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(Circulation Research. 2000;87:1019.)
© 2000 American Heart Association, Inc.

Cellular Biology

Calcium Modulation of Vascular Smooth Muscle ATP-Sensitive K⁺ Channels

Role of Protein Phosphatase-2B

Andrew J. Wilson¹, Rita I. Jabr¹, Lucie H. Clapp

From the Centre for Clinical Pharmacology, Department of Medicine, University College London, UK.

Correspondence to Dr Lucie H. Clapp, Centre of Clinical Pharmacology, Department of Medicine, University College London, 5 University St, London WC1E 6JJ, UK. E-mail l.clapp{at}ucl.ac.uk

	Abstract

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Abstract—ATP-sensitive K⁺ (K_ATP) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP]_i) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that K_ATP channels are insensitive to Ca²⁺, although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca²⁺ on whole-cell K_ATP current in rat aortic smooth muscle cells recorded in a physiological [ATP]_i and K⁺ gradient. Under control recording conditions, cells had a resting potential of -40 mV when bathed in 1.8 mmol/L Ca²⁺. The K_ATP channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP]_i from 3 to 0.1 mmol/L hyperpolarized cells to -60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca²⁺ levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca²⁺]_i showed that K_ATP current was maximally activated at 10 nmol/L [Ca²⁺]_i and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca²⁺-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by 20 to 25 mV. We report for the first time that K_ATP channels can be modulated by Ca²⁺ at physiological [ATP]_i and conclude that modulation occurs via protein phosphatase type 2B.

Key Words: calcium • K_ATP channel • protein phosphatase-2B • smooth muscle • whole-cell recording

	Introduction

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Potassium channels inhibited by intracellular adenosine-5'-triphosphate (K_ATP) and glibenclamide have been identified in many different cell types. In vascular smooth muscle, opening of these channels causes K⁺ efflux and membrane hyperpolarization leading to the closure of voltage-gated Ca²⁺ channels and, hence, vasorelaxation.¹ There is convincing evidence that K_ATP channels are the target for several endogenous vasoconstrictors (eg, angiotensin II) and vasodilators (eg, calcitonin gene–related peptide),^{1 2 3} all of which are capable of modulating intracellular Ca²⁺ levels ([Ca²⁺]_i).

Vascular K_ATP channels are inhibited at a half-maximal concentration of intracellular ATP ([ATP]_i) of between 10 and 300 µmol/L,¹ and because [ATP]_i in arterial smooth muscle is on the order of 1.5 to 3 mmol/L,⁴ it is anticipated that these channels would normally be closed under physiological conditions. However, several reports show that glibenclamide depolarizes intact blood vessels^{5 6} or isolated cells in the perforated patch configuration⁷ in vitro and decreases resting blood flow in vivo.⁸ These observations suggest that K_ATP channels are open and do indeed contribute to the resting membrane potential and maintenance of arterial tone in some smooth muscles.¹ Moreover, other factors, including various protein kinases, are known to regulate the activity of these channels presumably by controlling the degree of phosphorylation of the channel itself or a closely associated protein.^{1 9}

Classically, K_ATP channels have been investigated under conditions where [Ca²⁺]_i is buffered to low levels and external Ca²⁺ ([Ca²⁺]_o) has been removed. Such conditions have been used primarily because several single-channel studies¹ have indicated that K_ATP channels are insensitive to Ca²⁺ but also to minimize the activity of Ca²⁺-dependent K⁺ and chloride channels. To our knowledge, there has been only one report to date of Ca²⁺ modulation of K_ATP channels in freshly isolated smooth muscle cells, where a small conductance channel activated by micromolar [Ca²⁺]_i and inhibited by [ATP]_i was observed in excised patches in rat portal vein.¹⁰ In cultured porcine coronary myocytes, K_ATP channels with a high activity at a physiological [Ca²⁺]_o have been described,² although such a channel has not been reported in freshly isolated cells.¹ Whether Ca²⁺ regulates K_ATP channels in intact cells, where cytosolic regulatory processes would be preserved, remains to be determined. Therefore, the present study was designed to investigate the effects of [Ca²⁺] on whole-cell K_ATP current under a physiological [ATP]_i in isolated rat aortic smooth muscle cells (RASMCs).

	Materials and Methods

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RASMCs were enzymatically dissociated from thoracic aorta of male Sprague-Dawley rats.¹¹ Cells were superfused (0.5 mL/min) with HBSS containing, in mmol/L, NaCl 136.8, KCl 5.4, MgCl₂ 0.5, HEPES 7.5, NaHCO₃ 2.5, KH₂PO₄ 0.44, NaH₂PO₄ 0.65, MgSO₄·7H₂O 0.4, glucose 5.5, CaCl₂ 1.8, and phenol red 0.03 (pH 7.4 with NaOH). For nominally Ca²⁺-free conditions, CaCl₂ was substituted with MgCl₂. Pipettes had resistances of 2.5 to 4.5 M when filled with solution containing, in mmol/L, KCl 135, MgCl₂ 1.2, EGTA 1 or 10, HEPES 10, MgATP 0.1 or 3, and NaGTP 0.5 (pH 7.2 with KOH). Pipette solutions with known free [Ca²⁺] (1 to 300 nmol/L) were prepared by adding specified volumes of CaCl₂ (CaBuf software) to the 10 mmol/L EGTA pipette solution. RASMCs were continuously superfused with bath solution or drugs applied by pressure ejection from a multibarreled micropipette placed 500 µm from the cell.

Membrane potential and whole-cell currents were recorded under current or voltage clamp in the whole-cell configuration of the patch-clamp technique. Unless stated, all recordings were made at least 5 minutes after gaining cell access. Currents were recorded using either 100-ms depolarizing voltage steps of 10-mV increments (every 10 seconds) from -90 to +40 mV elicited from a holding potential of -80 mV or voltage ramps from -100 to +40 mV at a rate of 0.0875 V/s in cells held at -60 mV. The difference current was acquired by digital subtraction of the current trace obtained at the maximal effect of a given manipulation from its corresponding control.

Cyclosporin A, FK-506, and calcineurin autoinhibitory peptide were obtained from Affiniti-Research Products. All data are expressed as mean±SEM from n cells. Statistical difference between groups was calculated using either paired or Student’s unpaired t test. For multiple comparisons, a 1- or 2-way ANOVA with Bonferroni or Student-Newman-Keuls correction methods were used. A value of P<0.05 was considered statistically significant.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Effects of ATP, Glibenclamide, and Levcromakalim on Whole-Cell K⁺ Currents
Under control recording conditions, RASMCs superfused with HBSS and dialyzed with 1 mmol/L EGTA and 3 mmol/L ATP had a mean capacitance of 12.1±0.6 pF (n=36). The average resting potential at 0.5 to 1 minute after the establishment of whole-cell recording was -38.5±2.5 mV (n=40), which did not significantly change after 5 minutes (Table 1). In voltage clamp, a small instantaneous, time-independent background current was observed at negative potentials (Figure 1A). Superfusion with glibenclamide (10 µmol/L), an inhibitor of K_ATP channels, depressed this current (Figure 1 online, available at http://www.circresaha.org, and Figure 1B) and caused a small (9 mV) but significant depolarization of the membrane (Figure 3C). The effects of glibenclamide were fully reversible after several minutes of washout. In addition, the K_ATP channel opener levcromakalim (30 µmol/L) caused a small increase in current at all potentials (Figure 1B) and a modest hyperpolarization (9.0±1.3 mV; n=6; P<0.001).

View this table: [in this window] [in a new window]   Table 1. Effects of EGTA, ATP, and Ca2+ on Membrane Potential in RASMCs

View larger version (34K): [in this window] [in a new window]   Figure 1. Figure 1. Recordings of membrane currents evoked from a cell dialyzed with 3 (A) or 0.1 (B) mmol/L [ATP]i. Representative voltage-ramp currents in the presence of 3 (C) or 0.1 (D) mmol/L [ATP]i before and after superfusion with levcromakalim (Lev) (30 µmol/L) or glibenclamide (Glib) (10 µmol/L). E, Mean ILev at different potentials in cells dialyzed with 0.1 or 3 mmol/L [ATP]i. *P<0.05; **P<0.01.

View larger version (28K): [in this window] [in a new window]   Figure 3. Figure 3. Mean I-V relationship of instantaneous currents recorded from RASMCs under 3 different [Ca2+] conditions in the presence of 3 mmol/L [ATP]i. B, Mean I-V relationship of IGlib under all 3 conditions (n values are the same as in panel C). C, Mean resting membrane potentials measured from cells in the presence and absence of 3 (10 mmol/L EGTA) or 10 µmol/L Glib. *P<0.05; ***P<0.001.

To additionally characterize the background current, we assessed the effects of lowering [ATP]_i from 3 to 0.1 mmol/L. Such manipulations have previously been shown in many smooth muscle cell types to activate or enhance whole-cell K_ATP current and responses to K_ATP channel openers.^{1 12} Dialysis with 0.1 mmol/L [ATP]_i caused a large increase in the magnitude of the background current (Figure 1C), and cells were significantly more hyperpolarized (17 mV; P<0.001) compared with 3 mmol/L [ATP]_i (Table 1). Superfusion with levcromakalim also caused a larger hyperpolarization (16.9±2.2 mV; n=6, P<0.001) and a greater increase in current (Figures 1B and 1D). Indeed, the magnitude of the glibenclamide-sensitive current (I_Glib) and levcromakalim-sensitive current (I_Lev) (Figure 1E) were 2.5- to 3-fold larger in 0.1 compared with 3 mmol/L [ATP]_i. For example, at +10 mV, I_Glib increased from 33.8±6.1 pA (n=11) to 90.5±19.8 pA (n=5). Consistent with effects on K⁺ currents, the reversal potential of I_Glib and I_Lev was -77.6±1.2 mV (n=5) and -77.1±1 mV (n=6), respectively (Figure 1D), which is close to the predicted potassium equilibrium potential (E_K) under our recording conditions of -80.4 mV.

Modulation of Whole-Cell K⁺ Current by Ca²⁺
To assess the effects of Ca²⁺ on whole-cell K⁺ current and membrane potential, 2 sets of experiments were performed. Firstly, [Ca²⁺]_o was lowered to nominally Ca²⁺-free (0 [Ca²⁺]_o), and secondly, [Ca²⁺]_i was reduced by increasing the [EGTA]_i from 1 to 10 mmol/L. All other conditions remained the same. In 0 [Ca²⁺]_o, the magnitude of instantaneous background current was significantly increased at all potentials (Figure 2A) when compared with control conditions (Figure 1A). This was associated with a 20 mV membrane hyperpolarization (P<0.001, Table 1). Moreover, the resting potential remained stable, measuring -59.1±3.5 mV (n=8) and -55.1±3.2 mV (n=6) after 15 and 40 minutes of recording, respectively. Superfusion of glibenclamide (10 µmol/L) caused a complete inhibition of the increase in background current (Figure 2A) and reversed membrane hyperpolarization (Figure 3C). Furthermore, glibenclamide had no effect on time-dependent currents, which were apparent at positive potentials (Figure 2A). Similar results were also observed in cells bathed with HBSS containing 0.1 mmol/L Ca²⁺ (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 2. Figure 2. Recordings of membrane currents obtained in the absence (left) and presence (right) of glibenclamide (A) under different [Ca2+] conditions.

In another set of experiments, RASMCs dialyzed with 10 mmol/L [EGTA]_i displayed a current-voltage (I-V) relationship similar to that obtained for the 0 [Ca²⁺]_o condition (Figures 2B and 3A). Under these conditions, the background current became the predominant current observed. The appearance of this current was associated with similar membrane hyperpolarization to that recorded in 0 [Ca²⁺]_o (Table 1). In addition, the background current was highly sensitive to block by glibenclamide (P<0.001, Figures 2B and 3B), with 3 µmol/L inhibiting the instantaneous current measured from the I-V at 0 mV by 80% from 99.6±24.2 pA to 21.9±2.6 pA (n=10). This was associated with significant membrane depolarization (Figure 3C).

When comparing the mean instantaneous I-V relationships for the 3 different Ca²⁺ conditions (Figure 3A), there was significantly (P<0.05) less instantaneous current in 1.8 mmol/L [Ca²⁺]_o and 1 mmol/L [EGTA]_i compared with the low Ca²⁺ recording conditions. This is consistent with the observation that the magnitude of I_Glib was significantly less in control cells compared with recordings in 10 mmol/L [EGTA]_i or 0 [Ca²⁺]_o (Figure 3B). For example, at 0 mV, the size of I_Glib under control conditions was 20.2±6.7 pA (n=11) and with 10 mmol/L [EGTA]_i was 80.1±20.9 pA (n=10).

To estimate the [Ca²⁺]_i range that regulates K_ATP current, RASMCs were dialyzed with solutions containing different free [Ca²⁺]_i (1 to 300 nmol/L) and superfused with 1.8 mmol/L Ca²⁺. With 1 nmol/L [Ca²⁺]_i, RASMCs displayed voltage-ramp currents that were almost entirely inhibited by glibenclamide (Figure 4A), with the size of I_Glib at +10 mV being similar (89.3±10 pA, n=14) to that recorded from ramps in either the 0 [Ca²⁺]_o (85.6±11 pA, n=9) or 10 mmol/L [EGTA]_i (88.7±18 pA, n=9) condition. However, with 300 nmol/L [Ca²⁺]_i, the I-V relationship was substantially altered, with little current being activated at negative potentials. At positive potentials, current activated steeply and was noisy in appearance, being several hundred pA in size at +40 mV. This current was largely unaffected by glibenclamide (Figure 4B), I_Glib being only 16.6±3.9 pA (n=8) at +10 mV, but it was almost completely inhibited by 300 nmol/L paxilline (Figure 4B), a potent inhibitor of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel.¹³ In contrast, paxilline had only a small effect on currents recorded in 1 nmol/L [Ca²⁺]_i (Figure 4A) or 0 [Ca²⁺]_o (see Figure 2 online), nor did it affect resting membrane potential in low [Ca²⁺]_o (<300 nmol/L; see Figure 2 online). These latter results suggest that BK_Ca channels do not contribute to the resting potential, as has been observed in many smooth muscle types.^{14 15} The averaged voltage-ramp data for I_Glib under the different Ca²⁺ conditions is shown in Figure 4C, where current has been normalized to cell size. Analysis reveals that the magnitude of I_Glib is negatively correlated to the free [Ca²⁺]_i (Figure 4D), whereas the magnitude of the paxilline-sensitive current (I_Pax) was enhanced by increasing [Ca²⁺]_i (Figure 4E). Maximal activation of I_Glib occurred between 1 and 10 nmol/L [Ca²⁺]_i and was reduced by 50% at 100 nmol/L and 90% at 300 nmol/L. Unlike I_Glib, there was no significant difference in the magnitude of I_Pax at +10 mV in any [Ca²⁺]_i tested, although at +40 mV, a substantial increase in I_Pax was observed (P<0.001) with 300 nmol/L Ca²⁺ compared with lower [Ca²⁺]_i (Figure 4E).

View larger version (26K): [in this window] [in a new window]   Figure 4. Figure 4. Representative voltage-ramp currents obtained from an RASMC dialyzed with 1 (A) or 300 (B) nmol/L [Ca2+]i, before (a) and during superfusion with glibenclamide (Glib; 10 µmol/L; b) and paxilline (Pax; 300 nmol/L; c). C, Mean voltage-ramps of IGlib from RASMCs dialyzed with different [Ca2+]i. D, Mean IGlib measured at +10 mV (from voltage-ramps) under different [Ca2+]i. *P<0.05, **P<0.01, and ***P<0.001 when compared with 1 nmol/L Ca2+; P<0.05 when compared with 100 nmol/L Ca2+. E, Mean paxilline-sensitive currents (IPax) measured at +10 and +40 mV from voltage-ramps in cells dialyzed with different [Ca2+]i. P<0.01 when compared with 100 nmol/L Ca2+; P<0.01 when +10 mV compared with +40 mV.

The mean resting potentials of RASMCs in 1 to 10 nmol/L Ca²⁺ were similar to 0 [Ca²⁺] and 10 mmol/L EGTA conditions (Tables 1 and 2), as were the effects of glibenclamide on membrane potential (Table 2 and Figure 3C). At 100 nmol/L [Ca²⁺]_i, cells still had a resting potential of -57 mV, although they became substantially more depolarized (-32 mV) at 300 nmol/L, where glibenclamide did not produce any significant depolarization (Table 2).Thus, whole-cell K_ATP current and resting potential are steeply dependent on [Ca²⁺]_i in a range that is close to resting [Ca²⁺]_i recorded in smooth muscle of between 100 and 200 nmol/L.¹⁶

View this table: [in this window] [in a new window]   Table 2. Effects of [Ca2+]i on Membrane Potential of RASMCs in the Presence and Absence of 10 µmol/L Glibenclamide

Effects of Ca²⁺-Dependent Protein Phosphatase-2B Inhibitors
Because lower Ca²⁺ concentrations seemed to enhance K_ATP channel activity, in a separate group of experiments, we assessed the possibility that a Ca²⁺-dependent enzyme may be involved in the modulation of channel activity. It has been shown previously that L-type Ca²⁺ channels can be inhibited by intracellular Ca²⁺ via activation of calcineurin, the Ca²⁺-dependent protein phosphatase, type 2B (PP-2B),¹⁷ an enzyme known to be present in RASMCs.¹⁸ These results suggest that a dephosphorylating event causes Ca²⁺ channel inactivation to occur. Applying the same principle to our study, we postulated that under conditions where the [Ca²⁺]_i is low, the activity of PP-2B would be concomitantly reduced, leading to enhanced activity of K_ATP channels through increased phosphorylation of the channel.

Under control recording conditions of 3 [ATP]_i, 1 [EGTA]_i, and 1.8 [Ca²⁺]_o, where there was usually little background current, dialysis of RASMCs with cyclosporin A (10 µmol/L), an inhibitor of PP-2B,¹⁹ caused an increase of an instantaneous background current (Figure 5A), which was similar to that observed in the low [Ca²⁺] conditions (Figure 2). This increase was associated with significant membrane hyperpolarization from -42.0±2.7 mV at 30 seconds after gaining cell access to -58.6±1.6 mV after 5 minutes (n=9; P<0.001). This effect could be reversibly inhibited by glibenclamide (10 µmol/L) (Figure 6A). Furthermore, cyclosporin A induced an 3-fold increase in I_Glib over control conditions (Figure 6B). To confirm the specificity of cyclosporin A on PP-2B, we also investigated the effects of 2 structurally unrelated PP-2B inhibitors, FK-506²⁰ and the highly selective inhibitor calcineurin autoinhibitory peptide (CAP).²¹ In 4 of 8 cells, dialysis with FK-506 (5 to 10 µmol/L) for at least 5 minutes was associated with increased background currents (Figure 5B) and a significant hyperpolarization of the resting membrane potential (-57.6±5.9 mV, n=4). Again, this hyperpolarization was inhibited by glibenclamide (Figure 6A), and the magnitude of I_Glib was large (Figure 6B). With FK-506, the experiments were performed in the presence of paxilline (250 nmol/L), because we observed significant activation of BK_Ca current with this agent. Dialysis with CAP (10 µmol/L), like cyclosporin A, resulted in every cell tested becoming significantly hyperpolarized with time. The resting potential of cells was -31.8± 2.5 mV immediately on gaining cell access and hyperpolarized to -62.3±1.3 mV after 10 minutes (n=18; P<0.001), remaining stable for several minutes thereafter. This is in contrast to control cells, where the resting potential remained close to -40 mV during the first 5 minutes of recording (see above) and declined subsequently in a number of cells. Not only did glibenclamide cause substantial depolarization (33 mV) with CAP (Figure 6A), but we tended to record the largest basal K_ATP currents in this condition (Figure 5C).

View larger version (38K): [in this window] [in a new window]   Figure 5. Figure 5. A, Whole-cell membrane currents evoked from an RASMC superfused with 1.8 mmol/L [Ca2+]o and dialyzed with 1 mmol/L EGTA, 3 mmol/L ATP, and cyclosporin A (10 µmol/L) (top). Representative voltage-ramp recordings obtained from the same cell (bottom) in the absence (control) and presence of glibenclamide (Glib) (10 µmol/L). Experiments were repeated with FK-506 (10 µmol/L) (B) or calcineurin autoinhibitory peptide (10 µmol/L) (C) using the same protocol as in panel A.

View larger version (33K): [in this window] [in a new window]   Figure 6. Figure 6. A, Mean membrane potentials measured from RASMCs dialyzed with cyclosporin A (Cyclo A), FK-506, or CAP in the presence and absence of glibenclamide (Glib) (10 µmol/L). B, Mean IGlib measured at +10 mV (from voltage ramps) from RASMCs under different recording conditions. **P<0.01; ***P<0.001.

	Discussion

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It is widely assumed that smooth muscle K_ATP channels are largely insensitive to Ca²⁺, and so most studies to date have been carried out in low Ca²⁺.¹ However, we now provide direct evidence that basal K_ATP current can be regulated by Ca²⁺ in the nanomolar range in isolated smooth muscle cells. In a normal Ca²⁺ and K⁺ gradient and in the presence of a physiological concentration of intracellular ATP (3 mmol/L), the K_ATP channel inhibitor glibenclamide had a small but significant effect on membrane potential and current, suggesting that K_ATP channels do indeed contribute to the resting potential in rat aorta as previously reported.^{6 7} However, increasing [EGTA]_i or decreasing [Ca²⁺]_o increased a glibenclamide-sensitive background current by 2- to 4-fold and caused a large membrane hyperpolarization, effects similar to those observed in cells dialyzed with 1 to 10 nmol/L free [Ca²⁺]_i. Large K_ATP currents and hyperpolarized resting potentials could also be obtained in control cells dialyzed with specific inhibitors of PP-2B (cyclosporin A, FK-506, and CAP). Thus, our results demonstrate for the first time that smooth muscle K_ATP channels can be inhibited by Ca²⁺ under basal conditions through an action that is likely to be mediated by PP-2B.

The background current was identified as being carried by K_ATP channels on the basis of the following criteria. First, it was inhibited by glibenclamide in the low micromolar range, and I_Glib reversed within 5 mV of the predicted E_K under our recording conditions. It was essentially unaffected by paxilline, suggesting that the background current was not carried by BK_Ca channels. Second, the background current was sensitive to changes in [ATP]_i and increased in magnitude when [ATP]_i was lowered from 3 to 0.1 mmol/L. Third, the size of the levcromakalim-sensitive background current was larger when ATP was reduced in the pipette. These are very similar pharmacological characteristics to K_ATP current recorded in isolated cells from many smooth muscle types.¹ However, at this stage the molecular identity of the K_ATP channel subtype modulated by Ca²⁺ in rat aorta is unclear. Whether the whole-cell current is carried by glibenclamide-sensitive, nucleotide diphosphate K⁺ channels^{22 23} or the more classical type of K_ATP channel^{1 23 24} remains to be determined.

To our knowledge, the few studies that have assessed the Ca²⁺ sensitivity of K_ATP channels in excised patches or lipid bilayers have either reported no effect^{25 26 27} over the [Ca²⁺] range of 10 to 1000 nmol/L or an increase in activity above this concentration.^{2 10 26} In cultured porcine coronary myocytes, the sensitivity of channels to [ATP]_i was reported to decrease when [Ca²⁺]_o was raised from 1 to 100 µmol/L.² Our experimental observations are clearly different from all previous studies, because we report regulation in the opposite direction. Our findings are analogous to the situation in cardiac and skeletal myocytes, where K_ATP channels have a high activity when excised into Ca²⁺-free solutions but can be inhibited when Ca²⁺ is applied to the internal surface of the channel.^{28 29} However, the Ca²⁺ concentrations required to inhibit the channel are much higher (>10 µmol/L) than we estimate for our internal solutions. Although it is clearly difficult to predict the subsarcolemmal [Ca²⁺], we assume it to be higher than cytosolic Ca²⁺ levels, particularly in cells recorded under control conditions (1.8 mmol/L Ca²⁺ and 1 mmol/L EGTA). On the basis of the comparison of I_Glib recorded with pipette solutions of known [Ca²⁺]_i, we estimate that the [Ca²⁺] in the vicinity of channel is between 100 and 300 nmol/L in control cells and between 1 and 10 nmol/L for the low Ca²⁺ conditions (10 mmol/L [EGTA]_i and 0 [Ca²⁺]_o). However, it remains to be determined whether similar Ca²⁺ modulation or sensitivity of K_ATP channels can be observed in isolated patches. This is likely to be extremely difficult to assess because of the rapid run down and low density of K_ATP channels in smooth muscle.¹ Moreover, channel regulation may well be different in excised patches because of the absence of the intracellular matrix and cytosolic regulatory proteins. Thus, our results suggest that the level of Ca²⁺ buffering should be considered when studying K_ATP channels. Indeed, the differences in the ATP sensitivity of smooth muscle K_ATP channels that is observed experimentally¹ may well be related to different starting levels of Ca²⁺.

Evidence suggests that the activity of vascular K_ATP channels is governed by the degree and the site of phosphorylation and dephosphorylation.^{1 14} Clearly, [Ca²⁺]_i level plays an important role in regulating the activity of many Ca²⁺-dependent or -activated proteins in the cells, such as Ca²⁺-dependent protein kinase C (PKC) isoforms, Ca²⁺/calmodulin-dependent protein kinase II, and PP-2B.^{16 19} In smooth muscle cells, K_ATP channels can be opened by vasodilators stimulating protein kinase A (PKA) and closed by vasoconstrictors stimulating PKC.^{1 14} However, the effects of protein phosphatases on the modulation of K_ATP channel activity in smooth muscle by PKA and PKC is far from clear. In gallbladder smooth muscle cells, PKA-mediated activation of K_ATP by calcitonin gene–related peptide was shown to persist after treatment with okadaic acid (an inhibitor of Ca²⁺-independent PP-1 and PP-2A), suggesting that PP-1 and PP-2A dephosphorylate a PKA phosphorylation site.³ Similarly, okadaic acid was shown to prevent cardiac K_ATP channel rundown³⁰ and preserve PKC-induced K_ATP activation.³¹ Because these effects could be reversed by purified PP-2A, these studies suggested that PP-2A is responsible for dephosphorylating a PKC phosphorylation site. So far, the specific contribution of PP-2B to smooth muscle K_ATP channel regulation has not been investigated, although PP-2B protein or activity has been described in many cell types,¹⁹ suggesting that this enzyme may have important functional roles.

In this study we demonstrated that PP-2B is likely to be involved in the modulation of K_ATP channels by Ca²⁺. Dialysis with the highly selective PP-2B inhibitor, CAP, which has an IC₅₀ of 5 µmol/L against PP-2B,²¹ activated a large glibenclamide-sensitive current and significantly hyperpolarized the resting potential in control cells. This synthetic peptide is derived from a unique sequence in the autoinhibitory domain of calcineurin and has been reported to have a high degree of specificity for this enzyme, having no significant inhibitory effect on PP-1 or PP-2A or Ca²⁺/calmodulin-dependent protein kinase II.³² The specificity of this agent was additionally tested using 2 other structurally unrelated PP-2B inhibitors, cyclosporin A and FK-506, which inhibit the enzyme by first forming a complex with either cyclophilin A or FKBP12, respectively.²⁰ Both these inhibitors had effects similar to CAP, increasing whole-cell K_ATP current. However, we found that FK-506, and to a much lesser extent cyclosporin A, increased BK_Ca current, consistent with the known ability of FK-506 to increase [Ca²⁺]_i in bladder or A7r5 cells either by inhibiting the sarcoplasmic reticulum Ca²⁺ATPase or increasing Ca²⁺ release from the ryanodine receptor.^{33 34}

The effects of the PP-2B inhibitors on the K_ATP current and membrane potential that we observed were mimicked by recording current in low [Ca²⁺]_i (1 to 10 nmol/L). At this concentration, the activity of this enzyme should be negligible.¹⁹ Half-maximal activation of PP-2B by Ca²⁺ in the presence of calmodulin occurs at 300 to 500 nmol/L,²⁰ which is consistent with our observation that marked inhibition of the K_ATP current occurred on increasing [Ca²⁺]_i from 100 to 300 nmol/L. However, the regulation of PP-2B is complex, and several accessory proteins and kinases are known to modulate its activity, including PKC,¹⁹ so that the Ca²⁺ sensitivity in the intact cell may vary considerably between different cell types, possibly giving rise to variable basal K_ATP channel activity. The mechanism by which PP-2B would regulate K_ATP remains undetermined. Presumably, PP-2B regulates the degree of phosphorylation of an activation site on either the channel or a regulatory binding protein required for K_ATP channel opening, possibly one targeted by PKA. The situation seems different in cardiac cells, where the broad-spectrum protein phosphatase inhibitor, fluoride, had no effect on the Ca²⁺-induced inhibition of K_ATP channels in excised patches.²⁸ Moreover, in renal tubule cells, neither Ca²⁺ nor PP-2B affected rundown of K_ATP channel activity, whereas PP-2A accelerated this process.³⁵ This may suggest either a different K_ATP channel type or different channel regulation in smooth muscle. Although no clear role for PP-2B in regulating K_ATP channel in other tissues has been reported, PP-2B does seem to mediate Ca²⁺-induced inhibition of L-type Ca²⁺ channels in umbilical vein smooth muscle.¹⁷

In conclusion, we have demonstrated in rat aortic smooth muscle cells that Ca²⁺ is an important regulator of basal K_ATP channel activity within the physiological [Ca²⁺]_i range and provide evidence that this is mediated via PP-2B. Whether such regulation is specific to vascular smooth muscle remains to be determined. Our data strongly suggest that such a pathway may contribute to the action of vasodilator and vasoconstrictor agents on K_ATP channels either through kinase or Ca²⁺ regulation of PP-2B or from the ability of these agents to alter [Ca²⁺]_i levels directly.

	Acknowledgments

 

This study was supported by the Medical Research Council (MRC), UK, and the Special Trustees of the Middlesex and University College Hospitals. L.H.C. is an MRC Senior Research Fellow in Basic Science.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received May 30, 2000; revision received October 16, 2000; accepted October 17, 2000.

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