FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023732361> ?p ?o ?g. }

• W2023732361 endingPage "46565" @default.
• W2023732361 startingPage "46558" @default.
• W2023732361 abstract "Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 ± 0.5 pS (n = 11), between 0 and –200 mV, and was 9.6 ± 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of ∼2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl– secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16–28 mm if 11–25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139–146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+-K+-ATPase in Calu-3 cells. Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 ± 0.5 pS (n = 11), between 0 and –200 mV, and was 9.6 ± 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of ∼2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl– secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16–28 mm if 11–25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139–146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+-K+-ATPase in Calu-3 cells. The Calu-3 cell line (1Shen B.Q. Finkbeiner W.E. Wine J.J. Mrsny R.J. Widdicombe J.H. Am. J. Physiol. 1994; 266: L493-L501PubMed Google Scholar) is widely used as a model for human airway gland serous cells (2Haws C. Finkbeiner W.E. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1994; 266: L502-L512Crossref PubMed Google Scholar, 3Moon S. Singh M. Krouse M.E. Wine J.J. Am. J. Physiol. 1997; 273: L1208-L1219Crossref PubMed Google Scholar, 4Lee M.C. Penland C.M. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1998; 274: L450-L453Crossref PubMed Google Scholar, 5Devor D.C. Singh A.K. Lambert L.C. DeLuca A. Frizzell R.A. Bridges R.J. J. Gen. Physiol. 1999; 113: 743-760Crossref PubMed Scopus (239) Google Scholar, 6Cowley E.A. Linsdell P. J. Physiol. 2002; 538: 747-757Crossref PubMed Scopus (76) Google Scholar). When grown to confluence, Calu-3 cells form polarized monolayers and express CFTR 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; RT, reverse transcription.1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; RT, reverse transcription. apically at high levels (1Shen B.Q. Finkbeiner W.E. Wine J.J. Mrsny R.J. Widdicombe J.H. Am. J. Physiol. 1994; 266: L493-L501PubMed Google Scholar). In addition to expressing many typical serous cell markers (7Finkbeiner W.E. Carrier S.D. Teresi C.E. Am. J. Respir. Cell Mol. Biol. 1993; 9: 547-556Crossref PubMed Scopus (107) Google Scholar), Calu-3 cells are of special interest because they produce two kinds of anion secretion depending upon the mode of stimulation. When activated via agents that elevate [cAMP]i, Calu-3 cells secrete a HCO–3-rich fluid, whereas if activated by agents that elevate [Ca2+]i they secrete a Cl–-rich fluid (5Devor D.C. Singh A.K. Lambert L.C. DeLuca A. Frizzell R.A. Bridges R.J. J. Gen. Physiol. 1999; 113: 743-760Crossref PubMed Scopus (239) Google Scholar), although it still contains a large component of HCO–3 (4Lee M.C. Penland C.M. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1998; 274: L450-L453Crossref PubMed Google Scholar, 8Krouse M.E. Talbott J.F. Lee M.M. Joo N.S. Wine J.J. Am. J. Physiol. 2004; 287 (in press)Google Scholar). In either case the final step appears to be electrodiffusion of the anions through CFTR (4Lee M.C. Penland C.M. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1998; 274: L450-L453Crossref PubMed Google Scholar, 5Devor D.C. Singh A.K. Lambert L.C. DeLuca A. Frizzell R.A. Bridges R.J. J. Gen. Physiol. 1999; 113: 743-760Crossref PubMed Scopus (239) Google Scholar, 9Illek B. Yankaskas J.R. Machen T.E. Am. J. Physiol. 1997; 272: L752-L761PubMed Google Scholar). It has now been shown directly that anion secretion from Calu-3 cells drives the robust secretion of fluid at rates up to 10 μl/cm2/h (10Irokawa T. Krouse M.E. Joo N.S. Wu J.V. Wine J.J. Am. J. Physiol. Lung Cell Mol. Physiol. 2004; 287: L784-L793Crossref PubMed Scopus (23) Google Scholar).Recently, it was shown that apically secreted HCO–3 is partially neutralized by proton secretion under some conditions (8Krouse M.E. Talbott J.F. Lee M.M. Joo N.S. Wine J.J. Am. J. Physiol. 2004; 287 (in press)Google Scholar). The source of the protons appears to be an H+-K+-ATPase because it is ouabain-sensitive and requires apical K+ (8Krouse M.E. Talbott J.F. Lee M.M. Joo N.S. Wine J.J. Am. J. Physiol. 2004; 287 (in press)Google Scholar). This raises the issue of the source of the apical K+. An apical K+ channel in Calu-3 cells is suggested by results of Cowley and Linsdell (6Cowley E.A. Linsdell P. J. Physiol. 2002; 538: 747-757Crossref PubMed Scopus (76) Google Scholar), who showed that 16% of the Isc under basal conditions could be blocked with apical Ba2+, but nothing else is known about the properties of the putative apical K+ channels.In the present experiments, patch clamp methods and RT-PCR were used to characterize apical K+ channels in the apical membranes of polarized Calu-3 cells. In excised apical membrane patches, we found abundant copies of a single type of inwardly rectifying K+ channel (Kir). The Kir family of channels (reviewed in Ref. 11Bichet D. Haass F.A. Jan L.Y. Nat. Rev. Neurosci. 2003; 4: 957-967Crossref PubMed Scopus (207) Google Scholar) are homo- or heterotetramers. Each subunit has two transmembrane domains, a pore loop, and cytoplasmic N and C termini. At least 16 genes (KCNJ1–16) have been identified, giving rise to channels grouped into 7 subfamilies that differ in their channel signatures, distribution, and mode of activation. Kir channels differ from the previously identified basolateral K+ channels of Calu-3 cells, which are 6 transmembrane domain channels activated by elevations of [Ca2+]i and [cAMP]i, respectively. Transcripts for Kir4.2 were amplified in Calu-3 lysates, but the signature of the Calu-3 apical K+ channel did not exactly match previous descriptions of Kir4.2 channels, leaving its identity undetermined.MATERIALS AND METHODSCells and Cell Culture—Experiments were conducted using Calu-3 cells grown as previously described by Shen et al. (1Shen B.Q. Finkbeiner W.E. Wine J.J. Mrsny R.J. Widdicombe J.H. Am. J. Physiol. 1994; 266: L493-L501PubMed Google Scholar). Briefly, the Calu-3 cell line was obtained from the American Type Culture Collection (Rockville, MD). After thawing, cells were grown at 37 °C in T25 tissue culture flasks (Costar, Pleasanton, CA) containing a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, plus 15% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine in an atmosphere of 5% CO2, 95% O2. Cells were passaged at 1:8 dilution and plated at 5 × 105 cells/cm2 onto a 35-mm Petri dish coated with human placental collagen. Cells were grown to partially confluent islands and used after 1–7 days in culture. To ensure that recordings were made from apical membranes, we only include recordings from cells that were surrounded by other cells within the islands; cells located at edges of the islands were ignored.Patch Clamp Recording and Solutions—Cell attached and inside-out single channel patch clamp recordings were made at ∼23 °C. Pipettes were pulled from Prism LA16/SA16 Glass (Dagan Corp.) on a P-87 Brown-Flaming puller. Pipettes with smaller tips were pulled from thick wall glass (SA16 inner diameter = 0.75, outer diameter = 1.65 mm) and those with larger tips from thin wall glass (LA16 inner diameter = 1.10, outer diameter = 1.65 mm). The different tip sizes were used to record either single channels for kinetic analyses or multiple channels when averaged data was required. Currents were amplified and filtered at 500 Hz or 2 kHz with an Axopatch 1C amplifier. Data acquisition was controlled by pClamp 8.0 (Axon Instruments). Data were sampled and digitized at a rate of 5 kHz and stored on disk. Liquid junction potentials between patch pipette and bath solution were electrically nulled by adjusting the amplifier prior to making seals. Membrane voltage was held at –60 mV unless otherwise indicated.Pipettes were filled with a solution (in mm) of 150 KCl, 2.5 CaCl2, 2.5 MgCl2, and 10 HEPES, with pH adjusted to 7.3 with KOH, and osmolarity adjusted to 320 mosmol. The standard bath and cytosolic control solution was (in mm) 130 K-glutamate, 20 KCl, 0.5 EGTA, 2.5 MgCl2, 1 CaCl2, and 10 HEPES titrated to pH 7.3 with KOH. Osmolarity was adjusted to 320–340 mosmol. The free calcium level was 500 μm as computed by MaxChelator (www.stanford.edu/~cpatton/maxc.html). Buffer solutions were titrated with KOH to pH values of 7.3, 6.0, 6.7, or 8.0.Solution Changer—The valve-controlled solution changer consisted of a manifold with 4 inlet ports, each connected to a different solution reservoir, and one outlet port connected to a perfusion pipette. The manifold was located ∼2 mm from the tip of the perfusion pipette. Solutions were switched manually in less than 2 s. In this setup the patch and perfusion pipette have fixed positions, which assures identical access to each solution.Ussing Chamber Experiments—A small number of short-circuit (Isc) experiments were carried out with monolayers of Calu-3 cells grown for ∼4 weeks on Snapwell filters coated with human placental collagen. Cells were fed from the basolateral side and grown at the air interface. Inserts were mounted in an Ussing chamber (4 ml volume each chamber), the voltage was short circuited with a Physiologic Instrument VCC600, and the current was recorded on a computer using PCLab. Bicarbonate containing solutions were bubbled with 95% O2, 5% CO2 and held at 37 °C. A voltage pulse was passed across the monolayer every 20 s to measure monolayer resistance. After mounting the basolateral membrane was permeabilized with amphotericin B (100 μm) and the Isc across the remaining apical membrane was recorded in the presence of an 11:1 K+ gradient. The apical K+ conductance was probed with 5 mm Ba2+ added apically.Data Analysis—All-point amplitude histograms were constructed for selected traces to determine the amplitude of the unitary current. Histograms were least-squares fit with (N + 1) Gaussian functions (N, number of active channels in the patch; resolution N < 10). The resulting average peak-to-peak interval represented the unitary current (i). Po was determined through least-square fits of binomial distributions of the multiple Gaussians or by Po = I/iNT, where I is the integrated current and T is the total recording time. N was also determined by the peak current observed in a patch, divided by the unitary current obtained as above.The permeability ratio, PNa/PK, was estimated for the excised patches. The plot of reversal potential against [K+]o was fitted to the Goldman-Hodgkin-Katz voltage equation, taking Er obtained with 150 mm K+ in the pipette as reference. This gave an estimate of PNa/PK. The relationship between Er and [K+]o was fitted with the Goldman-Hodgkin-Katz equation, Er = (RT/F) ln [(PK[K+]o + PNa[Na+]o)/PK[K+]i] = (RT/F) ln [[K+]o/C + PNa/K (1-[K+]o/C)], where C = [K+]o + [Na+]o = [K+]i = 150 mm, and R, T, and F have their usual meanings.Reverse Transcription-PCR—Total RNA was extracted from Calu-3 cells using the RNeasy® Mini extraction kit (Qiagen, Valencia, CA) with the ribonuclease-free deoxyribonuclease step. Two-tube RT-PCR was performed using Sensiscript® reverse transcriptase (Qiagen) protocol and HotStarTaq® DNA polymerase (Qiagen) protocol. The Qiagen PCR protocol was scaled down from 100 to 40 μl. The final reaction mixture contained 1.5 mm Mg2+, 200 μm dNTP, and 0.4 μm concentrations of each primer. The primers used are described in Table I (12Dewson G. Conley E.C. Bradding P. BMC Genomics. 2002; 3: 22Crossref PubMed Scopus (6) Google Scholar).Table IKir primersPrimerSequenceSizeTmKir4.1 sense5′-ACTCACTGGAGCCTTCCTCTTCTC-3′59674Kir4.1 antisense5′-GTGTGAACTCGTAGCCCCAAAG-3′68Kir4.2 sense5′-TTCAAATCATACCCCCTGCATC-3′59864Kir4.2 antisense5′-ATAAGATGTTCGGCTCTGGCAG-3′66Kir5.1 sense5′-CAGCAGCTATCATATTATCAATGCG-3′122370Kir5.1 antisense5′-AGAGATTCTGTTTAAAGTCAGGAG-3′66 Open table in a new tab Reactions were heated to 95 °C for 15 min to activate HotStarTaq, then PCR amplification was performed for 40 cycles at 95 °C for 45 s, 58 °C for 45 s, and 72 °C for 60 s, followed by 72 °C for 10 min. The amplification products were electrophoresed on a 1.4% agarose gel containing 0.02% ethidium bromide and photographed. In positive controls, the sets of primers for Kir4.1, Kir4.2, and Kir5.1 (i.e. genes KCNJ10, KCNJ15, and KCNJ16, respectively) yielded amplified products of the expected size.Statistics—All data are expressed as mean ± S.E. unless otherwise indicated. Statistical difference was determined by Student's t test. A value of p < 0.05 was considered statistically significant.RESULTSFunctional K+Channels Expressed in the Apical Membrane—When cell-attached patches were formed on polarized Calu-3 cells using pipettes filled with 150 mm KCl, the most abundant channel activity observed was CFTR, which tended to mask other channel activity. However, immediately upon excision of inside-out patches into an ATP-free, 150 mm K-glutamate solution, K+ channel activity became apparent in ∼45% of patches made with smaller tipped (thick wall) pipettes and ∼100% of patches made with larger tipped (thin wall) pipettes. K+ channel activity was observed even when Ca2+ and Mg2+ levels in the bath were <100 nm. The relative numbers of cell-attached CFTR and excised Kir channels observed in two samples of patches made with thick wall pipettes is shown in Table II.Table IIChannel count statistics in patch clampKirCFTRPatches (n)169158Patches with channels (n)76 (45%)77 (49%)Channels (n)145221Channels per active patch (n)1.92.9 Open table in a new tab Each of these samples was made in conditions designed to optimize recording of Kir or CFTR, respectively. To establish more definitively that these two types of channel occur in the same apical membrane patch and to estimate their relative abundance, recordings were made under conditions in which each type of channel could be observed, and ramp voltages were used to isolate the two kinds of channel currents (Fig. 1, top trace). In 28 excised patches that had both channel types, we observed a total of 79 CFTR and 58 Kir channels. The mean ± S.D. number of channels was 2.8 ± 0.40 CFTR channels and 2.1 ± 0.25 Kir channels per patch, with a mean CFTR:Kir ratio of 1.8 ± 0.33.Unitary Conductance and Inward Rectification—In solutions containing symmetrical K+, unitary current traces at different membrane potentials revealed asymmetrical amplitudes characteristic of inward rectification (Figs. 1, lower trace, and 2A). The rectification was quantified in I–V curves taken from 8 to 11 patches (Fig. 2B). The average inward chord conductance taken between 0 and –200 mV was 24.4 ± 0.5 pS (n = 11) and the outward chord conductance taken between 0 and 50 mV was 9.6 ± 0.7 pS (n = 8). The magnitude of the unitary conductances and their rectification ratio of ∼2.5 are most similar to K+ channels of the Kir4.x family.Fig. 2Permeation properties of an inwardly rectifying K+ channel (Kir) in excised apical membrane patch of Calu-3 cells. A, raw unitary current traces recorded in inside-out patch at different membrane potentials. B, unitary I–V curve averaged from 8 to 11 patches, showing an inward rectification in symmetrical K+. The solid line represents a least-squares fit of 2nd order polynomial function. The mean inward chord conductance γi = 24.4 pS and the mean outward chord conductance γo = 9.6 pS. The S.E. was less than the size of the symbol for most points. Unitary I–V curves from three different levels of [K+]o were used to determine reversal potentials. C, the semi-logarithmic plot of relative Erversus [K+]o. The solid line is the least square fit by the Goldman-Hodgkin-Katz voltage equation to get the permeability ratio, PNa/K = 0.058. Linear fit gives 48.3 mV shifts in Er per 10-fold change in [K+]o. D, [K+]o-dependent unitary conductance changes fitted with a power function of g ∼ [K+]n, n = 0.49. These channel properties are consistent with those of Kir4.2 reported elsewhere.View Large Image Figure ViewerDownload (PPT)Selective K+Permeation—To determine permeation-selectivity between K+ and Na+, we recorded the single channel currents in the inside-out configuration with pipettes filled with different concentrations of K+ (substituted with Na+) to determine reversal potentials. Unitary I–V curves at three different [K+]o are shown in Fig. 2B. Each symbol represents the average from 4 to 11 patches ± S.E. At negative membrane potentials ≤–100 mV, the I–V curves for lower [K+]o are no longer linear, suggesting that [Na+]o blocks permeation. Low [K]o data were least squares fit with a 3rd order polynomial to extract reversal potentials from the linear portions. The relative reversal potentials (Er) with respect to that in symmetrical K+ were –56.1 and –17.1 mV at [K+]o of 10 and 50 mm KCl, respectively. A semi-logarithmic plot of the data shown in Fig. 2C, if fit with a line, gives a ∼48.3 mV shift in reversal potential for a 10-fold change in [K+]o. The relationship between the relative Er and [K+]o was non-linear curve-fitted with the Goldman-Hodgkin-Katz voltage equation. Least square fit of the relative Erversus [K+]o resulted a permeability ratio of PNa/K = PNa/PK = 0.058. Fig. 2D shows the [K]o-dependent unitary conductance changes. Single channel slope conductances g between –100 and –40 mV are 6.8 ± 0.6 pS (n = 3) with 10 mm, 14.0 ± 0.6 pS (n = 6) with 50 mm, and 24.6 ± 1.0 pS (n = 8) with 150 mm [K+]o. The relationship between unitary conductance g and [K+]o can be approximately fitted with a power function of g ∼ [K+]n with an exponent n = 0.49. This is nearly a perfect square root function, typical of K+ channels and in general agreement with values of 0.66 for Kir4.2 (13Pearson W.L. Dourado M. Schreiber M. Salkoff L. Nichols C.G. J. Physiol. 1999; 514: 639-653Crossref PubMed Scopus (69) Google Scholar) and 0.54 for Kir4.1 (14Shuck M.E. Piser T.M. Bock J.H. Slightom J.L. Lee K.S. Bienkowski M.J. J. Biol. Chem. 1997; 272: 586-593Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 15Kusaka S. Horio Y. Fujita A. Matsushita K. Inanobe A. Gotow T. Uchiyama Y. Tano Y. Kurachi Y. J. Physiol. 1999; 520: 373-381Crossref PubMed Scopus (34) Google Scholar).Expression of Kir4.2 mRNA in Calu-3 Cells—To further test the hypothesis that this channel contains Kir4.x subunits, we performed RT-PCR analysis of total RNA extracted from Calu-3 cells. Based on the electrophysiological data, we attempted to amplify three relevant Kir genes: Kir4.1, Kir4.2, and Kir5.1. Kir5.1 is known to form heteromultimers with Kir4 subunits, so it is important to know if it is expressed. RT-PCR analysis in Fig. 3 shows that among the mRNA species examined, Kir4.2 was the only transcript expressed in appreciable amounts in the Calu-3 cells. The following experiments were designed to provide a more detailed characterization of the Calu-3 apical K+ channel.Fig. 3RT-PCR evidence for expression of Kir4.2 but not Kir4.1 or Kir 5.1 in Calu-3 cells.View Large Image Figure ViewerDownload (PPT)Three Modes of Gating—The Kir channels remained active in multichannel patches for more than 30 min after excision, but the open probability (Po) fluctuated markedly. This complicated gating behavior is illustrated in Fig. 4A, a 35-min recording of a single Kir channel in 150 mm [KCl]o/[K-glutamate]i. Po(t) was computed from the normalized average of 5000 data points for every 10 s, and the resulting time-dependent Po was plotted (Fig. 4B). For this channel the mean overall was Po = 0.55 ± 0.27 (S.D.).Fig. 4Gating analysis revealed three distinct gating modes. A, a single channel current trace recorded with a clock of 200 μs for over 35 min at –60 mV. B, fluctuation in Po(t) computed from the box average of the above trace. C, A trace expanded from a part of the above trace, consists of several different gating modes. D, a trace expanded from the long bursts portion reveals very fast closures. E, an expanded trace from the portion of the short burst mode. F, Po distribution histogram with a bin width of 0.05. Peaks were least squares fitted with three Gaussians.View Large Image Figure ViewerDownload (PPT)An expanded portion of the single channel current trace (Fig. 4C) shows that the gating consisted of at least three modes that were captured sequentially in this sample, which shows a long burst, followed by short bursts and then the start of a long closure, which could last for several or tens of seconds. Fig. 4D further expands the long burst portion to show interruptions by very fast closures, and Fig. 4E details the short burst portion that includes multiple opening and closing events lasting a few tenths of a second.To further quantify multi-modal gating, we determined the Po for each gating mode. An intuitive and convenient way to illustrate this analysis is depicted in Fig. 4F. First, the single channel current trace was reduced by averaging every 2-s interval of the original data. Then the reduced data were binned to make an open probability (Po) distribution histogram with a bin width of 0.05. Three major peaks appeared in the histogram, and these were fitted with Gaussian distributions using least squares fits. The fitted peak with the lowest Po corresponded to the long closures and had an average Po = 0.10 and an area (representing the percent time in this mode) of 5.1. The middle peak corresponded to the short bursts and had an intermediate Po = 0.42 and an area of 31.4. The third peak, corresponding to the long bursts, had a Po = 0.80 and an area of 16.6. The middle peak was several folds wider (0.58) than the left (0.08) or right (0.16) peaks, suggesting that it might represent more than one gating mode.Fluctuations like those shown in Fig. 4 were sometimes superimposed upon a gradual decrease in Po that occurred overall during recordings of 10–60 min. This slow rundown was inconsistent from patch to patch, but when it happened it was characterized by a decrease in the long burst open time and an increase in the long closed time. The presence of 2 mm ATP in the bath did not prevent rundown.Characteristics of Cs+and Ba2+Block—Cs+ block from the extracellular surface was examined by recording channel activity in the inside-out configuration with pipettes containing various levels of Cs+. In the presence of [Cs+]o, current traces showed apparent reductions in unitary current amplitude as a result of fast block (Fig. 5A). I–V curves (Fig. 5B) revealed that the inward currents were blocked at hyperpolarized potentials, whereas outward currents were virtually unchanged.Fig. 5Cs+ blocks from the extracellular surface. A, unitary current amplitudes are reduced by the extracellular Cs+ block at potential –160 mV. B, I–V curves show the voltage-dependent Cs+ block at three concentration levels.View Large Image Figure ViewerDownload (PPT)Normalizing the Cs+-blocked I–V curves to the control I–V curves gave the remaining fractions in the presence of [Cs+]o shown in Fig. 5A. Error bars represent transformed S.E. Data were least square fitted with the Boltzmann function. The half-maximum potentials are –255, –158, and –84 mV for [Cs+] levels of 1, 5, and 20 mm, respectively. Fig. 6A depicts the Cs+-blocked currents normalized to that of the control versus [Cs+]o. The solid lines represent the least squares fit to the Hill equation. The fitted Kd(V) = 2.9, 3.6, 3.9, 5.4, 6.9, 11.6, 20.6, and 39.0 mm corresponding the membrane voltages from –200 to –60 mV with a step of 20, respectively. The voltage dependence of these Cs+ dissociation constants is plotted in the inset of Fig. 6B and fitted by a Woodhull-type exponential relationship that results in an electrical distance of δ = 0.69.Fig. 6Analysis of Cs+ block. A, voltage dependence of Cs+ block is illustrated by normalizing the Cs+ block I–V curves. Normalized remaining currents, caused by the Cs+ block, are fit with Boltzmann functions, resulting in voltages (V0.5) at which half the channels are open at different levels of [Cs+]. Error bars represent transformed S.E. ± mean. B, concentration-dependent Cs+ is illustrated by the fractional block (f), fitted with f = 1/{1 + (Kd(V)/[Cs+])H} shown as solid lines. H is the Hill coefficient ∼1. The fitted Cs+ dissociation constants, Kd(V), are plotted in the inset, which can be fitted with a simple exponential function defined by Woodhull: Kd(V) = Kd(0) × exp{–δFV/RT}, where δ is electrical distance and F/RT have their usual meanings.View Large Image Figure ViewerDownload (PPT)Ba2+ block of Kir was examined in the same way as Cs+ block, by filling the pipette with 0.5 mm Ba2+. In contrast to the Cs+ block, which reduced the unitary current, Ba2+ induced a voltage-dependent, discrete block (Fig. 7). It appears that at hyperpolarized potentials, Ba2+ caused an increase in the closed time. These results with blockers provide an additional signature for this channel.Fig. 7Voltage dependent Ba2+ block. Pipettes were filled with 0.5 mm Ba2+ to examine the Kir current blocked from the external surface. In contrast to the external Cs+ block, which reduced the unitary current, Ba2+ induced a voltage-dependent, discrete block. It appears that at hyperpolarized potentials, the closed time caused by barium block increases.View Large Image Figure ViewerDownload (PPT)The basolateral membranes of Calu-3 cells contain K+ channels that are strongly and directly activated by 1-EBIO and are inhibited by clotrimazole. To determine whether the apical K+ channel shared these properties, the apical K+ conductance was tested in Ussing chambers using 5 mm Ba2+ added to the apical chamber. The magnitude of the apical Ba2+ block (under conditions mentioned under “Materials and Methods”) was compared before and after 1-EBIO (500 μm, n = 8), and before and after clotrimazole (50 μm, n = 8). Neither compound caused a significant difference in the magnitude of the Ba2+-blockable K+ conductance.ATP Weakly Activated Kir—ATP was not required for channel opening, as shown by our routine observation of Kir activity in ATP-free conditions. Furthermore, when partial rundown was observed it was not prevented by the presence of 2 mm ATP. To look for more subtle effects of ATP on channel gating, we used larger tipped pipettes to obtain patches with multiple K+ channels, so that fluctuation-induced error would be reduced relative to the signal. Then, the multiple (n > 2) channel patches were rapidly and repeatedly switched among several ATP levels by moving them into different streams of a multibarrel perfusion outlet.Under these conditions, responses to ATP could be observed even in the raw traces (Fig. 8A). For analysis, control currents (0 ATP) from before and after a given ATP concentration were averaged (mean at –60 mV =–4.23 ± 0.29 pA, n = 29) and the percentage increases relative to the average were computed. This method minimized the remaining fluctuations and possible rundown. Data were compiled in this way for 6 patches, with 8–12 trials each (Fig. 8B). The data show a 35% increase in Kir currents for ATP levels of 2 or 10 mmversus 0 or 0.5 mm (p < 0.05 and p < 0.001, t test). Note that if a Po" @default.
• W2023732361 created "2016-06-24" @default.
• W2023732361 creator A5011348984 @default.
• W2023732361 creator A5021112469 @default.
• W2023732361 creator A5022593117 @default.
• W2023732361 creator A5046097549 @default.
• W2023732361 creator A5080722924 @default.
• W2023732361 date "2004-11-01" @default.
• W2023732361 modified "2023-10-18" @default.
• W2023732361 title "An Inwardly Rectifying Potassium Channel in Apical Membrane of Calu-3 Cells" @default.
• W2023732361 cites W1859154741 @default.
• W2023732361 cites W1881435564 @default.
• W2023732361 cites W1965559088 @default.
• W2023732361 cites W1971765032 @default.
• W2023732361 cites W1975201915 @default.
• W2023732361 cites W1985770721 @default.
• W2023732361 cites W1995200773 @default.
• W2023732361 cites W1999257065 @default.
• W2023732361 cites W2019251722 @default.
• W2023732361 cites W2019544462 @default.
• W2023732361 cites W2038482806 @default.
• W2023732361 cites W2038961327 @default.
• W2023732361 cites W2040680037 @default.
• W2023732361 cites W2045419222 @default.
• W2023732361 cites W2052211249 @default.
• W2023732361 cites W2054555777 @default.
• W2023732361 cites W2055547525 @default.
• W2023732361 cites W2056663194 @default.
• W2023732361 cites W2058747402 @default.
• W2023732361 cites W2064662837 @default.
• W2023732361 cites W2064953660 @default.
• W2023732361 cites W2071598381 @default.
• W2023732361 cites W2077400381 @default.
• W2023732361 cites W2083061901 @default.
• W2023732361 cites W2083963379 @default.
• W2023732361 cites W2087139802 @default.
• W2023732361 cites W2092552227 @default.
• W2023732361 cites W2102730957 @default.
• W2023732361 cites W2110188188 @default.
• W2023732361 cites W2129110633 @default.
• W2023732361 cites W2133400203 @default.
• W2023732361 cites W2142804053 @default.
• W2023732361 cites W2143389504 @default.
• W2023732361 cites W2156790391 @default.
• W2023732361 cites W2160846272 @default.
• W2023732361 cites W2182697993 @default.
• W2023732361 cites W2187694235 @default.
• W2023732361 cites W2214123781 @default.
• W2023732361 cites W2243408637 @default.
• W2023732361 cites W4234808089 @default.
• W2023732361 doi "https://doi.org/10.1074/jbc.m406058200" @default.
• W2023732361 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15328350" @default.
• W2023732361 hasPublicationYear "2004" @default.
• W2023732361 type Work @default.
• W2023732361 sameAs 2023732361 @default.
• W2023732361 citedByCount "20" @default.
• W2023732361 countsByYear W20237323612012 @default.
• W2023732361 countsByYear W20237323612013 @default.
• W2023732361 countsByYear W20237323612015 @default.
• W2023732361 crossrefType "journal-article" @default.
• W2023732361 hasAuthorship W2023732361A5011348984 @default.
• W2023732361 hasAuthorship W2023732361A5021112469 @default.
• W2023732361 hasAuthorship W2023732361A5022593117 @default.
• W2023732361 hasAuthorship W2023732361A5046097549 @default.
• W2023732361 hasAuthorship W2023732361A5080722924 @default.
• W2023732361 hasBestOaLocation W20237323611 @default.
• W2023732361 hasConcept C12554922 @default.
• W2023732361 hasConcept C127162648 @default.
• W2023732361 hasConcept C170493617 @default.
• W2023732361 hasConcept C178790620 @default.
• W2023732361 hasConcept C181911157 @default.
• W2023732361 hasConcept C185592680 @default.
• W2023732361 hasConcept C3018436504 @default.
• W2023732361 hasConcept C41008148 @default.
• W2023732361 hasConcept C41625074 @default.
• W2023732361 hasConcept C50254741 @default.
• W2023732361 hasConcept C517785266 @default.
• W2023732361 hasConcept C55493867 @default.
• W2023732361 hasConcept C76155785 @default.
• W2023732361 hasConcept C83743174 @default.
• W2023732361 hasConcept C86803240 @default.
• W2023732361 hasConcept C94924445 @default.
• W2023732361 hasConcept C95444343 @default.
• W2023732361 hasConceptScore W2023732361C12554922 @default.
• W2023732361 hasConceptScore W2023732361C127162648 @default.
• W2023732361 hasConceptScore W2023732361C170493617 @default.
• W2023732361 hasConceptScore W2023732361C178790620 @default.
• W2023732361 hasConceptScore W2023732361C181911157 @default.
• W2023732361 hasConceptScore W2023732361C185592680 @default.
• W2023732361 hasConceptScore W2023732361C3018436504 @default.
• W2023732361 hasConceptScore W2023732361C41008148 @default.
• W2023732361 hasConceptScore W2023732361C41625074 @default.
• W2023732361 hasConceptScore W2023732361C50254741 @default.
• W2023732361 hasConceptScore W2023732361C517785266 @default.
• W2023732361 hasConceptScore W2023732361C55493867 @default.
• W2023732361 hasConceptScore W2023732361C76155785 @default.
• W2023732361 hasConceptScore W2023732361C83743174 @default.
• W2023732361 hasConceptScore W2023732361C86803240 @default.

• next