Address correspondence to Vladimir V. Matchkov, The Water and Salt Research Center, Department of Physiology University of Aarhus Universitetsparken, Building 160, DK-8000 Aarhus C, Denmark. Fax: (45) 8612 9065; email: vvm{at}fi.au.dk
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ABSTRACT |
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Key Words: chloride channels calcium signaling calcium oscillations cyclic nucleotides
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INTRODUCTION |
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In the present work we have examined the characteristics of this Ca2+-activated current. Although this current was shown to be a Ca2+-activated Cl- current (ICl(Ca)), it has several characteristics that are distinct from previously described ICl(Ca) (Large and Wang, 1996; Frings et al., 2000
). The previously characterized current has properties such as time and voltage dependence, sensitivity to pharmacological blockers such as niflumic acid (Large and Wang, 1996
), a characteristic permeability sequence for halides (Frings et al., 2000
), and specific interactions with multivalent cations (Tokimasa and North, 1996
). However, the properties of the cGMP-dependent chloride current in smooth-muscle cells from mesenteric small arteries described here differs in many of these aspects. For comparison we have therefore also characterized ICl(Ca) from pulmonary arterial myocytes, where it has been characterized in detail previously (Yuan, 1997
; Greenwood and Large, 1999
; Greenwood et al., 2001
). The new channel seems to combine properties characteristic for different families of Cl channels. Brief preliminary reports of part of the present results have been given elsewhere (Matchkov et al., 2001
, 2003
).
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MATERIALS AND METHODS |
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Subsequently, in most experiments papain digestion (Schubert et al., 2001) was used to isolate smooth-muscle cells. The arteries were stored overnight at 4°C in a solution for enzymatic digestion of the arteries (for composition see below). Before experiments, the microtube with the vessels was incubated for 510 min at 37°C, after which the vessels were transferred to a low-calcium solution. Single cells were released by trituration with a polyethylene pipette into the experimental solution. This method consistently produces a high yield of relaxed smooth-muscle cells, which respond reversibly to vasoconstrictors and remain viable for at least 3 h (Clapp and Gurney, 1991
).
In experiments where BAY K8644 was used to activate calcium channels, cells were instead isolated by the collagenase-elastase method of Petkov (Petkov et al., 2001). This method produces smooth-muscle cells that express a high density of voltage-gated calcium currents.
Patch-clamp Recording
All experiments were made at room temperature (2224°C). Patch pipettes were prepared from borosilicate glass (PG15OT-7.5; Harvard Apparatus), pulled on a P-97 puller (Sutter Instrument Co.) and fire polished to achieve tip resistances in the range of 25 M.
Recordings were made with an Axopatch 200B amplifier (Axon Instruments, Inc.) in whole-cell configuration. Data were sampled at a rate of 2 kHz and filtered at 1 kHz. Data acquisition and analysis were done with the software package Clampex 7 for Windows (Axon Instruments, Inc.). Only cells with essentially no leak current (seal resistance >2 G) and a low access resistance (510 M
) were used, and the stability of these parameters was tested regularly during the course of the experiment. Series resistance and capacitive current were routinely compensated. Junction potentials were estimated using the junction potential calculator of pClamp 7 (Axon Instruments, Inc.) and data were corrected for these.
Currents were recorded at a holding potential of 60 mV. Current-voltage characteristics were obtained either from ramp protocols or from a voltage-step protocol. Voltage ramps were performed from 60 to +60 mV with a duration of 100 ms, unless specified otherwise. In the voltage-step protocol currents were evoked by pipette solutions containing calcium buffered at 900 nM (Fixed-Ca solution, see below). This technique has been used to activate ICl(Ca) in studies on smooth muscle (Greenwood et al., 2001; Britton et al., 2002
) and directly activates the chloride current at a clamped concentration of calcium. The current-voltage relationship of the activated current (after subtraction of baseline current) was determined by stepping the potentials (20-mV steps) between 90 and +90 mV, maintaining the voltage at each step for 2 s. Current amplitudes were averaged over the last 0.1 s before the end of the test pulse.
To obtain the relative permeabilities for anions under biionic conditions the current was activated by application of ionomycin in symmetrical chloride conditions. Current-voltage relationships were obtained by 2-s voltage steps covering the range 70 to +90 mV in 20-mV increments from a holding potential of 90 mV. The protocol was performed initially in symmetric, chloride-containing solution, and then repeated during extracellular superfusion with the substitute anion. Finally, chloride-containing solution was reapplied and a control measurement was made. Current values were taken from the last 0.1 s of the 2-s voltage step. Ionomycin was present throughout. Results were discarded from analysis if the initial and final measurements in the presence of chloride differed in characteristics.
Drug Application
Drugs were either applied to the bath or superfused locally over the patched cell (except experiments in biionic conditions, where drugs were applied in the solutions for flow exchange with the rate 5 ml/min; see above). For local superfusion, a specially designed electric manipulator (Danish Myo Technology) was used to position the application pipettes immediately before application and to move them out of the solution immediately afterwards. In this way seepage of drugs onto the cells before or after application was eliminated.
The standard protocol consisted of breaking the membrane and waiting 5 min for equilibration. Then a control recording was made, followed by a 10 min wash out. Finally, the drug was superfused for 1 min followed by another current recording in the continued presence of the drug.
Solutions
The solution used for enzymatic digestion of arteries contained (in mM): NaCl 110, KCl 5, MgCl2 2, KH2PO4 0.5, NaH2PO4 0.5, NaHCO3 10, CaCl2 0.16, EDTA 0.49, Na-HEPES 10, glucose 10, taurine 10 at pH 7.0, as well as 1.5 mg ml1 papain, 1.6 mg ml1 albumin, and 0.4 mg ml1 dl-dithiothreitol. The collagenase/elastase enzyme solution contained (in mM): NaCl 110, KCl 5.6, MgCl2 1.2, Na-HEPES 10, glucose 20, taurine 20, Na-pyruvate 5 at pH 7.4, as well as 1 mg ml1 collagenase type 1 (Worthington Biochemical Corporation), 4.85 µg ml1, elastase type I (Sigma-Aldrich), 1 mg ml1 soybean trypsin inhibitor, 1 mg ml1 albumin.
The active subunit of protein kinase G (PKG) was obtained by trypsinization of PKG as described by White et al. (1993). In brief, 10 µg PKG I
was dissolved in 300 µl water. Trypsin (1 µg) was added and incubated for 3 min at 30°C, after which 10 µg soybean trypsin inhibitor was added. This solution was added in a ratio of 1:30 to the pipette solution.
Compositions of solutions used in the patch-clamp experiments are given for bath solutions in Table I, and for pipette solutions in Table II. Free calcium concentration was estimated by the WebMaxC v2.22 buffer program (Chris Patton, Stanford University, CA). For the "low-calcium" (B3; Table I) and "low-calcium-Cs" (B4) solutions in the presence of 1 mM BaCl2 free calcium was estimated to be 19 µM. To determine the relative permeability to halides the CsCl in the extracellular solution was substituted with the corresponding cesium salt on an equimolar basis in 46 experiments for each ion species tested. In experiments under biionic conditions 3 M KCl salt-agar bridge was placed between the Ag/AgCl pellet and the bath solution.
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Statistics
All data are given as means ± SEM. Only one experimental recording was taken from each cell, thus n is the number of cells. Statistical analysis was performed using cells from at least three different isolations. Unpaired Student's t test was used for single comparisons, and one-way analysis of variance test with Bonferroni's post-test for multiple comparisons (GraphPad Prism v. 2.01; GraphPad Software). Nonlinear regression to the Hill equation was used for the analysis of concentration-effect curves. Linear regression was used to compare estimated and experimental changes in ECl, and in experiments for determination of the relative conductance of halides.
Relative permeability was determined by measuring the shift in reversal potential (Erev) upon changing the solution on one side of the membrane from one containing chloride ions (Cl) to another with the substitute anion (X). The permeability ratio was estimated using the Goldman-Hodgkin-Katz equation: PX/PCl = exp(ErevF/RT), where
Erev is the difference between the reversal potential with the test anion X and that observed with Cl, F is Faraday's constant, R is the gas constant, and T is temperature.
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RESULTS |
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Ion Selectivity
The inward current is unlikely to be a potassium current, since the potassium equilibrium potential (78.5 mV) is negative to the holding potential (60 mV). Also, the cGMP-sensitive inward current was insensitive to various K-channel antagonists: neither 100 nM charybdotoxin (n = 34) nor 100 nM iberiotoxin (n = 13), 1 µM apamin (n = 5), 10 mM 4-aminopyridine (n = 5), or 10 mM tetraethyl ammonium (n = 5) reduced the [Ca2+]i-activated inward current. In 6 of 10 experiments performed in the absence of potassium-channel blockers, a transient outward current was seen superimposed on the inward current: this outward current was most likely due to activation of a KCa channel, since it was never observed in the presence of 100 nM charybdotoxin (ChTX).
It is possible that Na+ ions carried the inward current (ENa +68 mV for B1:P1). However, neither partial nor complete replacement of extracellular sodium affected the amplitude of the current, indicating that sodium is not involved: the inward current amplitude was 8.66 ± 0.74 A F1 (n = 6; cell capacitance 15.8 ± 0.65 pF) in the presence of 145 mM Na+ (solutions B1:P1), 8.93 ± 0.62 A F1 (n = 4; cell capacitance 16.9 ± 2.59 pF) in the presence of 10 mM Na+ (the bath solution B1 was modified by substitution of 135 mM Na+ with NMDG; the pipette solution was P1), and 7.15 ± 0.48 A F1 (n = 5; cell capacitance 14.8 ± 1.71 pF) in the absence of Na+ (the bath solution B1 was modified by complete substitution of Na+ with NMDG; the pipette solution P1).
Calcium ions could also potentially carry inward current (ECa +89 mV for B1:P1). However, neither 1 µM nifedipine (n = 5) nor 10 µM mibefradil (n = 5) inhibited the inward current. Furthermore, superfusing the cell for 1 min with a Ca2+-free solution before caffeine application did not affect the current: the inward current amplitude was 8.12 ± 2.12 A F1 (n = 5; solutions B1:P1) in the control conditions and 8.79 ± 2.07 A F1 (n = 5; cell capacitance 12.8 ± 0.46 pF) after superfusion with Ca2+-free solution (solutions B5:P1). This finding not only demonstrates that the charge carrier is not calcium ions, but it also shows that the calcium that activates the current during caffeine stimulation is derived from an intracellular pool, and that the current under these conditions is not activated secondarily to calcium influx through, e.g., store-operated calcium channels.
A further candidate as charge carrier for an inward current is chloride. An involvement of chloride was tested in CsCl solutions by partially replacing chloride by aspartate in either the intracellular or extracellular solution. The replacement was such that it would move the equilibrium potential 20 mV in either direction. This caused corresponding shifts in the reversal potential of the current (Fig. 2). The current thus was determined to be carried by chloride ions.
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A 1-min incubation with 100 µM Zn2+ inhibited the calcium-activated inward current evoked either by a 10-mM caffeine application, by incubation with 0.4 µM ionomycin, or by an application of 1 µM BAY K 8644 (Fig. 4, A and B). Even in a concentration of 1 µM, Zn2+ strongly reduced the current (tested only with caffeine; Fig. 4 A). The effect of Zn2+ was specific for the chloride current, since KCa channel currents could still be activated by caffeine application if the experimental solutions did not contain K channel blockers (bath solution was B1 without BaCl2 and ChTX; pipette solution was P1): at +60 mV, outward current density was 159 ± 38 A F1 (n = 4; cell capacitance 17.9 ± 3.85 pF) in the absence of Zn2+ and 177 ± 40 A F1 (n = 4, NS; cell capacitance 17.8 ± 2.69 pF) in the presence of 100 µM Zn2+, and this was reduced to 0.6 ± 0.9 A F1 (n = 4; cell capacitance 15.3 ± 1.45 pF) by further addition of 100 nM ChTX. Thus, the effect of Zn2+ is an inhibition of the inward current evoked by calcium release, and not an inhibition of the calcium release itself.
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Also Ni2+ inhibited the calcium-activated inward current, although less potently then Zn2+. As shown in Fig. 4 E, the concentration-effect curve had an EC50 for Ni2+ of 27.5 µM with a Hill coefficient of 1.5. In contrast, Co2+ had little effect on the current at concentrations up to 100 µM. The current density was 6.28 ± 1.10 A F1 under control conditions, and after 1 min in the presence of 100 µM Co2+ it was 5.70 ± 1.07 A F1 (n = 4, NS; cell capacitance 17.8 ± 2.23 pF) when the membrane potential was held at 60 mV.
Anion Selectivity of the Current
We obtained the relative permeabilities for different anions under biionic conditions (see MATERIALS AND METHODS) based on symmetrical CsCl solution with extracellular ionomycin. Typical current-voltage recordings are shown in Fig. 5, AF. As is evident from the current-voltage curves, the chloride current did not show any voltage dependence or rectification. The relative permeability (PX/PCl) was calculated from the shift in Erev using the Goldman-Hodgkin-Katz equation and the relative conductance (GX/GCl) was measured for outward currents. The selectivity of the channel estimated from PX/PCl followed the sequence: SCN > Br > I > Cl > acetate > F >> aspartate (2.85:1.39:1.25:1:0.87:0.79:0.06). However, the relative conductances exhibited a different sequence: I > Br > Cl > acetate > F > aspartate = SCN (1.41:1.17:1:0.85:0.71:0.26:0.23). Based on these results we estimated the relative affinity of the anion for a binding site in the channel (Qu and Hartzell, 2000) as the ratio of the relative permeabilities to the relative conductances; this sequence was: SCN >> Br = F = Cl = acetate = I
aspartate.
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Cyclic GMP Dependence
As mentioned above, hardly any inward current could be evoked by caffeine in the absence of cGMP. Increasing concentrations of cGMP increased the amplitude of the current (Fig. 7 A; solutions B1:P1 with the concentrations of cGMP shown in Fig. 7 A added to the pipette solution). The concentration-effect curve was quite steep, with a Hill coefficient of 3.3 and an EC50 of
6.4 µM. Based on this concentration-effect relation, we routinely used 10 µM internal cGMP to activate the current near-maximally.
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The possible involvement of a kinase in the activation of the current was investigated by replacing the ATP normally present in the intracellular solution by the nonhydrolyzable AMP-PNP. As shown in Fig. 7 E, in the presence of 3 mM AMP-PNP <20% of the current could be evoked by caffeine. The lack of effect of the competitive inhibitor AMP-PNP in lower concentration (0.1 µM) may indicate sufficient production of ATP within the cell to sustain current activation.
Further support for the involvement of a kinase in activating the current was obtained by studying the effects of ATP--S, which can replace ATP as a kinase substrate. The thiol group of ATP-
-S is ineffective as a substrate for phosphatases (Yount, 1975
), leading to what functionally appears as irreversible phosphorylation. Including ATP-
-S in the pipette solution caused the appearance of an inward current over the first few minutes after breaking into the cell (Fig. 7 F). When this current had stabilized, addition of 8Br-cGMP had no further effect (not depicted). The current was inhibited by 100 µM Zn2+. Thus, ATP-
-S activated the cGMP-dependent current as would be expected if a kinase was involved.
We also examined whether PKG was capable of activating the current, using the active subunit of PKG isolated as described in MATERIALS AND METHODS. When this was added to a pipette solution without cGMP, activation of a Zn2+-sensitive current was observed after breaking into the cell (Fig. 7 F). Activation was not seen if ATP was replaced by 3 mM AMP-PNP in the pipette solution (unpublished data). Adding 8Br-cGMP in the presence of active PKG did not enhance the current further (unpublished data). This demonstrates that the current can be activated by PKG.
Calcium-activated Chloride Current in Pulmonary Artery Myocytes
To confirm that the characteristics of the cGMP-dependent current are not merely a reflection of our experimental conditions, we also recorded the previously well-characterized ICl(Ca) from pulmonary arteries (Salter et al., 1995; Greenwood and Large, 1999
; Greenwood et al., 2001
). This current could be evoked by caffeine in the absence of cGMP (Fig. 8 A, solution B1:P1 without cGMP in the pipette solution). The density of the whole-cell current at 60 mV in the control condition was 6.16 ± 0.74 A F1 (n = 14; capacitance 18.5 ± 1.5 pF) and was not enhanced by 300 µM 8Br-cGMP (5.91 ± 1.06 A F1, n = 5; capacitance 19.8 ± 1.8 pF). Zn2+ at a concentration of 100 µM, which eliminates the cGMP-dependent ICl(Ca) of mesenteric artery myocytes, did not change the current in the pulmonary artery myocytes (current density 6.93 ± 1.53 A F1, n = 5; capacitance 15.8 ± 1.6 pF). Also in contrast to the cGMP-dependent current in the mesenteric artery smooth-muscle cells, the current in the pulmonary artery myocytes was inhibited (n = 5) by 100 µM niflumic acid (Fig. 8).
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DISCUSSION |
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The most prominent channel activated by calcium release in vascular smooth-muscle is the large-conductance calcium-activated potassium channel (ZhuGe et al., 1998; Smani et al., 2001
). Such a current was also observed in the present study, but at holding potentials of 60 mV the dominating current was an inward current. Using a combination of ion substitution experiments and blockers it was possible to conclude that the current was a Cl current since the current reversal potential closely followed the Cl equilibrium potential, whereas extracellular removal of either Na+ or Ca2+ had no effect on the current.
The pharmacological and biophysical characteristics of this Ca2+-activated, cGMP-dependent Cl current were investigated and will be discussed in relation to the previously described Ca2+-activated Cl current in smooth muscle (Wang et al., 1992; Large and Wang, 1996
; Greenwood and Large, 1999
).
cGMP Dependence
The cGMP-dependent ICl(Ca) was evoked by calcium release from the SR in response to caffeine application, through influx of Ca2+ induced by ionomycin or BAY K 8644, and by direct activation with high-calcium pipette solution. In every case the cGMP dependence was apparent and the EC50 value of 6 µM (determined with caffeine induced activation) is similar to that of cyclic nucleotidegated channels elsewhere (Müller et al., 1998
, 2001
), and substantially lower than that for cGMP modulation of calcium-activated potassium currents (Schubert and Nelson, 2001
). Recalculation of data on cGMP content in mesenteric arteries (Liu et al., 2002
) with data on smooth-muscle cell water content (Aalkjaer and Mulvany, 1983
) suggest that concentrations in the range 24 µM may be physiological. This agrees well with the concentration range over which the present channel is regulated. Since the current was elicited by four different means of increasing intracellular Ca2+ it is unlikely that the role of cGMP is due to an effect on the Ca2+ release or Ca2+ kinetics. In particular since ionomycin could elicit the current in the presence of ryanodine it is unlikely that cGMP acts through modification of Ca2+ release from the SR. Therefore, a more likely site of action of cGMP is on the channel or a channel-related protein. Moreover, since current activation seemed to require phosphorylation, was sensitive to PKG inhibitors, and could be evoked by PKG, we believe that the effect of cGMP on the current is mediated through PKG.
There is no previous report on a cGMP-dependent ICl(Ca) in smooth muscle. In cat tracheal smooth-muscle, cGMP inhibits a Ca2+-activated Cl current, an effect not mediated by G kinase (Waniishi et al., 1998). That current further differs from the present current in having a higher sensitivity to Cl-channel blockers and being insensitive to 500 µM Zn2+. Indirect evidence from rat aorta, based on the influence of the chloride gradient on contractile responses to noradrenaline in the presence and absence of endothelium, also points to an inhibitory role of cGMP on chloride currents (Lamb and Barna, 1998
). In portal vein smooth-muscle cells, both enhancement and inhibition of the volume-sensitive chloride current by cGMP have been reported (Ellershaw et al., 2000
). Cyclic AMP generally has been reported to activate chloride channels, especially the CFTR (Chen et al., 2001
), but had no effect on the present current. Interestingly, the epithelial CFTR channel was also shown to be activated by 100 µM cGMP via PKG (Lin et al., 1992
). However, that channel had a different permeability sequence from the one studied here, and could also be activated by PKA.
In addition to the cGMP-dependent current, a cGMP-independent current was detected in the smooth-muscle cells from the rat mesenteric arteries, and this was the only current detected in the smooth-muscle cells from pulmonary arteries. This indicates that not all vascular beds may have a prominent cGMP-dependent ICl(Ca) and also shows that the cGMP dependence was not a consequence of our experimental approach. The fact that the conventional ICl(Ca) was more clearly detected with high calcium in the pipette than after caffeine application may indicate that the two currents differ in their calcium sensitivity or are differently affected by calcium release.
ICl(Ca) has been reported to be more stable in experiments using the permeabilized patch technique than in conventional whole-cell experiments, where it tends to show significant run-down (Janssen and Sims, 1992; Large and Wang, 1996
). This suggests the presence of a soluble factor of importance for maintaining channel activity. In our previous study (Peng et al., 2001
) using permeabilized patches, the current studied here could be activated even after some time in the absence of 8Br-cGMP. In the present study using conventional whole-cell experiments the current was not detectable 5 min after breaking the membrane in the absence of cGMP. This suggests that the permeabilized cells retained some endogenous cGMP, which was sufficient for partial activation of the current. However, we did not observe such a pronounced run-down in the pulmonary myocytes, where the current was totally insensitive to cGMP. This makes it unlikely that the run-down of the conventional ICl(Ca) can be explained by washout of cGMP.
Relative Anion Selectivity
The relative permeability for all Ca2+-activated chloride channels depends on the ionic radius, and although slightly variable the typical sequence is I > Br > Cl > F (Amédée et al., 1990; Arreola et al., 1996
; Clapp et al., 1996
; Frings et al., 2000
). The permeabilities obtained in our experiments under biionic conditions, while not identical, were similar (Br > I > Cl > F). The sequence Br > I > Cl > F is typical for a system with an I/Cl selectivity ratio close to 1, while the sequence I > Br > Cl > F is characteristic for systems with an I/Cl ratio higher than 1 (Wright and Diamond, 1977
). Our data had I/Cl ratios of 1.25 (permeability) and 1.41 (conductance), and the obtained selectivity sequences thus fit the theoretical predictions well (Wright and Diamond, 1977
). The data are consistent with selectivity being primarily based on energy of hydration.
Thiocyanate had a high permeability but a low conductance through the channel, indicating that it may bind with high affinity within the channel. Thiocyanate shows a similar high affinity for the GABA receptor of spinal neurons (Bormann et al., 1987). It also shows high affinity for the Xenopus oocyte calcium-activated chloride channel (Qu and Hartzell, 2000
), although both its relative permeability and conductance in the oocyte channel are somewhat higher than in our data.
The fact that both cGMP-dependent and cGMP-independent Cl currents were present in the same smooth-muscle cells opens the possibility that the data for anion selectivity for the cGMP-dependent current might be contaminated by the cGMP-independent current. We never observed the conventional, cGMP-independent current in experiments using ionomycin to elevate [Ca2+]i under control conditions, and therefore used this protocol in the substitution experiments. It is possible that a contaminating current may have become visible in the presence of a substitute ion. However, none the substitutes used here give substantially larger currents than chloride through the conventional, cGMP-independent current (Qu and Hartzell, 2000), with the possible exception of aspartate. Even though we cannot entirely exclude a contribution of the conventional current to the permeability experiments, we therefore believe any such contribution to be small.
Effect of Cl Channel Blockers
There are no specific high-affinity blockers for Ca2+-activated Cl channels available presently. Niflumic acid has been shown in several studies to be an effective inhibitor in micromolar concentrations (Pacaud et al., 1989; Hogg et al., 1994
; Lamb et al., 1994
; Kirkup et al., 1996
). The cGMP-dependent ICl(Ca) was sensitive to both niflumic acid, DIDS, and IAA-94, but the potency of these drugs was low. However, some Ca2+-activated Cl channel clones are either insensitive or have a low sensitivity to niflumic acid (Cunningham et al., 1995
). This has been suggested to be due to a dual effect of niflumic acid to simultaneously enhance and block ICl(Ca) by binding to an external site, probably close to the mouth of the chloride channel (Piper et al., 2002
). Whether this can explain the low sensitivity of the cGMP-dependent ICl(Ca) to niflumic acid is unknown. The fact that not only niflumic acid, but also DIDS and IAA-94 exhibited a low potency could suggest that the structure of the channel may differ from other Ca2+-activated Cl channels. The experiments in Figs. 6 and 8 demonstrate clearly the difference in sensitivity to niflumic acid between the cGMP-dependent and -independent ICl(Ca).
Cation Sensitivity
ICl(Ca) is known to be sensitive to block by several multivalent cations, such as gadolinium, lanthanum, cadmium, cobalt, and nickel applied to the extracellular side of the membrane, whereas Zn2+ has little effect (Tokimasa and North, 1996; Waniishi et al., 1998
). Lack of sensitivity to Zn2+ was also found in our experiments on pulmonary arterial myocytes (Fig. 8) and for the cGMP-independent ICl(Ca) in mesenteric artery smooth-muscle cells. In contrast, the cGMP-dependent ICl(Ca) had a high sensitivity to block by Zn2+ and was unaffected by cobalt. This pattern is known for voltage-gated chloride channels (Kurz et al., 1997
; Chen, 1998
), and suggests that the cGMP-dependent ICl(Ca) in this respect is more like these chloride channels.
Voltage and Time Dependence
The cGMP-dependent ICl(Ca) did not display the voltage dependence commonly seen for other ICl(Ca) (Ishikawa and Cook, 1993; Arreola et al., 1996
; Large and Wang, 1996
; Greenwood et al., 2001
). The voltage dependence of ICl(Ca) depends on the calcium concentration and at very high intracellular calcium concentrations little rectification of ICl(Ca) is seen in parotid glands (Arreola et al., 1996
). However, in some experiments we fixed intracellular calcium at 900 nM where we found no rectification, while the cGMP-insensitive current in the same experiments exhibited outward rectification (Fig. 6). Even though the voltage independence was unexpected, similar observations have previously been reported for ICl(Ca) in hepatocytes (Koumi et al., 1994
) and in neuronal tissue (Krause and Welsh, 1990
; Hallani et al., 1998
).
Comparison with other Channels
The cGMP-dependent ICl(Ca) is similar to the classical, cGMP-independent ICl(Ca) in terms of being calcium dependent, but distinct in terms of lack of voltage and time dependence, and in terms of its sensitivity to pharmacological inhibitors. Its high sensitivity to inhibition by divalent cations seems reminiscent of voltage-activated Cl channels. However, many of these characteristics are similar to what has been reported recently for members of the bestrophin family (Qu et al., 2003; Tsunenari et al., 2003
). Whether this similarity is only superficial or points to structural similarities remains to be determined.
Physiological Role
Since the chloride equilibrium potential of mesenteric artery smooth-muscle cells is about 30 mV (Videbaek et al., 1990; Chipperfield and Harper, 2000
), opening of these channels in a resting smooth-muscle with a membrane potential in the range of 50 to 60 mV will cause depolarization, calcium influx, and contraction (Pacaud et al., 1992
; Wang et al., 1992
; Lepretre et al., 1994
; Kirkup et al., 1996
). In the arteries studied here, calcium release is not essential for depolarization in response to noradrenaline, and the transmembrane gradient for chloride has little influence on the membrane potential at steady-state, whether at rest or during maximal activation (Nilsson et al., 1998
). The cGMP-dependent ICl(Ca) therefore is not likely to be important for the tonic contractile response to receptor activation. In contrast, the current has characteristics that could suggest that it has importance for smooth-muscle synchronization as we have suggested previously (Peng et al., 2001
). Synchronization is essential for coordinating oscillations in [Ca2+]i in vasomotion, and we have suggested that this is brought about by changes in membrane potential subsequent to released intracellular calcium activating the cGMP-dependent current (Peng et al., 2001
). In this way this current may generate intermittent depolarizations maintaining synchrony between the individual smooth-muscle cells in the vessel wall. At the same time, by promoting continuous variations in tone, formation of the latch state is prevented, enabling the smooth-muscle to respond rapidly to, e.g., neural stimuli. Being regulated by two important pathways, [Ca2+]i and NO/cGMP, this current is potentially important in integrating endothelial and smooth-muscle control of vascular function.
It may seem surprising that cGMP promotes an inward current leading to depolarization and vasoconstriction rather than the vasodilatation normally associated with increased cGMP. However, it should be pointed out that the net effect of elevating cGMP in mesenteric small arteries is hyperpolarization and relaxation. How these apparently opposing effects of cGMP on membrane events are orchestrated under physiological conditions is currently unknown.
In summary, the present study characterized a novel Ca2+-activated Cl channel with an obligatory requirement for cGMP in its activation by intracellular calcium. This channel has distinct properties reminiscent of characteristics of both classical Ca2+-activated and voltage-gated Cl channels. The channel is characterized by a high sensitivity to inhibition by Zn2+ but a low sensitivity to inhibition by conventional chloride channel blockers and exhibits no rectification, and is thus different from the ICl(Ca) of pulmonary artery myocytes (Greenwood et al., 2001; this study). Further molecular characterization of the channel(s) responsible for this current could provide insight into structural elements of importance for Cl channel function and for regulation of vascular function in general.
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ACKNOWLEDGMENTS |
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Lawrence G. Palmer served as editor.
Submitted: 6 November 2003
Accepted: 29 December 2003
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REFERENCES |
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