical membrane. We deter-mined previously that under these conditions the apicalmembrane is the rate-limiting step during Cl − secretion(Illek et al., 2000). The mucosal Cl − -free solution hadthe following composition: 120mM Na-gluconate, 20mMNaHCO 3 , 5mM KHCO 3 , 1.2mM NaH 2 PO 4 , 5.6mM glu-cose, 2.5mM Ca(gluconate) 2 , and 1.2mM MgSO 4 . Theserosal Cl − -containing solution had the following com-position: 120mM NaCl, 25mM NaHCO 3 , 5mM KCl,1.2mM NaH 2 PO 4 , mM 5.6 glucose, 2.5mM CaCl 2 , and1.2mM MgCl 2 . The cAMP-elevating agonist forskolin(Calbiochem, LaJolla, CA) was prepared as a 20mM stock solution in dimethylsulfoxide and an aliquot was added tothe serosal compartment to give a final concentration of 20  M.  N  -phenyl anthranylic acid (DPC, Aldrich, Milwau-kee, WI) was prepared as a 200mM stock in ethanol andused at 5mM at the end of all experiments to fully block Cl − secretion. SB-300 and SP-303 were dissolved in wa-ter as 100mM stock solutions. Aliquots were added to themucosal compartment to give final concentrations of 5, 50and 100  M.Dose-dependent block of   I  sc  was analyzed usingMichaelis–Menten (MM) kinetics. Blocked  I  sc  (in %) wasplotted versus concentration ( c ) and fitted to the MM equa-tion of the form  I  sc  = (I  max × c)/(K B + c) , which yields thehalfmaximal effective blocker concentration,  K  B , and themaximally blocked current,  I  max . Data have been expressedas mean  ±  S.E.;  n  refers to the number of experiments.Statistical analysis was performed using ANOVA analysis(StatView, Abacus Concepts, Berkeley, CA). Probabilitiesof   P  <  0.05 were considered significant. 2.4. Patch clamp recordings Single T84 cells were whole cell patch-clamped as de-scribed previously (Illek and Fischer, 1998). Briefly, cellswere patch-clamped in an open, heated (to 37 ◦ C) chamber(volume  =  500  l) on the stage of an inverted micro-scope. The membrane potential was continuously clampedto  − 60mV (EPC7, ALA Scientific Instruments Inc., West-bury, NY) and pulsed every 20s for 1s to − 50mV to mon-itor membrane conductance,  G m . Bath solution contained(in mM): 140mM  N  -methyl- d -glucamine (NMDG)Cl,10mM glucose, 10mM sucrose, 10mM HEPES (  N  -[2-hydroxyethyl]piperazine-  N  ′ -[2-ethanesulfonic acid]), 1.7mM CaCl 2 , 1mM MgCl 2 , pH 7.3. The pipette (i.e., theintracellular) solution contained: 140mM NMDGCl, 5mMNMDG–EGTA (ethyleneglycol-bis-(b-aminoethyl ether)  N  ,  N  ,  N  ′ ,  N  ′ -tetraacetic acid), 1mM MgCl 2 , 2mM HEPES,1mM glucose, 1mM Mg-ATP, 0.1mM Li-GTP, pH 7.3.With these solutions the major current carrier is chloride.Bath solution was hypertonic with respect to pipette solu-tion to prevent cell swelling (Worrell et al., 1989). Cl − cur-rents were activated by stimulation of the cell with 10  Mforskolin in the bath solution. SP-303 and SB-300 wereadded to the bath. Current–voltage step protocols were ap-pliedbeforeandafteradditionofblocker.Themembranepo-tential was clamped to the target voltages ( − 80 to + 30mV)in 10mV increments using pClamp software (Axon Instru-ments Inc., Foster City, CA). Voltage-dependence of theblocked  G m  was determined from the numerical differenceof current–voltage-step protocols recorded before and afterblocker addition. Currents were reported as current densitiesnormalized to cell membrane capacitance ( C  m , a measurefor the membrane area).  C  m  was determined from currenttransients elicited by a voltage pulse. Average  C  m  of T84cells was 27 . 1 ± 2 . 7pF ( n = 17). 3. Results 3.1. Inhibition of cAMP-stimulated chloride secretionacross monolayers of T84 cells by SB-300 The expedited extraction of the bark latex of   Croton lech-leri  yielded a novel extract (named SB-300) which con-tained on average 70 . 6  ±  7 . 2% by weight SP-303 ( n  = 56; mean ± S . D . ). The efficacy of SB-300 on blocking in-testinal Cl − secretion was studied on forskolin-stimulatedT84 monolayers in Ussing chambers. Under control condi-tions  I  sc  =  36 . 7 ± 3 . 2  A/cm 2 and  R T  =  441 ± 65  cm 2 ( n = 12). Addition of the cAMP-elevating agonist forskolin(10  M) increased  I  sc  to 125.0 ± 9.4  A/cm 2 and decreased  R T  to 279 ± 27  cm 2 ( n = 7) After the forskolin-stimulatedCl − currents had stabilized, cumulative doses of SB-300were added to the mucosal Ussing chamber reservoir re-sulting in final concentrations of 5, 50 and 100  M. Theforskolin-stimulated  I  sc  was dose-dependently blocked by  354  H. Fischer et al./Journal of Ethnopharmacology 93 (2004) 351–357  (A)(B) Fig. 1. Effect of SB-300 on cAMP-stimulated Cl − secretion across T84 monolayers. (A) Transepithelial Cl − secretion was stimulated by the cAMP agonistforskolin (10  M). Mucosal addition of SB-300 inhibited forskolin-stimulated  I  sc  dose-dependently. Subsequent addition of DPC (5mM) further blocked  I  sc . On this compressed time scale the single 2-mV pulses are not discernible. (B) Dose-dependent inhibition of forskolin-stimulated Cl − currents. Linerepresents fit to Michaelis–Menten kinetics yielding a  K  B  of 4.8  ±  0.8  M and  I  max  = 92.2  ±  3.0%. Values represent means  ±  S.E. of six individualT84 monolayers. (A)(B) Fig. 2. Effect of SP-303 on cAMP-stimulated Cl − secretion across T84 monolayers. (A) Typical  I  sc  recording; current was activated with 10  Mforskolin and blocked by SP-303. In this experiment 50  M SP-303 blocked 93% of the forskolin-stimulated  I  sc . (B) Dose-dependent inhibition of forskolin-stimulated Cl − currents by SP-303. Line represents fit to Michaelis–Menten kinetics yielding a halfmaximal inhibitory constant of   K  B  = 4.1 ±  1.3  M and  I  max  of 98  ±  7.2%. Values represent mean  ±  S.E. of three individual T84 monolayers treated with each dosage. SB-300 (Fig. 1A). Subsequent addition of the Cl − channelblocker DPC (5mM) produced a small further decrease in  I  sc . Apical exposure of the intestinal cells to 5  M SB-300decreased forskolin-stimulated Cl − currents from 125.0  ± 9.4 to 79.4 ± 10.6  A/cm 2 ( n = 6) corresponding to an av-erage inhibition of forskolin-stimulated Cl − currents by 46.0 ± 8.8%. Exposure to 50  M SB-300 blocked  I  sc  to 46.1 ± 10.5  A/cm 2 correspondingto90.5 ± 9.8%inhibition.Max-imal blocker effects were seen at 100  M. Kinetic analysisof dose-dependent current inhibition by SB-300 is shown inFig. 1B. Data were fitted with MM kinetics yielding a  K  B of 4.8 ± 0.8  M and  I  max  = 92.2 ± 3.0%. 3.2. Comparison of SB-300 to SP-303 standards The potency of the novel SB-300 extract was comparedto the standard compound SP-303. Fig. 2A shows the inhi-bition of forskolin-stimulated  I  sc  by 5 and 50  M SP-303.As reported previously (Gabriel et al., 1999),  I  sc  was alsoeffectively blocked by SP-303 in our experiments. Thedose-dependent blocker effects of SP-303 are shown inFig. 2B. Fit of the dose-response relation resulted in  K  B = 4.1 ± 1.3  M and  I  max  of 98 ± 7.2%.Fig. 3 summarizes the inhibitory effects of SB-300 com-pared to the standard SP-303 at 5  M (Fig. 3A) and 50  M(Fig. 3B) concentrations. At both concentrations SB-300showed effects not different from the SP-303 standards (A) (B) Fig. 3. Comparison of Cl − current inhibition by SB-300 and two differentbatches of SP-303. (A) Inhibitory effects of SB-300 and SP-303 areexpressed as percent block of forskolin-stimulated current (  I  fsk  ). Inhibitoryeffects were not different between SB-300 and SP-303 standards at 5  M(ANOVA,  P  = 0.70). (B) Similarly, at 50  M blocker effects were notdifferent between SB-300 and SP-303 (ANOVA,  P  = 1.0). Numbers inparentheses note number of experiments.   H. Fischer et al./Journal of Ethnopharmacology 93 (2004) 351–357   355 (ANOVA), resulting in a total average of current inhibitionfor all three extracts of 50.3  ±  5.3% ( n  = 12) at 5  M,and 91.2  ±  6.0% ( n  = 10) at 50  M. Therefore, SB-300blocked transepithelial Cl − secretion with a similar efficacyas the SP-303 standard. 3.3. Whole cell patch clamp experiments To identify the biophysical properties of the Cl − conduc-tance blocked by SB-300 and SP-303, single T84 cells wereinvestigated with the whole cell patch clamp technique un-der conditions where only Cl − current was measured (seeSection 2). T84 cells were continuously clamped to − 60mVand pulsed every 20s to  − 50mV. The voltage-dependenceof the measured Cl − conductance was determined by clamp-ing the membrane potential from − 80 to + 30mV in 10-mVincrements. Cl − currents were continuously recorded. Un-stimulated T84 cells expressed a specific membrane con-ductance ( G m ) of 19.9  ±  5.6pS/pF ( n  = 6), which was (A)(B)(C) Fig. 4. Effect of SB-300 on forskolin-stimulated whole cell Cl − currentsin single T84 cells. (A) Continuous whole cell current recording. Specificcurrent density (in pA/pF) is plotted over time. T84 cell was pre-stimulatedwith 10  M forskolin. Membrane potential was clamped to  − 60mV andpulsed every 20s to  − 50mV. Current–voltage step protocols (from  − 80to  + 30mV in 10mV increments) were applied before and after additionof 50 and 100  M drug (at , , ). Fifty and 100  M of SB-300were added to the bath where indicated. (B) SB-300-blocked currentswere calculated by subtracting currents recorded at from currents atand the currents are voltage-independent. (C) Current–voltage relationof SB-300-blocked current. Line shows linear regression, which yieldeda SB-300-blocked  G m  of 215 pS/pF and  C  m  of 13 pF. In this recording G m  was 362 pS/pF before blocker addition (at ). increased to 348 ± 209pS/pF by 10  M forskolin. Fig. 4Ashows a typical recording of the inhibitory effect of SB-300on forskolin-stimulated whole cell chloride currents. Subse-quent additions of 50 and 100  M SB-300 to the bath solu-tion blocked whole cell Cl − currents with a  t  1 / 2  of  ∼ 1minsuggesting that SB-300 acts from the extracellular side. Be-fore and after drug addition current–voltage relations wererecorded, as depicted in Fig. 4A with circled 1 to circled 3.The voltage-dependence of the SB-300-sensitive Cl − cur-rent is shown in Fig. 4B. The SB-300 blocked Cl − currentwas time- and voltage-independent and the current–voltagerelation was linear (Fig. 4C), which are typical character-istics of Cl − currents mediated by the CFTR Cl − channel.Blocked  G m  = 215pS/pF, which was 59.6% of total  G m (362 pS/pF). On average, 50  M SB-300 inhibited 63  ± 8.5% ( n = 3) of whole cell  G m .Fig. 5 shows the inhibition of forskolin-stimulated wholecell Cl − currents by the standard SP-303. Addition of 50  MSP-303 decreased  G m  from 584 to 100pS/pF, i.e., by 82.8%.On average, 50  M SP-303 blocked 83  ±  0.6% ( n  =  2)of whole cell  G m . SP-303-blocked currents were time- andvoltage-independent (Fig. 5B), and the current–voltage rela-tion was linear (Fig. 5C). The voltage-dependent properties (A)(B)(C) Fig. 5. Effect of SP-303 on forskolin-stimulated whole cell Cl − currentsin single T84 cells. (A) SP-303 was added at 50  M to the bath solution.Conditions as in Fig. 4. (B) SP-303-blocked currents were calculated bysubtracting currents recorded at from currents at . Membrane potentialwas clamped from − 80 to + 30mV. The currents are voltage-independent.(C) Current–voltage relation of SP-303-blocked current; linear regressionyielded a SP 303-blocked  G m  of 484 pS/pF and  C  m  of 51 pF. In thisrecording  G m  was 584 pS/pF before blocker addition (at ).
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