FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2123832242> ?p ?o ?g. }

• W2123832242 endingPage "18429" @default.
• W2123832242 startingPage "18423" @default.
• W2123832242 abstract "We examined the voltage-dependent block of Ca2+-activated Cl− channels by anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and niflumic acid (NFA) in excised inside-out and outside-out patches fromXenopus oocytes. The fraction of the voltage field (δ) experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from the outside. δ was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for NFA. Because the voltage dependence of the drugs differed, the order of potency was also voltage-dependent. At +100 mV the order of potency was NFA > A9C > DIDS > DPC (Ki (μm) = 10.1, 18.3, 48, and 111, respectively). Because the drugs are hydrophobic, they can cross the bilayer when applied from the inside and block the channel from the outside. The equilibrium geometries of the blockers were determined by molecular modeling and compared with their blocking positions (δ). This analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 × 0.75 nm, because C(CN)3−, which has these dimensions, permeates. The external opening is at least 0.60 × 0.94 nm, because DPC has these dimensions and penetrates the channel ∼30%. We examined the voltage-dependent block of Ca2+-activated Cl− channels by anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and niflumic acid (NFA) in excised inside-out and outside-out patches fromXenopus oocytes. The fraction of the voltage field (δ) experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from the outside. δ was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for NFA. Because the voltage dependence of the drugs differed, the order of potency was also voltage-dependent. At +100 mV the order of potency was NFA > A9C > DIDS > DPC (Ki (μm) = 10.1, 18.3, 48, and 111, respectively). Because the drugs are hydrophobic, they can cross the bilayer when applied from the inside and block the channel from the outside. The equilibrium geometries of the blockers were determined by molecular modeling and compared with their blocking positions (δ). This analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 × 0.75 nm, because C(CN)3−, which has these dimensions, permeates. The external opening is at least 0.60 × 0.94 nm, because DPC has these dimensions and penetrates the channel ∼30%. Ca2+-activated Cl− cystic fibrosis transmembrane conductance regulator current 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid diphenylamine-2-carboxylic acid niflumic acid anthracene-9-carboxylic acid N-methyl-d-glucamine Ca2+-activated Cl−(Cl(Ca))1 channels play important roles in physiological processes, including epithelial secretion, repolarization of the cardiac action potential, regulation of vascular tone, olfactory transduction, and neuronal excitability (1Frings S. Reuter D. Kleene S.J. Prog. Neurobiol. ( Oxf. ). 1999; 60: 247-289Crossref Scopus (201) Google Scholar, 2Greger R. Pfluegers Arch. Eur. J. Physiol. 1996; 432: 579-588Crossref PubMed Scopus (50) Google Scholar, 3Begenisich T. Melvin J.E. J. Membr. Biol. 1998; 163: 77-85Crossref PubMed Scopus (84) Google Scholar, 4Large W.A. Wang Q. Am. J. Physiol. 1996; 271: C435-C454Crossref PubMed Google Scholar). Xenopus oocytes have long served as a model system for studying Cl(Ca) channels because these channels are the predominant channel type natively expressed in this cell and because they are expressed at extremely high levels (0.5 mA/cm2) (5Dascal N. CRC Crit. Rev. Biochem. 1987; 22: 317-387Crossref PubMed Scopus (518) Google Scholar). Recently, we have been investigating the mechanisms of anion permeation (6Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (151) Google Scholar), gating (7Kuruma A. Hartzell H.C. J. Gen. Physiol. 2000; 115: 59-80Crossref PubMed Scopus (158) Google Scholar), and regulation (8Hartzell H.C. Machaca K. Hirayama Y. Mol. Pharmacol. 1996; 51: 683-692Crossref Scopus (30) Google Scholar, 9Machaca K. Hartzell H.C. Biophys. J. 1998; 74: 1286-1295Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 10Machaca K. Hartzell H.C. J. Biol. Chem. 1999; 274: 4824-4831Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Machaca K. Hartzell H.C. J. Gen. Physiol. 1999; 113: 249-266Crossref PubMed Scopus (37) Google Scholar) of Ca2+-activated Cl− channels in Xenopus oocytes, where these channels play a key role in fast block to polyspermy (12Jaffe L.A. Cross N.L. Annu. Rev. Physiol. 1986; 48: 191-200Crossref PubMed Scopus (67) Google Scholar).Xenopus oocyte Cl(Ca) channels have many features in common with Cl(Ca) channels in epithelial cells, cardiac myocytes, and vascular smooth muscle cells (see references in Ref. 6Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (151) Google Scholar), and we think that elucidating the mechanisms of operation of Xenopusoocyte channels will provide important insights into the function of these channels in other tissues. The roles of Cl(Ca) channels in human disease are not yet firmly established. However, there are reasons to think that they may be involved in diseases as diverse as cystic fibrosis and cardiac arrhythmias. For example, there appears to be a reciprocal relationship between the level of expression of CFTR and Cl(Ca) channels in airway epithelial cells. Cells from the airway of cystic fibrosis patients can secrete in response to elevations of intracellular Ca2+(13Boucher R.C. Cheng E.H.C. Paradiso A.M. Stutts M.J. Knowles M.R. Earp H.S. J. Clin. Invest. 1989; 84: 1424-1431Crossref PubMed Scopus (117) Google Scholar, 14Willumsen N.J. Boucher R.C. Am. J. Physiol. 1989; 256: C226-C233Crossref PubMed Google Scholar), and ICl(Ca) is up-regulated in the airway of CFTR knockout mice (15Clarke L.L. Grubb B.R. Yankaskas J.R. Cotton C.U. McKenzie A. Boucher R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 479-483Crossref PubMed Scopus (311) Google Scholar). The up-regulation ofICl(Ca) in the airway of CFTR knockout mice can apparently compensate for the lack of CFTR and ameliorate the lung pathology in this mouse model (16Grubb B.R. Vick R.N. Boucher R.C. Am. J. Physiol. 1994; 266: C1478-C1483Crossref PubMed Google Scholar). Furthermore, overexpression of CFTR in cultured airway epithelial cells from cystic fibrosis patients results in a decrease in Cl(Ca) current (17Johnson L.G. Boyles S.E. Wilson J. Boucher R.C. J. Clin. Invest. 1995; 95: 1377-1382Crossref PubMed Scopus (164) Google Scholar). In the heart, the transient outward current (Ito), which plays an important role in repolarization of the cardiac action potential, is composed of several components, one of which (Ito2) is mediated by Cl(Ca) channels (18Hiraoka M. Kawano S. J. Physiol. 1989; 410: 187-212Crossref PubMed Scopus (146) Google Scholar, 19Zygmunt A.C. Robitelle D.C. Eddlestone G.T. Am. J. Physiol. 1997; 273: H1096-H1106PubMed Google Scholar, 20Papp Z. Sipido K. Callewaert G. Carmeliet E. J. Physiol. 1995; 483: 319-330Crossref PubMed Scopus (39) Google Scholar). Changes in Ito2 can alter cardiac rhythmicity by affecting action potential duration (19Zygmunt A.C. Robitelle D.C. Eddlestone G.T. Am. J. Physiol. 1997; 273: H1096-H1106PubMed Google Scholar, 21Kawano S. Hiraoka M. J. Mol. Cell. Cardiol. 1991; 23: 681-693Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Recently, it has been shown that dogs that are genetically prone to cardiac sudden death have an abnormal Ito (22Freeman L.C. Pacioretty L.M. Moise N.S. Kass R.S. Gilmour Jr., R.E. J. Cardiovasc. Electrophysiol. 1997; 8: 872-883Crossref PubMed Scopus (38) Google Scholar), implying that Cl(Ca) channels play a role in sudden cardiac death. Cl(Ca) channels also contribute to the arrhythmogenic transient inward current (Iti) in some species (22Freeman L.C. Pacioretty L.M. Moise N.S. Kass R.S. Gilmour Jr., R.E. J. Cardiovasc. Electrophysiol. 1997; 8: 872-883Crossref PubMed Scopus (38) Google Scholar, 23Zygmunt A.C. Am. J. Physiol. 1994; 267: H1984-H1995PubMed Google Scholar, 24Zygmunt A.C. Goodrow R.J. Weigel C.M. Am. J. Physiol. 1998; 275: H1979-H1992PubMed Google Scholar, 25Han X. Ferrier G.R. J. Mol. Cell. Cardiol. 1996; 28: 2069-2084Abstract Full Text PDF PubMed Scopus (18) Google Scholar). During Ca2+ overload, Iti can trigger oscillatory afterpotentials resulting in serious cardiac arrhythmias (26Hiraoka M. Kawano S. Hirano Y. Furukawa T. Cardiovasc. Res. 1998; 40: 23-33Crossref PubMed Scopus (91) Google Scholar, 27January C.T. Fozzard H.A. Pharmacol. Rev. 1988; 40: 219-227PubMed Google Scholar). Cl− channels clearly can participate in arrhythmogenesis, because anion substitution or pharmacologic Cl− channel blockade protects against reperfusion and ischemia-induced arrhythmias (28Curtis M.J. Garlick P.B. Ridley P.D. J. Mol. Cell. Cardiol. 1993; 25: 417-436Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 29Ridley P.D. Curtis M.J. Circ. Res. 1992; 70: 617-632Crossref PubMed Scopus (56) Google Scholar, 30Tanaka H. Matsui S. Kawanishi T. Shigenobu K. J. Pharmacol. Exp. Ther. 1996; 278: 854-861PubMed Google Scholar). Understanding the nature of the pore of this channel is an important step in elucidating how ions permeate anion-selective channels in general and also in developing reagents that can be used to block or activate these channels. In our previous study (6Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (151) Google Scholar), we concluded that the pore of the Cl(Ca) channel must be at least 0.74 nm in one dimension, because the pseudo-halide anion C(CN)3, which is 0.33 × 0.75 nm in its smallest cross section, is permeant through the channel. In the present study (6Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (151) Google Scholar), we have examined the voltage dependence and sidedness of block of several classical Cl−channel blockers and have related these to the molecular size and structure of the blocking molecules. These studies provide insights into the functional geometry of the permeation pathway of this channel. Stage V–VI oocytes were harvested from adult Xenopus laevis females (Xenopus I) as described by Dascal (5Dascal N. CRC Crit. Rev. Biochem. 1987; 22: 317-387Crossref PubMed Scopus (518) Google Scholar). Xenopuswere anesthetized by immersion in tricaine (1.5 g/liter). Ovarian follicles were removed, cut into small pieces, and digested in normal Ringer's solution with no added calcium containing about 2 mg/ml collagenase type IA (Sigma) for 2 h at room temperature. The oocytes were extensively rinsed with normal Ringer's, placed in L-15 medium (Life Technologies, Inc.), and stored at 18 °C. Oocytes were used 1–6 days after isolation. For excised patch experiments, oocytes were placed in a hypertonic solution (200 mm potassium aspartate, 20 mm KCl, 1 mm MgCl2, 10 mm EGTA, 10 mm HEPES, pH 7.2 with KOH) for 1–10 min to facilitate manual removal of the vitelline membrane, and then they were placed in a standard solution (see “Solutions” below) until use. Recordings were performed using the inside-out and outside-out excised patch configurations of patch clamp technique. Pipettes were made of borosilicate glass (Sutter Instrument Co.), pulled by a Sutter P-2000 puller, and fire-polished. Patch pipettes had resistances of 6–10 megohms. Unless noted, they were filled with a standard solution (see “Solutions”), which was always the same as the solution in the bath. The bath was grounded via a 3-M KCl-agarose bridge connected to a Ag-AgCl reference electrode. Solution changes were performed by positioning the patch at the end of a battery of sewer pipes having 100-μm internal diameter connected to the gravity feed solution containers. Patches were usually obtained from the animal hemisphere of oocytes, because Cl(Ca) channels are concentrated here (9Machaca K. Hartzell H.C. Biophys. J. 1998; 74: 1286-1295Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The patch was typically held at 0 mV, and current was measured in response to a 200-ms voltage ramp from −100 to +100 mV. Patch clamp data were acquired by an Axopatch 200A amplifier controlled either by Clampex 8.1 via a Digidata 1322A analog-to-digital and digital-to-analog converter (Axon Instruments) or by Curcap 3.0 and a Challenger DB voltage stimulator (W. Goolsby, Emory University). Symmetrical solutions containing ∼150 mm Cl− were used. In different experiments, the cation was either Na+ or NMDG, as indicated in the figure legends. Na+ was always used in experiments with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) because of possible interaction of DIDS and NMDG. The NMDG standard solution contained 150 mm NMDG-Cl, 10 mm NMDG-HEPES, 4 mm MgCl2, pH 7.3. Zero Ca2+solution contained 10 mm EGTA. High Ca2+solution contained 10 mm EGTA titrated with Ca2+ to give ∼50 μm free Ca2+. The standard Na+-containing solution contained 150 mm NaCl, 10 mm HEPES, pH 7.2. Zero Ca2+ solution contained 10 mm EGTA, and the high Ca2+ solution contained 0.1 mmCaCl2 with no added EGTA. With inside-out patches, to be certain that the currents recorded were Ca2+-dependent, we always measured the current in a zero Ca2+ solution at the start and end of the experiment. If the Ca2+-independent current was >5% of the total current in Ca2+, the patch was discarded. For outside-out patches, it was not practical to change the Ca2+ bathing the cytosolic face. Therefore, with outside-out patches, the identity of the current as a Cl−current was verified at the start and end of an experiment by bathing the external face of the patch in solution in which all Cl− was replaced with SO4−. Patches were not analyzed if the outward current under these conditions was >5% of the current with symmetrical Cl−. Diphenylamine-2-carboxylic acid (DPC) and niflumic acid (NFA) were from Sigma; anthracene-9-carboxylic acid (A9C) was from Aldrich; DIDS was from Molecular Probes. DIDS from Sigma and Molecular Probes differed significantly in their color, and some preliminary experiments with Sigma DIDS yielded irreproducible results. DIDS (Molecular Probes) was suspended in water at 0.3m as a stock before working solutions were made. Other compounds were dissolved in Me2SO at 0.3m as stocks to keep the [Me2SO] in working solutions < 0.1%. For the calculations and graphical presentation, we used Origin 6.0 software (Microcal). Curve fitting was performed using the iterative algorithms in Origin. Results are presented as means ± S.E., and n refers to the number of patches in each experiments. The equilibrium geometries of the blockers used were calculated using both MMFF94 molecular mechanics models and Hartree-Fock molecular orbital calculations with the 3–21G* basis set (31Hehre W.J., Yu, J. Klunzinger P.E. Lou L. A Brief Guide to Molecular Mechanics and Quantum Chemical Calculations. Wavefunction Inc., Irvine, CA1998Google Scholar). Calculations were performed using PC Spartan Pro software (Wavefunction, Inc., Irvine, CA) run on an Intel Pentium-III-based PC running Windows 2000. To determine the minimal cross-sectional dimensions of the molecule, the molecule was rotated manually to fit it into the smallest possible rectangle. The center-to-center atomic distances were measured using utilities in PC Spartan Pro; the molecular dimensions were calculated by adding the van der Waals radii of the terminal atoms to the measured center-to-center distances. In the cases of C(CN)3 and A9C, the geometries were confirmed by comparison to crystal structure data available in the Cambridge Crystallographic Database. The goal of these experiments was to characterize the inhibition of Ca2+-activated Cl− channels by various Cl− channel blockers. Excised patches in either inside-out or outside-out configurations were pulled from stage VIXenopus oocytes. The solutions on the inside and outside of the patch were always the same, except that 0.1 mmCaCl2 was added to the cytosolic face to activate Cl(Ca) channels. The effects of the blockers applied to either the cytosolic face of the membrane in inside-out patches or the extracellular face in outside-out patches were then determined. Fig.1 A showsI-V curves of ICl(Ca) in an outside-out excised patch from Xenopus oocytes recorded with symmetrical 150 mm Cl− solutions. TheI-V curve in the absence of A9C was approximately linear (Fig. 1 A). The current was a Cl−current, because the outward current was completely blocked by replacement of extracellular Cl− by impermeant anions such as SO4−, and replacing Na+with NMDG had little effect on Erev (data not shown, but see Ref. 6Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (151) Google Scholar). The outward current was blocked in a concentration- and voltage-dependent manner by addition of A9C to the extracellular face of the membrane (Fig. 1 A). The inward current was only slightly affected. In Fig. 1 B, the currents from Fig. 1 A were expressed as a fraction of the current in the absence of A9C (I/IA9C = 0) and plotted as a function of voltage. Relative current amplitude decreased with depolarization, confirming that inhibition of Cl− current by A9C was voltage-dependent. The data in Fig. 1 B were replotted in Fig. 1 C for each voltage as a function of [A9C]. These data were fitted to an equation of the formI/IA9C=0=Imin+(Imax−Imin)/{1+([A9C]/Ki)n},Equation 1 where Imax and Iminare the maximum and minimum current amplitudes, Kiis the concentration of A9C required to reduce the current amplitude to (Imax + Imin)/2, andn is the slope factor. Ki was determined for each potential and plotted in Fig. 1 D. The apparentKi decreased ∼10-fold per 100-mV depolarization. The Ki at 0 mV was estimated to be 158 μm, and the Ki at +100 mV was 18.3 μm. From Fig. 1 D, one can estimate the fraction of the voltage field experienced by the blocking particle at its blocking site from the equation derived by Woodhull (32Woodhull A.M. J. Gen. Physiol. 1973; 61: 687-708Crossref PubMed Scopus (1232) Google Scholar, 33Hille B. Ion Channels of Excitable Membranes. 2nd Ed. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar),logKi(V)=logKi(0mV)+(zδFV/2.303RT)Equation 2 where log Ki (V) is theKi at each voltage, Ki (0 mV) is the Ki at 0 mV, z is the electronic charge of the blocking particle, δ is the fraction of the voltage field sensed by the blocker from the outside of the membrane,R, F, and T have their usual thermodynamic meanings, and V is voltage. The solid line is the best fit of the antilog of Equation 2 to the data. δ was estimated to be ∼0.6 the distance of the voltage field from the extracellular side. The inhibition of currents by voltage-dependent blockers is often time-dependent; as membrane potential is changed, the current changes as the blocker accumulates or is removed from the blocking site. Fig. 2 showsICl(Ca) currents in response to 100-ms duration voltage pulses from a holding potential of 0 mV to voltages between +100 and −100 mV. In the absence of A9C (Fig. 2 A), outward currents were time-independent. Inward currents at the most negative potentials exhibited a slow decrease in current; currents decreased <10% in 100 ms. I-V curves were plotted for the current at the beginning (squares) and at the end (circles) of the 100-ms pulses (Fig. 2 B). Both curves were approximately linear. In the presence of 10 μm A9C (Fig. 2 C), the outward currents exhibited pronounced time dependence. The currents decayed with time as A9C block developed (Fig. 2 C), and theI-V curves measured at the beginning and end of the voltage pulses diverged at positive potentials. Increasing the [A9C] increased both the rate and the degree of block of the outward current (Fig. 2, E–H). The decay of the current upon stepping from 0 to +100 mV was fitted to a single exponential. The time constants were concentration-dependent; τ = 42.5 ± 1.2 ms at 10 μm A9C, 16.4 ± 1.1 ms at 30 μmA9C, and 11.6 ± 0.4 ms at 100 μm A9C (n = 3 for each). These data are consistent with a model in which A9C enters the pore of the channel and blocks it in a voltage-dependent manner. To determine whether A9C also blocked from the inside, we obtained inside-out patches (Fig.3). Surprisingly, A9C applied from the cytoplasmic side of the patch also blocked outward current (Fig.3 A). If A9C had blocked the channel from the inside in a similar manner to block from the outside, we would have expected A9C applied on the cytoplasmic surface to block inward, not outward, current. Woodhull analysis of the voltage-dependent block showed that δ = 0.6 (Fig. 3 B). Thus, A9C blocked outward current at the same site in the channel with the same voltage dependence whether it was applied from the inside or outside. The most logical interpretation of these data is that A9C blocks only from the outside but that it crosses the lipid membrane and gains access to the outside when applied from the inside. If this is true, we might expect that the apparent Ki for block by A9C applied on the inside would be larger than when applied from the outside, because the actual concentration of A9C on the outside when applied from the inside would be lower than expected. In support of this idea, the Ki for A9C applied to the inside at 0 mV was 945 μm and at +100 mV was 103.7 μm, ∼5–6-fold greater than the Ki for A9C applied from the outside at the same voltage. In addition, DPC, which has a similar structure to A9C, has been shown to cross lipid membranes readily at neutral pH (see references and “Discussion” in Ref.34Schultz B.D. Singh A.K. Devor D.C. Bridges R.J. Physiol. Rev. 1999; 79 Suppl. 1: 109-144Crossref Scopus (256) Google Scholar). A similar analysis was performed for DPC (Fig.4). In outside-out patches DPC blocked outward current in a dose-dependent manner with an apparentKi at 0 mV of 323 μm and at 100 mV of 111 μm. In the experiment illustrated, there was also a small effect on inward current, but it was difficult to determine whether this was a true inhibitory effect or was related to channel rundown, which occurs in some patches. Woodhull analysis of DPC is presented in Fig. 4, B–D. The voltage dependence of the relative current (I/IDPC = 0) in Fig.4 B shows that the block by DPC is clearly voltage-dependent. However, the voltage dependence is less than that of A9C (Fig. 1, C and D). δ was estimated to be 0.3. These data show that DPC blocks in a voltage-dependent manner but that the blocking site is not as deep in the pore as the A9C blocking site. The kinetics of voltage-dependent block by DPC were faster than block by A9C (Fig. 5). Fig. 5,A and B, shows current traces in response to a series of voltage pulses from 0 mV to voltages between −100 mV and +100 mV for 0 and 300 μm DPC, respectively. Fig.5 C shows current traces from the voltage pulses from −100 to +100 mV on a faster time scale for four different DPC concentrations. As [DPC] was increased, a more pronounced time-dependent tail current was observed at the onset of the pulse. The time constant was faster than 1 ms. TheI-V curves showed clearly increasing inward rectification with increasing DPC concentration (Fig.5 D). DPC, like A9C, can cross the membrane when applied from the inside in inside-out patches and block the channel from the outside (Fig.6). DPC applied from the inside blocked outward current with a Ki of 212 μm at 100 mV, which was about twice the Ki for inhibition from the outside. The estimated δ was 0.2, which was similar to the estimate for DPC applied from the outside. As noted above, DPC has been shown to partition readily into lipid membranes at neutral pH (34Schultz B.D. Singh A.K. Devor D.C. Bridges R.J. Physiol. Rev. 1999; 79 Suppl. 1: 109-144Crossref Scopus (256) Google Scholar). DIDS applied to the extracellular side decreased outward Cl− current in a dose-dependent manner (Fig. 7 A). Block was clearly voltage-dependent (Fig. 7 B). The apparentKi at 0 mV was 562 μm and at +100 mV was 48 μm. δ was estimated to be 0.3 (Fig. 7,C and D). 100 μm DIDS applied to the cytoplasmic side blocked inward current >50% and outward current ∼30%, but the block required >1 min to develop (Fig.8). In contrast, DIDS (and the other blockers used here) applied to outside-out patches produced a steady-state block within several seconds. Because this slowness of block suggested that the mechanisms were significantly different from block from the outside, we did not analyze these results quantitatively. NFA has been widely used to block Ca2+-activated Cl− channels inXenopus oocytes (35White M.M. Aylwin M. Mol. Pharmacol. 1990; 37: 720-724PubMed Google Scholar). In outside-out patches, NFA applied to the extracellular face blocked both inward and outward current (Fig.9 A). The apparentKi was 12.9 μm at 0 mV and 10.1 μm at +100 mV (Fig. 9 D). Although plots ofI/INFA = 0 versus Vm (Fig. 9, B and C) showed some curvature at the highest NFA concentrations, the voltage dependence was rather small. Woodhull analysis (Fig. 9, Cand D) showed that the apparent Ki did not change significantly between 20 mV (11.6 μm) and 100 mV (10.1 μm). The best linear fit to the data in Fig.9 D yielded a small slope that translated into an estimate for δ of 0.1. NFA applied to the cytoplasmic face of inside-out patches also blocked both inward and outward current (Fig.10 A) with aKi of 53.7 μm at +100 mV (Fig.10 B). This Ki is about 4 times greater than Ki for NFA applied from the outside. We do not know whether block of NFA from the inside involves NFA crossing the membrane and blocking from the outside. However, because NFA is very closely related to DPC, we presume that it crosses the membrane readily. There was no voltage dependence to the block (Fig.10 C). The data on the block of Ca2+-activated Cl− channels inXenopus oocytes by various pharmacological agents is summarized in Table I. In outside-out patches, block of Cl− current with externally applied blockers exhibited an order of potency of NFA > A9C > DIDS > DPC. Block by NFA was not voltage-dependent, but the blocks by A9C, DIDS, and DPC were voltage-dependent. NFA blocked both inward and outward current, whereas the other blockers blocked only outward current significantly. Block by all four blockers was always reversible under these conditions.Table IVoltage-dependent block of Cl(Ca) currentsOutside-outInside-outKi (100 mV)Ki (0 mV)δKi (100 mV)Ki(0 mV)δA9C18.3 ± 3.6 (4)158 ± 17 (4)0.6 (3)103.7 ± 28.5 (3)945 ± 162 (3)0.6 (3)DPC111 ± 9.9 (3)323 ± 56 (3)0.3 (3)212.7 ± 8.2 (3)422 ± 20 (3)0.2 (3)DIDS48 ± 7.7 (3)562 ± 39 (3)0.3 (3)N/A 1-aN/A, not applicable.N/AN/ANFA10.1 ± 3.7 (4)12.9 ± 3 (4)<0.1 (3)53.7 ± 4.8 (3)N/AN/AThe numbers in the parentheses show n (the number of patches in each experiment); Ki is in μm. δ is the fraction of the voltage field experienced by the blocker.1-a N/A, not applicable. Open table in a new tab" @default.
• W2123832242 created "2016-06-24" @default.
• W2123832242 creator A5037972336 @default.
• W2123832242 creator A5073473903 @default.
• W2123832242 date "2001-05-01" @default.
• W2123832242 modified "2023-09-26" @default.
• W2123832242 title "Functional Geometry of the Permeation Pathway of Ca2+-activated Cl− Channels Inferred from Analysis of Voltage-dependent Block" @default.
• W2123832242 cites W1819465909 @default.
• W2123832242 cites W1894088205 @default.
• W2123832242 cites W1991369024 @default.
• W2123832242 cites W2012280093 @default.
• W2123832242 cites W2020754695 @default.
• W2123832242 cites W2021973679 @default.
• W2123832242 cites W2023141575 @default.
• W2123832242 cites W2034209106 @default.
• W2123832242 cites W2043954515 @default.
• W2123832242 cites W2048680763 @default.
• W2123832242 cites W2058626030 @default.
• W2123832242 cites W2059189028 @default.
• W2123832242 cites W2078956864 @default.
• W2123832242 cites W2085739173 @default.
• W2123832242 cites W2095090966 @default.
• W2123832242 cites W2102581316 @default.
• W2123832242 cites W2108425243 @default.
• W2123832242 cites W2124654729 @default.
• W2123832242 cites W2130076730 @default.
• W2123832242 cites W2131375257 @default.
• W2123832242 cites W2133464980 @default.
• W2123832242 cites W2140765579 @default.
• W2123832242 cites W2149624003 @default.
• W2123832242 cites W2159763620 @default.
• W2123832242 cites W2171674075 @default.
• W2123832242 cites W2199386566 @default.
• W2123832242 cites W2277417740 @default.
• W2123832242 cites W2405726434 @default.
• W2123832242 cites W2410214285 @default.
• W2123832242 doi "https://doi.org/10.1074/jbc.m101264200" @default.
• W2123832242 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11279188" @default.
• W2123832242 hasPublicationYear "2001" @default.
• W2123832242 type Work @default.
• W2123832242 sameAs 2123832242 @default.
• W2123832242 citedByCount "59" @default.
• W2123832242 countsByYear W21238322422012 @default.
• W2123832242 countsByYear W21238322422013 @default.
• W2123832242 countsByYear W21238322422014 @default.
• W2123832242 countsByYear W21238322422015 @default.
• W2123832242 countsByYear W21238322422016 @default.
• W2123832242 countsByYear W21238322422017 @default.
• W2123832242 countsByYear W21238322422018 @default.
• W2123832242 countsByYear W21238322422019 @default.
• W2123832242 countsByYear W21238322422021 @default.
• W2123832242 countsByYear W21238322422022 @default.
• W2123832242 crossrefType "journal-article" @default.
• W2123832242 hasAuthorship W2123832242A5037972336 @default.
• W2123832242 hasAuthorship W2123832242A5073473903 @default.
• W2123832242 hasBestOaLocation W21238322421 @default.
• W2123832242 hasConcept C12554922 @default.
• W2123832242 hasConcept C185592680 @default.
• W2123832242 hasConcept C2524010 @default.
• W2123832242 hasConcept C2777210771 @default.
• W2123832242 hasConcept C33923547 @default.
• W2123832242 hasConcept C41625074 @default.
• W2123832242 hasConcept C50670333 @default.
• W2123832242 hasConcept C55493867 @default.
• W2123832242 hasConcept C86803240 @default.
• W2123832242 hasConceptScore W2123832242C12554922 @default.
• W2123832242 hasConceptScore W2123832242C185592680 @default.
• W2123832242 hasConceptScore W2123832242C2524010 @default.
• W2123832242 hasConceptScore W2123832242C2777210771 @default.
• W2123832242 hasConceptScore W2123832242C33923547 @default.
• W2123832242 hasConceptScore W2123832242C41625074 @default.
• W2123832242 hasConceptScore W2123832242C50670333 @default.
• W2123832242 hasConceptScore W2123832242C55493867 @default.
• W2123832242 hasConceptScore W2123832242C86803240 @default.
• W2123832242 hasIssue "21" @default.
• W2123832242 hasLocation W21238322421 @default.
• W2123832242 hasOpenAccess W2123832242 @default.
• W2123832242 hasPrimaryLocation W21238322421 @default.
• W2123832242 hasRelatedWork W1963779027 @default.
• W2123832242 hasRelatedWork W1964339357 @default.
• W2123832242 hasRelatedWork W1987436683 @default.
• W2123832242 hasRelatedWork W2009888578 @default.
• W2123832242 hasRelatedWork W2062208111 @default.
• W2123832242 hasRelatedWork W2068799778 @default.
• W2123832242 hasRelatedWork W2108399540 @default.
• W2123832242 hasRelatedWork W2567818495 @default.
• W2123832242 hasRelatedWork W3004622189 @default.
• W2123832242 hasRelatedWork W308961640 @default.
• W2123832242 hasVolume "276" @default.
• W2123832242 isParatext "false" @default.
• W2123832242 isRetracted "false" @default.
• W2123832242 magId "2123832242" @default.
• W2123832242 workType "article" @default.