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• W2022253491 abstract "Activation of Ca2+-dependent Cl- currents (ICl(Ca)) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca2+-dependent phosphorylation suppresses ICl(Ca) in pulmonary artery myocytes, and the aim of the present study was to determine the role of the Ca2+-dependent phosphatase calcineurin on chloride channel activity. Immunocytochemical and Western blot studies with isoform-specific antibodies revealed that the α and β forms of the CaN catalytic subunit are expressed in PA cells but that only the α variant translocated to the cell periphery upon a rise in intracellular [Ca2+]. ICl(Ca) evoked by pipette solutions containing a [Ca2+] set at 500 nm was considerably larger when the pipette solution included constitutively active CaN containing the α catalytic isoform. This stimulatory effect was lost by boiling the enzyme or by the inclusion of a specific CaN inhibitory peptide and was not shared by the inclusion of the β form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed ICl(Ca) evoked by 500 nm Ca2+ when the current amplitude was relatively large but was ineffective in cells with smaller currents. In perforated patch recordings, cyclosporin A consistently inhibited ICl(Ca) evoked as a consequence of Ca2+ influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of ICl(Ca) is enhanced by Ca2+-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific. Activation of Ca2+-dependent Cl- currents (ICl(Ca)) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca2+-dependent phosphorylation suppresses ICl(Ca) in pulmonary artery myocytes, and the aim of the present study was to determine the role of the Ca2+-dependent phosphatase calcineurin on chloride channel activity. Immunocytochemical and Western blot studies with isoform-specific antibodies revealed that the α and β forms of the CaN catalytic subunit are expressed in PA cells but that only the α variant translocated to the cell periphery upon a rise in intracellular [Ca2+]. ICl(Ca) evoked by pipette solutions containing a [Ca2+] set at 500 nm was considerably larger when the pipette solution included constitutively active CaN containing the α catalytic isoform. This stimulatory effect was lost by boiling the enzyme or by the inclusion of a specific CaN inhibitory peptide and was not shared by the inclusion of the β form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed ICl(Ca) evoked by 500 nm Ca2+ when the current amplitude was relatively large but was ineffective in cells with smaller currents. In perforated patch recordings, cyclosporin A consistently inhibited ICl(Ca) evoked as a consequence of Ca2+ influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of ICl(Ca) is enhanced by Ca2+-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific. Smooth muscle cells actively accumulate chloride resulting in an equilibrium potential ∼30 mV less negative than the resting membrane potential (1Chipperfield A.R. Harper A.A. Prog. Biophys. Mol. Biol. 2000; 74: 175-221Crossref PubMed Scopus (142) Google Scholar). Consequently, the activation of Cl- channels leads to Cl- efflux and membrane depolarization. Ca2+-dependent Cl- currents (ICl(Ca)) 1The abbreviations used are: ICl(Ca), Ca2+-dependent Cl- currents; CaN, calcineurin; CaMKII, calcium/calmodulin-dependent kinase II; CsA, cyclosporin A; pF, picofarad; PA, pulmonary artery; TRITC, tetramethylrhodamine isothiocyanate; BAPTA, 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid.1The abbreviations used are: ICl(Ca), Ca2+-dependent Cl- currents; CaN, calcineurin; CaMKII, calcium/calmodulin-dependent kinase II; CsA, cyclosporin A; pF, picofarad; PA, pulmonary artery; TRITC, tetramethylrhodamine isothiocyanate; BAPTA, 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid. have been recorded from a wide number of smooth muscle cells where they have been implicated in agonist-induced and spontaneous contractions (2Large W.A. Greenwood I.A. Piper A.S. Curr. Top. Membr. 2002; 53: 99-118Crossref Scopus (8) Google Scholar, 3Large W.A Wang Q. Am. J. Physiol. 1996; 271: C435-C454Crossref PubMed Google Scholar). In all of the smooth muscle cells, the generation of ICl(Ca) has an obligatory requirement for increased intracellular [Ca2+] with a threshold of ∼200 nm (4Pacaud P. Loirand G. Grégoire G. Mironneau C. Mironneau J. Pflügers Arch. Eur. J. Physiol. 1992; 421: 125-130Crossref PubMed Scopus (67) Google Scholar, 5Wang Y.X. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14918-14923Crossref PubMed Scopus (97) Google Scholar). However, in tracheal and arterial smooth muscle cells, the activation of ICl(Ca) was augmented by inhibitors of Ca2+/calmodulin-dependent kinase, (5Wang Y.X. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14918-14923Crossref PubMed Scopus (97) Google Scholar, 6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar) and internal dialysis with constitutively active CaMKII suppressed ICl(Ca) in pulmonary artery myocytes (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar). These data revealed an inhibition of channel activity contemporaneously with the generation of the current. However, these studies did not take into account other possible Ca2+-dependent pathways. The aim of the present study was to assess whether ICl(Ca) activity in rabbit pulmonary artery (PA) myocytes is also influenced by Ca2+-dependent dephosphorylation. Calcineurin (CaN) is a heterodimeric serine/threonine protein phosphatase that is involved in a number of cellular responses (7Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 8Klee C.B. Ren H. Wang X.T. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 9Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1079) Google Scholar). CaN is composed of a catalytic subunit (CaNA) that is activated by Ca2+ binding to its regulatory subunit (CaNB) and by the binding of the Ca2+/calmodulin complex (Ca2+/CaM). The catalytic subunit of calcineurin (CaNA) exists in three distinct isoforms (α, β, and γ), each encoded by a separate gene, and isoform-specific substrates have been identified (10Perrino B.A. Wilson A.J. Ellison P. Clapp L.H. Eur. J. Biochem. 2002; 269: 3540-3548Crossref PubMed Scopus (43) Google Scholar). Western blot analysis indicated that the α and β isoforms are expressed in pulmonary arteries but that only the CaNA-α isoform translocates from the cytosol to the membrane following an elevation of intracellular Ca2+ concentration. Intracellular dialysis with constitutively active CaNA-α produced a large enhancement of ICl(Ca) elicited by 500 nm Ca2+-containing pipette solutions that was attenuated by the co-dialysis with a peptide inhibitor of CaN. In comparison, CaNA-β had no stimulatory effect on ICl(Ca) in PA myocytes. Consequently, through the use of recombinant calcineurin, we show that the regulation of ICl(Ca) by this phosphatase is highly dependent on the calcineurin isoform. This study, in association with our earlier work with CaN inhibitors in coronary artery smooth muscle cells (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar), reveals the crucial influence of calcineurin on calcium-activated chloride channels and highlights the complex pathways that govern Ca2+-dependent Cl- activity in vascular myocytes. Single Cell Electrophysiology—Cells were prepared from the main and second branch pulmonary arteries isolated from New Zealand White rabbits (2-3 kg) as described previously (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar, 11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar, 12Piper A.S. Greenwood I.A. Large W.A. J. Physiol. (Lond.). 2002; 593: 117-131Google Scholar, 13Piper A.S Greenwood I.A. Br. J. Pharmacol. 2003; 138: 31-38Crossref PubMed Scopus (27) Google Scholar). After isolation, cell were stored in a low Ca2+-physiological salt solution at 4 °C and used within 6 h. The composition of the modified physiological salt solution was as follows: NaCl (120 mm); NaHCO3 (25 mm) (pH 7.4 after bubbling with 95% O2, 5% CO2 gas); KCl (4.2 mm); KH2PO4 (1.2 mm); MgCl2 (1.2 mm); CaCl2 (0.05 mm); and glucose (11 mm). ICl(Ca) were recorded predominantly in the whole-cell voltage clamp mode and were evoked directly by pipette solutions containing free Ca2+ set at 500 nm. For experiments in which ICl(Ca) was elicited by 500 nm Ca2+, the external solution contained: NaCl (126 mm); Hepes-NaOH (10 mm), pH 7.4; glucose (20 mm); CaCl2 (1.8 mm); MgCl2 (1.2 mm); and tetraethylammonium chloride (10 mm). The pipette solution contained: tetraethylammonium chloride (20 mm); CsCl (106 mm); Hepes (5 mm); BAPTA (10 mm); MgATP (3 mm); GTP (0.2 mm); and MgCl2 (0.42 mm) and pH was set to 7.2 by the addition of CsOH. Free [Ca2+] was set by adding an appropriate amount of CaCl2 (7.3 mm) determined by the EQCAL buffer program (Biosoft, Ferguson, MO) and was independently verified using a Ca2+-sensitive electrode (Thermo Orion, Model 93-20, Beverly, MA) and calibrated Ca2+ solutions available from a commercial source (World Precision Instruments, Inc., CALBUF-2, Sarasota, FL). Sustained Ca2+-activated Cl- currents generated by this technique have been characterized fully in previous studies (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar, 11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar, 12Piper A.S. Greenwood I.A. Large W.A. J. Physiol. (Lond.). 2002; 593: 117-131Google Scholar). The main voltage step protocol used in these experiments was the same as that used in earlier studies to characterize ICl(Ca). In the perforated patch experiments, the pipette solution contained CsCl (126 mm), Hepes (5 mm), EGTA (5 mm), amphotericin (300 μg ml-1 from a 60 mg ml-1 stock in dimethyl sulfoxide), and pH set to 7.2 by the addition of CsOH. For those experiments, the external solution was identical to that used in experiments in which ICl(Ca) was evoked by 500 nm Ca2+ as described above. All of the enzymes, MgATP, BAPTA, ionomycin, and ML-7, were purchased from Sigma. Cyclosporin A was purchased from Calbiochem (EMD Biosciences, San Diego, CA), and calcineurin inhibitory peptide (CaN-AIP) was from Biomol Research Laboratories (Plymouth Meeting, PA). Synthesis of Constitutively Active Calcineurin—Constitutively active calcineurin isoforms were created by introducing stop codons into the cDNA for the catalytic subunit, CaNA, causing the translated CaNA subunits to truncate immediately C-terminal of the CaM-binding domain and delete the auto-inhibitory domain. All methodologies for cDNA manipulation, baculovirus screening, and purification of CaN using monolayer cultures of Sf21 cells have been described previously (7Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 10Perrino B.A. Wilson A.J. Ellison P. Clapp L.H. Eur. J. Biochem. 2002; 269: 3540-3548Crossref PubMed Scopus (43) Google Scholar, 14Perrino B.A. Fong Y.L. Brickey D.A. Saitoh Y. Ushio Y. Fukunaga K. Miyamoto E. Soderling T.R. J. Biol. Chem. 1992; 267: 15965-15969Abstract Full Text PDF PubMed Google Scholar). Immunofluorescence and Confocal Imaging—The immunocytochemical detection of CaN isoforms in single PA myocytes was performed as described for coronary artery myocytes by Ledoux et al. (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar) using polyclonal goat anti-calcineurin A-α and A-β antibodies (Santa Cruz Biotechnology) both at a dilution of 1:50. These antibodies are reported by Santa Cruz Biotechnology to recognize a C-terminal epitope in human, rat, and mouse CaNA-α or CaNA-β, respectively. However, the amino acid sequences of rabbit CaNA-α (GenBank™ accession number AAN23152) and CaNA-β (GenBank™ accession number AAN23153) are identical to the human CaNA-α and CaNA-β amino acid sequences, so it is very possible that these antibodies specifically recognized rabbit CaNA-α and CaNA-β in our experiments. Cells were imaged at rest and after stimulation with ionomycin and 500 nm Ca2+ to raise intracellular [Ca2+] and create an internal environment similar to the conditions of the electrophysiological experiments. Contraction of the myocytes was prevented by incubation with the myosin light chain kinase inhibitor, ML-7 (3 μm). The primary antibodies were diluted in phosphate-buffered saline containing 1% normal donkey serum and 0.04% Triton X-100. Negative control experiments were performed by repeating the above steps in the absence of primary antibodies. Coverslips containing the cells were washed three times in phosphate-buffered saline and exposed to a Cy5-coupled anti-mouse antibody (Alexa 647; Molecular Probes) and a TRITC-coupled anti-rabbit antibody (Alexa 546; Molecular Probes) at a dilution of 1:400 for 1 h in the dark at room temperature. Solutions of both secondary antibodies were prepared in goat and diluted in 1% normal donkey serum and 0.04% Triton X-100 (Jackson Immunoresearch Laboratories, Inc.). The bar graphs shown in Fig. 2, C and D, are the mean ± S.E. ratio of membrane/cytosol fluorescence intensity for CaNA-α and CaNA-β taken from a cross-sectional line scan of an arbitrary region of the cell located outside the nuclear region. Fluorescence intensity of ∼5-10 pixels wide on the two sides of the membrane spanned by the line scan was averaged and normalized to averaged fluorescence intensity in the cytoplasm. Western Blot Analysis of Rabbit Pulmonary Artery Smooth Muscle—50 μg of protein from the 1000 × g supernatant of homogenized rabbit PA smooth muscle was separated by 10% SDS-PAGE and electroblotted onto nitrocellulose sheets. Protein concentrations were determined by the Bradford assay using bovine γ-globulin as standard. The blots were probed with Santa Cruz Biotechnology goat anti-CaNA-α or goat anti-CaNA-β antibodies (1:10,000 dilution) followed by horseradish peroxidase-conjugated rabbit anti-goat IgG (1:50,000 dilution) antibodies. Immunodetection was carried using ECL Advance from Amersham Biosciences, and the TIFF images were collected with a CCD camera imaging system (Labworks, UVP Inc.). Densitometry was carried out using Un-Scan-It (Silk Scientific). Statistics—All of the data are the mean ± S.E. of n cells from at least two different animals. Significance was taken with p values below 0.05. Endogenous Expression of Distinct Isoforms of Calcineurin A—Western blot analysis was performed using tissue lysates derived from endothelium-denuded pulmonary arteries. Antibodies selective for CaNA-α and CaNA-β revealed that both isoforms were expressed, and a comparison with purified CaN standards showed that ∼0.56 μg of CaNA-α and 0.3 μg of CaNA-β were present (Fig. 1). Consequently, PA smooth muscle expresses both the α and β variants of CaNA but the α appears to be slightly more abundant. The use of a CaN antibody that was nonspecific for these isoforms revealed that this enzyme translocated to the membrane of coronary artery myocytes upon a rise of intracellular [Ca2+] (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar). We used antibodies that were specific for the α and β isoforms of CaNA to examine the cellular distribution of the individual isoforms in freshly dissociated PA myocytes under resting conditions and after raising intracellular [Ca2+]. Under control conditions, both isoforms appeared evenly distributed between the cytosol and membrane (Fig. 2A). After the cells were exposed to a medium containing 80 nm ionomycin, 3 μm ML-7 (to inhibit myosin light chain kinase and thus contraction; see Ref. 11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar) and 500 nm Ca2+ designed to raise [Ca2+]i, CaNA-α translocated toward the plasma membrane, whereas CaNA-β remained for the most part distributed homogenously throughout the cytoplasm and membrane (Fig. 2A). Consequently, the ratio of optical density of immunofluorescence intensity for CaNA-α labeling at the membrane over cytosol was ∼2.5 regardless of whether the immunodetection involved single (Fig. 2C) or dual (Fig. 2D) antibody experiments. The preferential localization of CaNA-α was not observed when the extracellular solution did not contain Ca2+. These results reveal that in PA cells CaN translocation in response to an elevation of intracellular Ca2+ concentration is isoform-specific. Effect of CaN Inhibition on Ca2+-activated Cl- Channels in PA Myocytes—The influence of endogenous CaN-mediated dephosphorylation on ICl(Ca) was investigated using the CaN-specific blocker, cyclosporin A (CsA), which is a neutral lipophilic cyclic undecapeptide that binds to cyclophilin A to form a complex that suppresses CaN activity by interfering with the active site of the catalytic domain (8Klee C.B. Ren H. Wang X.T. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 9Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1079) Google Scholar). Initial experiments were performed using the perforated patch variant of the whole-cell recording configuration, and ICl(Ca) was elicited by promoting Ca2+ influx through voltage-dependent calcium channels as characterized in previous studies on various types of smooth muscle cell (e.g.15Akbarali H.I. Giles W.R. J. Physiol. (Lond.). 1993; 460: 117-133Crossref Scopus (67) Google Scholar, 16Greenwood I.A Large W.A. Pflügers Arch. Eur. J. Physiol. 1996; 432: 970-979Crossref PubMed Scopus (31) Google Scholar, 17Lamb F.S. Volk K.A. Shibata E.F. Circ. Res. 1994; 75: 742-750Crossref PubMed Scopus (76) Google Scholar). Calcium channels were opened by depolarization from the holding potential of -60 mV, and ICl(Ca) was manifest as an outward current at test potentials positive to the theoretical chloride equilibrium (∼0 mV) and as a slowly declining inward current upon repolarization to the holding potential (Fig. 4A). The application of 2 μm CsA for 180 s had a small insignificant effect (p = 0.09) on the voltage-gated calcium current at +10 mV (Fig. 3, A and B) but produced a 39 ± 10% inhibition of the inward Cl- tail current at -60 mV (n = 5; p = 0.006, Fig. 3C) that was associated with an increase in its rate of decay (Fig. 3D).Fig. 3Effects of CsA on ICl(Ca) recorded using the perforated patch recording configuration. Panel A shows currents recorded in the perforated patch configuration and evoked by step depolarization from -60 mV to +10 mV in the absence or presence of 2 μm CsA. This generated a rapidly activating dihydropyridine-sensitive Ca2+ current (ICa(L)) and a slowly declining ICl(Ca) upon repolarization to -60 mV. Scalars represent 100 ms and 50 pA. The mean effects of CsA on the amplitude of ICa(L) at +10 mV and the magnitude of inward ICl(Ca) tail current and its rate of decay at -60 mV are shown in panels B-D. In panel C, the tail current was measured as the amplitude of the current obtained by extrapolating an exponential fit to time = 0 after return to -60 mV. Data are the mean of five cells from four animals.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To isolate an effect of CsA on CaN-mediated dephosphorylation of the underlying Cl- channel as opposed to possible effects on the voltage-dependent calcium channels or Ca2+-homeostatic mechanisms, experiments were performed where ICl(Ca) was evoked directly by pipette solutions containing 500 nm Ca2+ and 3 mm ATP. With this pipette solution, the rupture of the cell membrane in PA myocytes to achieve whole-cell mode resulted in a large inward current at the holding potential of -50 mV and the generation of time-dependent outward relaxations following depolarization to +70 mV (Fig. 4). Under the ionic conditions used, these currents represent sustained ICl(Ca) that have been characterized extensively in vascular smooth muscle cells including PA myocytes (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar, 11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar, 12Piper A.S. Greenwood I.A. Large W.A. J. Physiol. (Lond.). 2002; 593: 117-131Google Scholar, 13Piper A.S Greenwood I.A. Br. J. Pharmacol. 2003; 138: 31-38Crossref PubMed Scopus (27) Google Scholar). In accord with previous observations (12Piper A.S. Greenwood I.A. Large W.A. J. Physiol. (Lond.). 2002; 593: 117-131Google Scholar), the amplitude of ICl(Ca) at -50 mV declined progressively to a steady-state level over ∼2 min following rupture that was associated with a reduction in the amplitude of the outward relaxation at +70 mV (Fig. 4, A and B). Following the initial period of rundown, the amplitude of ICl(Ca) remained at a constant level ∼20% of the initial amplitude for the remainder of the experiment. Currents were elicited in cells bathed in normal external solution and alternated with cells incubated in 2 μm CsA for 10 min before rupturing the membrane seal to gain whole-cell access. Incubation of cells in CsA significantly attenuated the amplitude of ICl(Ca) generated by 6 min of cell dialysis with 500 nm Ca2+ in seven cells from three animals (Fig. 4D), and the mean late current at +70 mV was 501 ± 70 and 263 ± 51 pA in the absence and presence of CsA (p = 0.043). In an additional study, CsA had no apparent effect on the amplitude of ICl(Ca) (Fig. 4E). However, in these cells, the control current was significantly smaller (see Fig. 4F) that was associated with a more prominent rundown of ICl(Ca) upon membrane rupture. These data show that CsA was able to inhibit sustained ICl(Ca), and the effectiveness of this agent was proportional to the amplitude of the control currents. Overall, these experiments show that the suppression of endogenous CaN by CsA diminishes the amplitude of ICl(Ca) in PA smooth muscle cells but also suggest that CaN is particularly labile in the whole-cell configuration resulting in variable effects of the phosphatase inhibitor. Effect of Dialysis with Constitutively Active Forms of CaN—To circumvent any variable influence due to endogenous CaN and to shift the cellular status in favor of dephosphorylation, we undertook experiments using pipette solutions enriched with constitutively active recombinant CaN isoforms. The inclusion of CaNA-α in a pipette solution containing 500 nm Ca2+ attenuated the initial rundown observed upon rupture of the cell membrane (Fig. 5A), and this was followed by a progressive enhancement of current amplitude over the next 20 min. Consequently, intracellular dialysis with CaNA-α augmented considerably the amplitude of ICl(Ca) (Fig. 5, B and C). After a 6-min recording, the mean current at the end of a step to +90 mV was 17 ± 5 and 47 ± 8 pA pF-1 in the absence and presence of 500 nm CaNA-α, respectively (n = 8 and 6). The increase in ICl(Ca) amplitude was associated with an increase in the rate of current activation at positive potentials and a slowing of current decline at -80 mV (Fig. 5D). The inclusion of CaNA-α that had been boiled for 15 min to reduce enzyme activity failed to increase ICl(Ca) (mean current at +90 mV after a 2-min recording time was 16 ± 6 pA pF-1 compared with 7.4 ± 2 pA pF-1 under control conditions; Fig. 5C). Similar to previous studies (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar, 12Piper A.S. Greenwood I.A. Large W.A. J. Physiol. (Lond.). 2002; 593: 117-131Google Scholar), currents evoked by 500 nm Ca2+ in the absence and presence of CaNA-α reversed (Erev) close to the theoretical chloride equilibrium potential (+2 mV) at +6 ± 2 and +6 ± 1 mV (n = 4), respectively. Replacement of external NaCl with sodium thiocyanate shifted Erev to -40 ± 1 and -42 ± 5 mV, respectively (Fig. 6). These data show that the large current recorded in the presence of CaNA-α was not due to the de novo activation of a contaminating current but was due to an enhanced activation of ICl(Ca). The stimulatory effects of CaNA-α were abolished by co-dialysis with CaN-AIP (Fig. 7A), a peptide inhibitor of CaN that mimics the auto-inhibitory domain (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar, 10Perrino B.A. Wilson A.J. Ellison P. Clapp L.H. Eur. J. Biochem. 2002; 269: 3540-3548Crossref PubMed Scopus (43) Google Scholar). In these experiments, ICl(Ca) at +90 mV with CaNA-α alone was 68 ± 14 pA pF-1 (n = 6), but in combination with 100 μm CaN-AIP, the mean current at +90 mV was 19 ± 4 pA pF-1 (n = 5). In contrast to the striking data with CaNA-α, the β isoform failed to enhance ICl(Ca). In these experiments, a pipette solution containing 500 nm CaNA-α was alternated with one containing 500 nm CaNA-β and the mean ICl(Ca) values at +90 mV were 56 ± 11 and 15 ± 4 pA pF-1, respectively (n = 9 both groups, Fig. 7B). These data not only reveal a high degree of isoform specificity but also show that the stimulation produced by CaNA-α was not the result of a nonspecific effect due to the cell dialysis of a foreign protein. This point was supported by the observation that CaNA-α did not enhance currents evoked by pipette solutions containing 10 mm BAPTA with no added Ca2+ (effectively zero Ca2+, Fig. 7D). These experiments also reveal that CaN-dependent dephosphorylation “alone” cannot stimulate ICl(Ca) and establish that CaN is a crucial regulator of Cl- channel activity but is “not” the impetus for channel activation. The findings of the present study reveal CaN to be an important regulator of ICl(Ca) in PA smooth muscle cells and demonstrate that the modulation of the underlying channels is mediated solely by CaNA-α. Our data show that the inhibition of endogenous CaN with the specific agent, cyclosporin A, reduced the amplitude of ICl(Ca) evoked either directly by pipette solutions containing 500 nm Ca2+ or as a consequence of Ca2+ influx through voltage-dependent Ca2+ channels. These data are consistent with our previous observation in coronary cells that endogenous CaN is an important regulator of ICl(Ca) in vascular smooth muscle cells (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar). The enrichment of the pipette solution with a constitutively active form of CaNA-α augmented markedly the amplitude of ICl(Ca) allied with a considerable change in the voltage-dependent kinetics. Remarkably, this effect was not shared by the β isoform of CaNA, although Western blot analysis revealed that both α and β isoforms are expressed at significant levels in the PA. Consistent with a specific role of CaNA-α for regulating ICl(Ca) in PA cells was the observation that this isoform translocated toward the plasma membrane following elevation of intracellular Ca2+ levels, a property not shared by CaNA-β. Specific Regulation of ICl(Ca) by CaNA-α in Pulmonary Artery Smooth Muscle Cells—CaN-dependent dephosphorylation influences a number of cellular processes including the regulation of transcription factors, synaptic vesicle recycling, and cardiac muscle hypertrophy (8Klee C.B. Ren H. Wang X.T. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 9Rusnak F. Mertz P. Physiol. Rev. 2000; 80: 1483-1521Crossref PubMed Scopus (1079) Google Scholar). CaN has also been shown to modulate a number of cation channels including voltage-dependent and ATP-sensitive K+ channels in smooth muscle cells (18Wilson A.J. Jabr R.I. Clapp L.H. Circ. Res. 2000; 87: 1019-1025Crossref PubMed Scopus (45) Google Scholar, 19Amberg G.C. Koh S.D. Perrino B.A. Hatton W.J. Sanders K.M. Am. J. Physiol. 2001; 281: C2020-C2028Crossref Google Scholar), but data on Cl- channels are sparse. Recently, we reported that inhibitors of CaN reduced the amplitude of ICl(Ca) in coronary artery myocytes in a Ca2+-dependent manner and that these agents decreased the apparent binding affinity of the Cl- channel for Ca2+ (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar). This study shows that the CaN inhibitor CsA reduces the amplitude of ICl(Ca) in PA cells activated as a consequence of Ca2+ influx through voltage-dependent calcium channels or evoked directly by pipette solutions containing 500 nm free Ca2+. Moreover, CsA was observed to be relatively ineffective on currents recorded from cells where the control currents were relatively small. These data suggest that the activity of CaN may vary among populations of myocytes but that the level of activity appears to be a crucial determinant of Cl- current amplitude. Taken together with our previous findings with CaMKII inhibitors (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar) in PA cells, these results implicate a contemporaneous modulation of the channel protein or a closely associated regulatory subunit by phosphorylation and dephosphorylation mechanisms. A similar contra-parallel regulation also exists in coronary artery smooth muscle cells, but there seems to be differences in Ca2+ sensitivity that probably reflect a tissue-specific pattern of expression of the kinases and phosphatases regulating ICl(Ca). Consistent with this idea, CaMKII inhibition stimulates ICl(Ca) evoked by 500 nm Ca2+ in PA cells but has no effect on this current in coronary artery cells (6Greenwood I. Ledoux J. Leblanc N. J. Physiol. (Lond.). 2001; 534: 395-408Crossref Scopus (96) Google Scholar), although effects are observed when the channel is stimulated by 1 μm free Ca2+ (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar). However, peptide and inorganic inhibitors of CaN consistently inhibited ICl(Ca) elicited by 500 nm Ca2+ in coronary artery myocytes (11Ledoux J. Greenwood I.A. Villeneuve L. Leblanc N. J. Physiol. (Lond.). 2003; 552: 701-714Crossref Scopus (36) Google Scholar). Consequently, CaN-mediated dephosphorylation in PA myocytes may be subjugated by an overwhelming activity of CaMKII (and perhaps other kinases). The rundown of ICl(Ca) activity seen in PA cells following activation is likely to reflect a change in the kinase/phosphatase balance in the vicinity of the channel. This hypothesis was corroborated by the use of constitutively active CaN. It is worth stressing that in perforated patch experiments CsA consistently suppressed inward tail ICl(Ca) and altered deactivation kinetics without influencing peak ICa(L) significantly. These data lend support to the notion that ICl(Ca) may be physiologically regulated by CaN in conditions minimizing intracellular dialysis and infer that CaN may have a relatively greater impact on ICl(Ca) regulation when the stimulating rise in [Ca2+]i is transient. Under these conditions, the activation of both CaMKII and CaN by Ca2+/CaM would be less than that observed with a sustained rise in [Ca2+]i; however, as CaN has a greater Ca2+ sensitivity than CaMKII (8Klee C.B. Ren H. Wang X.T. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 20Abraham S.T. Benscoter H. Schworer C.M. Singer H.A. J. Biol. Chem. 1996; 271: 2506-2513Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), the influence of the phosphatase is likely to dominate. Unique Regulation of ICl(Ca) by Calcineurin A-α—One novel finding of our study was the isoform-specific enhancement of ICl(Ca) produced by the inclusion of a constitutively active form of CaN. Whereas both CaNA-α and CaNA-β were shown to be expressed in pulmonary arteries, only intracellular dialysis with CaNA-α modulated ICl(Ca), although the α and β forms of the catalytic A domain are 81% identical at the amino acid level (21Guerni D. Klee P. Adv. Protein Phosphatase. 1991; 6: 391-410Google Scholar) and have a similar Ca2+ dependence. Co-application of CaNA-α with a peptide fragment analogous to the auto-inhibitory domain confirmed that the effects of CaNA-α were due to a specific phosphatase action. Moreover, immunocytochemical experiments revealed that only endogenous CaNA-α, but not CaNA-β, translocated toward the membrane under conditions mimicking our patch clamp experiments with internal Ca2+ clamped at 500 nm. CaN heterodimers containing CaNA-α and CaNA-β catalytic subunits exhibit a similar Ca2+ dependence, as this is conferred by the B subunit, but display different substrate affinities and catalytic activities in vitro (10Perrino B.A. Wilson A.J. Ellison P. Clapp L.H. Eur. J. Biochem. 2002; 269: 3540-3548Crossref PubMed Scopus (43) Google Scholar). Moreover, trans-genetic approaches have revealed specific functions for the CaNA isoforms. For example, CaNA-β is required for T-cell proliferation as well as cardiac hypertrophy (22Bueno O.F. Brandt E.B. Rothenberg M.E. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9398-9403Crossref PubMed Scopus (155) Google Scholar, 23Bueno O.F. Wilkins B.J. Tymitz K.M. Glascock B.J. Kimball T.F. Lorenz J.N. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4586-4591Crossref PubMed Scopus (217) Google Scholar), whereas CaNA-α(-/-) mice display hyperphosphorylated τ-proteins in the brain and altered post-synaptic de-potentiation in the hippocampus (24Kayyali U.S. Zhang W. Yee A.G. Seidman J.G. Potter H. J. Neurochem. 1997; 68: 1668-1678Crossref PubMed Scopus (94) Google Scholar, 25Zhuo M. Zhang W. Son H. Mansuy I. Sobel R.A Seidman J. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4650-4655Crossref PubMed Scopus (149) Google Scholar). This study is the first to show isoform-selective effects on a native ion channel and suggest that the expression of the α isoform of CaNA is the crucial determinant of the phosphorylation status of the Ca2+-activated Cl- channel. Consequently, the relative expression of CaNA-α or differences in the α:β ratio of CaN heterodimers between different vascular smooth muscle tissues would be expected to alter the level of ICl(Ca) regulation by this phosphatase. This exquisite property allows the regulation of ICl(Ca) by CaN to be fine-tuned by the cell through alterations in the composition of CaN heterodimers. Moreover, the inability of CsA to suppress ICl(Ca) when the control amplitude was relatively small suggests strongly that the generation of ICl(Ca) was reliant upon the level of CaN activity. The precise mechanism conferring specificity of CaNA-α on ICl(Ca) cannot be deduced from our data and will require further investigation. Fig. 8A shows the basic features of the structural domains of CaN and the percentage of amino acid sequence identity of the rat brain α and β isoforms (26Kuno T. Takeda T. Hirai M. Ito A. Mukai H. Tanaka C. Biochem. Biophys. Res. Commun. 1989; 165: 1352-1358Crossref PubMed Scopus (86) Google Scholar). The greatest sequence divergence between these two isoforms is observed at the N and C termini (<30%) and to a lesser extent at the Linker I region (60%). An interesting characteristic of the β isoform is the presence of 10 proline residues at the N terminus, a sequence not shared by the α isoform (Fig. 8B). Such a sequence could make this domain a target of interacting proteins, which could obstruct or limit access of the phosphatase to the target protein. Future studies will be undertaken to compare the effects of various chimeric constructs of the two isoforms on the anion current. As yet, the molecular nature of the protein underlying ICl(Ca) remains undefined (27Britton F. Ohya S. Horowitz B. Greenwood I.A. J. Physiol. (Lond.). 2002; 539: 107-117Crossref Scopus (51) Google Scholar) and therefore precise information as to how dephosphorylation heightens ICl(Ca) activity can only be speculated. Our data show unequivocally that Ca2+-dependent dephosphorylation does not gate the opening of the Cl- channel by a rise in [Ca2+]i as CaNA-α failed to generate ICl(Ca) when the internal solution contained 10 mm BAPTA only (i.e. pipette [Ca2+] was in the low nanomolar range). The channel properties underlying the kinetics of ICl(Ca) elicited by the technique used in this study have been characterized in non-muscle cells (28Arreola J. Melvin J.E. Begenisich T. J. Gen. Physiol. 1996; 108: 35-47Crossref PubMed Scopus (137) Google Scholar, 29Kuruma A. Hartzell H.C. J. Gen. Physiol. 2000; 115: 59-80Crossref PubMed Scopus (158) Google Scholar). The voltage-dependent outward relaxation reflects an increase in open probability due to an increase in the binding affinity for Ca2+ and a slower rate of channel closure, whereas the exponentially declining inward current at negative potentials is a simple approximation of the channel deactivation. As CaNA-α increased the rate of activation at positive potentials and slowed that rate of decay at negative potentials, these observations suggest that the removal of phosphate groups either increases the apparent binding affinity or slows the rate of channel closure. These questions will be addressed in future experiments. This study shows that CaN is a crucial regulator of ICl(Ca) in PA cells and that this regulation exhibits a high degree of isoform selectivity. However, the effects of CaN rest in a delicate balance with the suppressive effects of CaMKII and probably other kinases and it is the relative contribution of these enzymes that dictates the amplitude of ICl(Ca). A corollary to this point is that the relative dominance of CaMKII and CaN probably differs between smooth muscles and will alter with different [Ca2+]i. In view of its greater sensitivity to Ca2+-CaM, CaN would be expected to dominate ICl(Ca) regulation at lower [Ca2+]i, whereas CaMKII will predominate when [Ca2+]i is raised (i.e. during agonist stimulation). A necessary caveat to this generalized hypothesis is that other Ca2+-independent phosphatases that have not been tested in this study may also regulate ICl(Ca)." @default.
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• W2022253491 title "Calcineurin Aα but Not Aβ Augments ICl(Ca) in Rabbit Pulmonary Artery Smooth Muscle Cells" @default.
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