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J. Biol. Chem., Vol. 279, Issue 37, 38830-38837, September 10, 2004

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Calcineurin A but Not A Augments I_Cl(Ca) in Rabbit Pulmonary Artery Smooth Muscle Cells^*

Iain A. Greenwood, Jonathan Ledoux¶||, Amy Sanguinetti¶, Brian A. Perrino**, and Normand Leblanc¶

From the Department of Basic Medical Sciences, Pharmacology & Clinical Pharmacology, St. George's Hospital Medical School, London SW17 ORE, United Kingdom, the ^¶Department of Pharmacology, Center of Biomedical Research Excellence (COBRE), and the ^**Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0270

Received for publication, June 4, 2004 , and in revised form, June 30, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activation of Ca²⁺-dependent Cl^- currents (I_Cl(Ca)) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca²⁺-dependent phosphorylation suppresses I_Cl(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 and forms of the CaN catalytic subunit are expressed in PA cells but that only the variant translocated to the cell periphery upon a rise in intracellular [Ca²⁺]. I_Cl(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 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 form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed I_Cl(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 I_Cl(Ca) evoked as a consequence of Ca²⁺ influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of I_Cl(Ca) is enhanced by Ca²⁺-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Smooth muscle cells actively accumulate chloride resulting in an equilibrium potential 30 mV less negative than the resting membrane potential (1). Consequently, the activation of Cl^- channels leads to Cl^- efflux and membrane depolarization. Ca²⁺-dependent Cl^- currents (I_Cl(Ca))¹ have been recorded from a wide number of smooth muscle cells where they have been implicated in agonist-induced and spontaneous contractions (2, 3). In all of the smooth muscle cells, the generation of I_Cl(Ca) has an obligatory requirement for increased intracellular [Ca²⁺] with a threshold of 200 nM (4, 5). However, in tracheal and arterial smooth muscle cells, the activation of I_Cl(Ca) was augmented by inhibitors of Ca²⁺/calmodulin-dependent kinase, (5, 6) and internal dialysis with constitutively active CaMKII suppressed I_Cl(Ca) in pulmonary artery myocytes (6). 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 I_Cl(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 (7-9). 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 (, , and ), each encoded by a separate gene, and isoform-specific substrates have been identified (10). Western blot analysis indicated that the and isoforms are expressed in pulmonary arteries but that only the CaNA- isoform translocates from the cytosol to the membrane following an elevation of intracellular Ca²⁺ concentration. Intracellular dialysis with constitutively active CaNA- produced a large enhancement of I_Cl(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- had no stimulatory effect on I_Cl(Ca) in PA myocytes. Consequently, through the use of recombinant calcineurin, we show that the regulation of I_Cl(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 (11), 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 (6, 11-13). 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). I_Cl(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 I_Cl(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 (6, 11, 12). The main voltage step protocol used in these experiments was the same as that used in earlier studies to characterize I_Cl(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 I_Cl(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%20Research%20Laboratories">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 (7, 10, 14).

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. (11) using polyclonal goat anti-calcineurin A- and A- 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- or CaNA-, respectively. However, the amino acid sequences of rabbit CaNA- (GenBank^TM accession number AAN23152 [GenBank] and CaNA- (GenBank^TM accession number AAN23153 [GenBank] are identical to the human CaNA- and CaNA- amino acid sequences, so it is very possible that these antibodies specifically recognized rabbit CaNA- and CaNA- 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- and CaNA- 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.

View larger version (38K): [in this window] [in a new window]   FIG. 2. Immunostaining of CaNA- and CaNA- in freshly isolated PA myocytes. Panels A and B show representative images of immunocytochemistry experiments on PA myocytes using antibodies specific for CaNA- (green) and CaNA- (red). In panel A, cells were bathed in a normal extracellular solution, whereas in B, the cells had been preincubated in the external solution containing 80 nM ionomycin, 3 µM ML-7, and 500 nM Ca2+ for 15 min prior to fixation to raise intracellular [Ca2+]. Quantification of data from the experiments shown in A and B is shown in panels C and D. Panel C represents the mean results of experiments where CaNA- and CaNA- antibodies were applied individually (CaNA-:Control (n = 4), 500 nM Ca2+ (n = 6) and CaNA-:Control (n = 5), 500 nM Ca2+ (n = 5)). Panel D shows the mean ± S.D. data from dual labeling experiments (Control (n = 4), 500 nM Ca2+ (n = 5)). For panels C and D, the open and solid bars refer, respectively, to myocytes exposed to normal external solution (1.8 mM Ca2+) labeled with the CaNA- or CaNA- antibodies, whereas the right-slanted and left-slanted hatched bars correspond to myocytes exposed to 500 nM of free Ca2+ solution containing 80 nM ionomycin and 3 µM ML-7, which were dually labeled with CaNA- and CaNA- antibodies.

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.

	RESULTS

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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- and CaNA- revealed that both isoforms were expressed, and a comparison with purified CaN standards showed that 0.56 µg of CaNA- and 0.3 µg of CaNA- were present (Fig. 1). Consequently, PA smooth muscle expresses both the and variants of CaNA but the 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⁺] (11). We used antibodies that were specific for the and 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. 11) and 500 nM Ca²⁺ designed to raise [Ca²⁺]_i, CaNA- translocated toward the plasma membrane, whereas CaNA- 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- 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- 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.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Western blot analysis of CaNA- and CaNA- from PA smooth muscle. Western blot analysis was performed using antibodies specific for CaNA- or CaNA- on PA smooth muscle lysates (lane 1, 50 µg) and purified CaNA- (1 µg) or CaNA- (0.5 µg) expressed in Sf9 cells (lane 2).

View larger version (23K): [in this window] [in a new window]   FIG. 4. Variable effects of cyclosporin A on sustained ICl(Ca) evoked by 500 nM Ca2+ in PA myocytes. A shows representative ICl(Ca) evoked by 500 nM Ca2+ recorded under control conditions at different times upon achieving whole cell access (t = 0, 40, and 80 s). Voltage-dependent relaxations were generated by depolarization from -50 to +70 mV followed by repolarization to -80 mV. Scalars show 250 ms and 200 pA. B shows the mean rundown of ICl(Ca) over the first 2 min of recording. The vertical axis shows the normalized amplitude of the outward relaxation as defined in panel A. The horizontal axis is the time after the rupture of the membrane. Each point is the mean of 22 cells ± S.E. C shows a family of ICl(Ca) currents evoked by 500 nM Ca2+ in the absence and presence of 2 µM CsA. Scalars represent 2.5 pApF-1 and 500 ms. Cells were held at -50 mV and stepped to potentials between -90 and +110 mV. Panels D and E show the mean current voltage relationship for currents evoked by 500 nM Ca2+ in the absence () and presence () of 2 µM CsA. D shows data from an initial study taken from eight cells isolated from three animals. E shows data from a second study again involving cells from two animals (n = 6 cells). F is a comparison of control currents measured in the absence of CsA during studies 1 (filled triangles) and 2 (open triangles). All of the data shown in panels D-F were collected after 6 min of cell dialysis. All of the points are the mean ± S.E. of 6-8 cells.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Effects 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.

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- 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- augmented considerably the amplitude of I_Cl(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-, respectively (n = 8 and 6). The increase in I_Cl(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- that had been boiled for 15 min to reduce enzyme activity failed to increase I_Cl(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).

View larger version (25K): [in this window] [in a new window]   FIG. 5. Effects of constitutively active CaN on ICl(Ca). Panel A shows the time course of changes of ICl(Ca) activated by 500 nM Ca2+ in the absence () or presence of 500 nM CaNA- (). The point of membrane rupture is shown by an asterisk. The vertical axis shows the amplitude of ICl(Ca) recorded at the end of a 750-ms depolarization to +90 mV normalized to cell size. The horizontal axis is the recording time following cell access. Panel B shows examples of ICl(Ca) activated by 500 nM Ca2+ in the absence (Control) or presence of 500 nM CaNA- 6 min after achieving whole cell access. Cells were held at -50 mV and stepped to potentials between -90 and +110 mV. C shows the mean amplitude of ICl(Ca) at the end of the test pulse in the presence of CaNA- (), boiled CaNA- (), or under control conditions (). Each point is the mean ± S.E. of 7, 12, and 4 cells, respectively, with error bars denoting the mean ± S.E. D shows the time constant for current activation at +130 mV and current decay at -80 mV in the absence (stripes) and presence (open) of CaNA-. For panels C and D, the data were all collected after 6 min of cell dialysis. Each column is the mean ± S.E. of 7-9 cells.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Reversal potential of currents evoked in the presence of CaNA- The reversal potential of currents evoked by 500 nM Ca2+ in the absence (A and B) or presence of (C and D) CaNA- were determined using a two-pulse protocol as shown at the bottom. All of the recordings were obtained after 15 min of cell dialysis. Cells were bathed initially in an external solution containing 126 mM NaCl (A and C) and then replaced by one containing 126 mM sodium thiocyanate (B and D). Arrows indicate the potential at which the current was close to the reversal potential.

View larger version (25K): [in this window] [in a new window]   FIG. 7. Specificity of the effects of CaNA- on ICl(Ca). Panel A shows that co-application of a CaN inhibitory peptide (CaN-AIP, ) suppressed the stimulatory effect of CaNA- () on ICl(Ca). Each point is the mean ± S.E. of 6-7 cells. B shows the mean amplitude of ICl(Ca) activated by 500 nM Ca2+ (n = 9) recorded at the end of a 1.5-s test step to different voltages ranging between -90 and +130 mV in the presence of constitutively active CaNA- () or CaNA- (). Panel C shows the amplitude of ICl(Ca) activated by 500 nM Ca2+ at the end of a test pulse to +90 mV normalized to cell size under different recording conditions. The control and CaNA- data were pooled from a number of experiments looking at different comparisons. Each bar is the mean ± S.E. of at least four cells. Panel D shows that CaNA- () had no effect on currents generated with pipette solutions containing 10 mM BAPTA and no added Ca2+. For all of the panels, data were consistently obtained after 6 min of cell dialysis. Each point is the mean ± S.E. of five cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Specific Regulation of I_Cl(Ca) by CaNA- 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 (8, 9). CaN has also been shown to modulate a number of cation channels including voltage-dependent and ATP-sensitive K⁺ channels in smooth muscle cells (18, 19), but data on Cl^- channels are sparse. Recently, we reported that inhibitors of CaN reduced the amplitude of I_Cl(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⁺ (11). This study shows that the CaN inhibitor CsA reduces the amplitude of I_Cl(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 (6) 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 I_Cl(Ca). Consistent with this idea, CaMKII inhibition stimulates I_Cl(Ca) evoked by 500 nM Ca²⁺ in PA cells but has no effect on this current in coronary artery cells (6), although effects are observed when the channel is stimulated by 1 µM free Ca²⁺ (11). However, peptide and inorganic inhibitors of CaN consistently inhibited I_Cl(Ca) elicited by 500 nM Ca²⁺ in coronary artery myocytes (11). Consequently, CaN-mediated dephosphorylation in PA myocytes may be subjugated by an overwhelming activity of CaMKII (and perhaps other kinases). The rundown of I_Cl(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 I_Cl(Ca) and altered deactivation kinetics without influencing peak I_Ca(L) significantly. These data lend support to the notion that I_Cl(Ca) may be physiologically regulated by CaN in conditions minimizing intracellular dialysis and infer that CaN may have a relatively greater impact on I_Cl(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 (8, 20), the influence of the phosphatase is likely to dominate.

Unique Regulation of I_Cl(Ca) by Calcineurin A-—One novel finding of our study was the isoform-specific enhancement of I_Cl(Ca) produced by the inclusion of a constitutively active form of CaN. Whereas both CaNA- and CaNA- were shown to be expressed in pulmonary arteries, only intracellular dialysis with CaNA- modulated I_Cl(Ca), although the and forms of the catalytic A domain are 81% identical at the amino acid level (21) and have a similar Ca²⁺ dependence. Co-application of CaNA- with a peptide fragment analogous to the auto-inhibitory domain confirmed that the effects of CaNA- were due to a specific phosphatase action. Moreover, immunocytochemical experiments revealed that only endogenous CaNA-, but not CaNA-, translocated toward the membrane under conditions mimicking our patch clamp experiments with internal Ca²⁺ clamped at 500 nM. CaN heterodimers containing CaNA- and CaNA- 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 (10). Moreover, trans-genetic approaches have revealed specific functions for the CaNA isoforms. For example, CaNA- is required for T-cell proliferation as well as cardiac hypertrophy (22, 23), whereas CaNA-(-/-) mice display hyperphosphorylated -proteins in the brain and altered post-synaptic de-potentiation in the hippocampus (24, 25). This study is the first to show isoform-selective effects on a native ion channel and suggest that the expression of the isoform of CaNA is the crucial determinant of the phosphorylation status of the Ca²⁺-activated Cl^- channel. Consequently, the relative expression of CaNA- or differences in the : ratio of CaN heterodimers between different vascular smooth muscle tissues would be expected to alter the level of I_Cl(Ca) regulation by this phosphatase. This exquisite property allows the regulation of I_Cl(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 I_Cl(Ca) when the control amplitude was relatively small suggests strongly that the generation of I_Cl(Ca) was reliant upon the level of CaN activity. The precise mechanism conferring specificity of CaNA- on I_Cl(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 and isoforms (26). 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 isoform is the presence of 10 proline residues at the N terminus, a sequence not shared by the 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.

View larger version (19K): [in this window] [in a new window]   FIG. 8. Amino acid sequence homology between CaNA- and CaNA- Panel A shows the basic structural domains of calcineurin and the percentage of amino acid sequence identity for the rat brain CaNA- and CaNA- based on the report by Kuno et al. (26). N-term and C-term, N- and C-terminal ends, respectively; CnB-BD, calcineurin B-binding domain; CaM-BD, calmodulin-binding domain; Inh-D, auto-inhibitory domain. Panel B shows the alignment of the amino acid sequence of the N and C termini of rat brain CaNA- and CaNA-. The numbers above or below the sequences indicate the linear sequence order of the amino acid for each isoform. Identical amino acids are highlighted by boxes. Notice the long sequence of proline residues at the N terminus of CaNA-, which is not present in CaNA-.

This study shows that CaN is a crucial regulator of I_Cl(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 I_Cl(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 I_Cl(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 I_Cl(Ca).

	FOOTNOTES

 
^* The work was supported in part by grants from the CIHR (Grant MOP-10863), the Western Affiliate of the American Heart Association (Grant 0355060Y), and COBRE (Grant NCRR 5 P20 RR15581) of the University of Nevada School of Medicine (Reno, NV) (to N. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Wellcome Trust Career Development Fellowship (53794).

^|| Supported by a doctoral studentship award from the Canadian Institutes of Health Research (CIHR).

Supported by National Institutes of Health Grants DK-57168 and NS-36318 and an innovation award from the American Diabetes Association.

To whom correspondence should be addressed: Dept. of Pharmacology, Mail Stop 318, COBRE, University of Nevada School of Medicine, Reno, NV 89557-0270. Tel.: 775-784-1420; Fax: 775-784-1620; E-mail: NLeblanc{at}med.unr.edu.

¹ The abbreviations used are: I_Cl(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.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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