INTRODUCTION

Mesangial cells, which are excitable and have contractile properties similar to smooth muscle, regulate the glomerular filtration rate by modulating the capillary surface area (1-3). Recent patch-clamp studies of human mesangial cells in culture have shown that the mechanism and ion-selective channels involved in maintaining contractile tone are similar or identical to those of vascular smooth muscle (4, 5).

Large calcium-activated potassium channels (BK_Ca),¹ characterized in mesangial cells (5, 6) and vascular smooth muscle (7), are not involved in setting resting potential but respond in a negative feedback manner to agonist-induced increases in contractile tone. Agonists such as angiotensin II elevate intracellular calcium and depolarize the membrane potential, producing an activation of BK_Ca. The hyperpolarizing membrane potential inhibits further entry of cell calcium by inactivating voltage-gated calcium channels. The gain in this feedback mechanism is increased by smooth muscle relaxants such as nitric oxide and atrial natriuretic peptide that, via cGMP-activated kinase, lower the voltage and calcium thresholds for activating BK_Ca (8, 9). However, activation of BK_Ca "on cell" by vasorelaxants or Bt₂cGMP is followed by an inactivation or run-down phase, in which BK_Ca returns to base line 20 s after peak activity.

Substrate regulation by phosphorylation is a dynamic balance between the forward kinase phosphorylation and the reverse dephosphorylation by a protein phosphatase. Several studies have now shown that vasorelaxants activate BK_Ca through guanylyl cyclase and cGMP-dependent protein kinase in both smooth muscle and mesangial cells (9-11); however, the role of protein phosphatase has been addressed only very recently (9, 12). Two such studies on tracheal smooth muscle and neurohypophyseal cells supported the notion that cGMP stimulated BK_Ca by activating protein phosphatase 2A (12, 13), which activated BK_Ca by dephosphorylation. In contrast, our laboratory previously showed that the mesangial BK_Ca was activated by cGMP-activated protein kinase in the presence of okadaic acid, an inhibitor of protein phosphatases 1 and 2A (9, 13), suggesting that these phosphatases inactivated, rather than activated, the mesangial BK_Ca.

The present studies were performed to elucidate the signaling pathways involved in the run-down phase following activation of BK_Ca by cGMP-dependent protein kinase. Using the cell-attached configuration and established phosphatase inhibitors, we specifically investigated the specific endogenous protein phosphatase involved in regulating BK_Ca. Using inside-out patches, we then determined the regulation of BK_Ca by protein phosphatases 1 and 2A.

EXPERIMENTAL PROCEDURES

Human mesangial cells were isolated originally by Abboud and co-workers (14) and cultured using standard techniques. Mesangial cells were plated in Waymouth culture medium, pH 7.4, supplemented with 15 mM HEPES buffer, 2.0 mM glutamine, 0.66 unit/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin (100 units/ml), streptomycin (100 µg/ml), and fetal calf serum (17% v/v). All experiments were performed using subpassages 6-10. During this generation span, mesangial cells maintained a constant phenotype and typical smooth muscle-like spindle shape (2).

Cells were subcultured onto individual 22 × 22 mm glass coverslips (Fisher) and maintained for 1-2 days at 37 °C and 5% CO₂ in a humidified tissue culture incubator (Queue Scimetrics Inc., Missouri City, TX). The subconfluent cells were placed into an RC-24 perfusion chamber (Warner Instrument Corp., Hamden, CT) and perfused with a physiological salt solution (135 mM NaCl, 5 mM KCl, and 1.0 mM CaCl₂, pH 7.4).

Mesangial cells were prepared for analysis of single BK_Ca channels using standard patch-clamp techniques previously described (5, 15). Current recordings were made after obtaining gigaohm seals with the patch electrode on the surface of the cell (cell attached) or after withdrawing the patch (excised, inside out). The unitary current (i), defined as zero for the closed state, was determined as the mean of the best fit Gaussian distribution of the amplitude histograms. Channels were considered in an open state when the current was >(n  1/2)i and <(n + 1/2)i, where n is the maximum number of current levels observed. The probability of a channel existing in an open state (P_o) is defined as the time spent in the open state divided by the total time of the recording. In all cases, V_p implies the holding potential relative to the pipette.

In all cell-attached experiments, the pipette solution contained 140 mM KCl plus 10 mM HEPES buffer, pH 7.4, and the bath solution contained 135 mM NaCl, 5 mM KCl, 1 mM CaCl₂, and 10 mM HEPES, pH 7.4. The free Ca²⁺ concentration of the bath, initially 1.0 mM, was adjusted to lower concentrations by buffering with EGTA as described previously (16). In excised patches, the bathing solution was replaced with 140 mM KCl, 1 µM CaCl₂, 10 mM HEPES, pH 7.4. Okadaic acid, cantharidic acid, and calyculin A, established inhibitors of PP1 and PP2A, were used to establish the role of endogenous protein phosphatase in regulation of BK_Ca. While monitoring BK_Ca channels in cell-attached patches, these inhibitors were added to the bathing solution in concentrations nonspecific for inhibition of PP1 and PP2A. The holding potential (V_p) was either 80 mV or 0 mV, which resulted in outward and inward currents, respectively. The effects of these inhibitors on the cGMP-dependent activation of BK_Ca was determined by adding inhibitor either in the continued presence of 10 µM dibutyryl cGMP (Bt₂cGMP) or at least 30 s before the addition of Bt₂cGMP. To determine if the endogenous protein phosphatase was either 1 or 2A, cells were incubated in either 5 nM okadaic acid (specific for PP2A) or 10 nM calyculin A for 10-30 min before obtaining a seal. Specific inhibition of BK_Ca by either exogenous PP2A or PP1 was determined in inside-out patches with 140 mM KCl in the bath and holding potentials of either 40 mV or 40 mV for outward or inward currents, respectively. BK_Ca were activated either by Bt₂cGMP plus MgATP at a holding potential of 40 mV or by Bt₂cGMP plus MgATP plus cGMP-activated protein kinase at a holding potential of 40 mV. PP2A (0.5 unit/ml) or PP1 (1 unit/ml) was added in the continued presence of Bt₂cGMP plus MgATP with or without cGMP-activated protein kinase. Groups were compared for statistical significance using the paired t test or the ANOVA plus the Student-Newman-Keuls test as appropriate. Cyclic GMP-activated kinase was purchased from Promega. All other chemicals used in this study were purchased from Sigma or Calbiochem.

RESULTS

Experiments were performed to determine if the run-down phase after adding Bt₂cGMP was the result of either PP1 or PP2A. Fig. 1A shows the effects of cantharidic acid (500 nM), a nonspecific inhibitor of PP1 and PP2A, on the rundown of Bt₂cGMP-activated BK_Ca in cell-attached patches in the absence of a phosphatase inhibitor (upper tracing) and with the simultaneous addition of cantharidic acid (lower tracing). The P_o of BK_Ca increased from <0.01 to 0.85 after 10 s with the addition of 10 µM Bt₂cGMP and then returned to base line in the next 90 s; after 2 min the P_o was <0.01. However, when Bt₂cGMP and cantharidic acid were added together, BK_Ca was activated to a P_o that was sustained at 0.78 after 2 min. Note that the channel amplitude diminished after addition of cantharidic acid. A decrease in the amplitude of BK_Ca is the result of a decrease in electrochemical potential due to the combination of a decrease in the intracellular potassium concentration and the hyperpolarization of the membrane potential as BK_Ca is activated in the cell membrane (8, 17).

Fig. 1B shows current tracings of BK_Ca in a cell-attached patch, illustrating the BK_Ca activity at a V_p of 80 and 0 mV with cantharidic acid and cantharidic acid plus Bt₂cGMP. In the control, BK_Ca were quiescent at 0 mV, and the P_o was <0.001 at 80 mV. Approximately 2 min after the addition of 500 nM cantharidic acid, the P_o increased to 0.084 at 80 mV and 0.005 at 0 mV. Note the difference in open probability between 80 and 0 mV, demonstrating the voltage dependence of BK_Ca. In the continued presence of cantharidic acid, Bt₂cGMP further increased BK_Ca after 2 min to a sustained P_o of 0.40 and 0.035 at 80 and 0 mV, respectively. Thus, cantharidic acid eliminated the rundown in the response of BK_Ca to Bt₂cGMP. Moreover, the effects of Bt₂cGMP and phosphatase inhibitor were potentiating and independent of the holding potential.

A summary of the separate and combined effects on BK_Ca of cantharidic acid and Bt₂cGMP are shown in Fig. 1C. Two minutes after the separate additions of Bt₂cGMP and 500 nM cantharidic acid, the open probability of BK_Ca was 0.011 ± 0.006 and 0.16 ± 0.05, respectively. Two minutes after the addition of Bt₂cGMP plus cantharidic acid, the P_o increased to a significantly higher value of 0.48 ± 0.10. This value was more than twice the value of the sum of the separate additions, suggesting a potentiated effect of Bt₂cGMP and cantharidic acid.

Fig. 2 shows the effects of 100 nM okadaic acid and 100 nM calyculin A on cGMP-activated BK_Ca in cell-attached patches. At these concentrations, the phosphatase inhibitors are nonspecific for PP2A and PP1 and are effective within 120 s. As shown in Fig. 2A (V_p = 0 mV), BK_Ca activated within 5 s from <0.001 to 0.10 in response to 10 µM Bt₂cGMP and then returned to base line after approximately 5 more seconds. In the continued presence of Bt₂cGMP, the addition of okadaic acid reactivated BK_Ca to a greater and sustained value of 0.83 after 120 s. Similar results were obtained with 100 nM calyculin A (Fig. 2B, V_p = 80 mV). The P_o of BK_Ca increased from <0.001 to 0.42 within 5 s after the addition of Bt₂cGMP and then returned to base line within the next 20 s. However, after the addition of Bt₂cGMP plus 100 nM calyculin A, the P_o increased and remained at 0.65. The increase in channel amplitude at 0 mV and the reduction in amplitude at 80 mV shows that the membrane potential is hyperpolarizing, as BK_Ca is activated by either okadaic acid or calyculin A. 

Low concentrations of okadaic acid (5 nM) and calyculin A (10 nM) were used to determine if the endogenous phosphatase was PP1 or PP2A. Although IC₅₀ values for phosphatase inhibition for okadaic acid and calyculin A are dependent on the substrate and purity of inhibitor, in general okadaic acid is at least 30-fold more potent for PP2A than PP1 (18, 19), and calyculin A is 30-fold more potent than okadaic acid for inhibiting PP1 (19, 20). Therefore, if PP1 were the endogenous phosphatase, it would be expected that BK_Ca would be activated by calyculin A more than okadaic acid. If PP2A were the endogenous phosphatase, it would be expected that BK_Ca would be activated more by okadaic acid than calyculin A.

In the experiments of Fig. 3, okadaic acid (5 nM) and calyculin A (10 nM) were added to the bathing medium 20-50 min before obtaining a cell-attached patch. As shown by the upper current tracing of Fig. 3A, in the presence of okadaic acid, BK_Ca was active (P_o = 0.063) after obtaining the seal at 80 mV. The addition of Bt₂cGMP increased the P_o further to a sustained value of 0.32. As shown in Fig. 3B, BK_Ca was relatively quiescent (P_o < 0.005) after the addition of calyculin A and was activated transiently to 0.227 on the addition of Bt₂cGMP. This rundown to base line approximately 20 s after peak activation was similar to control experiments (see Fig. 1A). These results are summarized in Fig. 3B. In control cells, the P_o of BK_Ca was increased by Bt₂cGMP to a value of 0.19 ± 0.04 after 5 s and ran down to base line after 120 s (n = 6). After incubation with okadaic acid, the basal P_o was 0.18 ± 0.07 and increased to 0.29 ± 0.05 and 0.25 ± 0.05 after 5 and 120 s, respectively (n = 4). After incubation with calyculin A (n = 4), the P_o increased from a basal of 0.002 ± 0.001 to 0.20 ± 0.05 after 5 s and returned near the base line value (0.008 ± 0.006) after 120 s. Using the ANOVA plus the Student-Newman-Keuls test, the effects of okadaic acid at basal and Bt₂cGMP (120 s) were significantly greater than the respective P_o values for control and calyculin A. These results indicate that BK_Ca are maintained quiescent in cell-attached patches by endogenous PP2A. Moreover, specific inhibition of PP2A (by 5 nM okadaic acid) prevents the rundown after activation of BK_Ca by Bt₂cGMP in cell-attached patches.

The inside-out patch configuration was used to determine the effects of exogenous PP2A and PP1 on BK_Ca. Fig. 4 shows the inactivation of cGMP-activated BK_Ca by PP2A. As shown in the continuous tracing of Fig. 4A, dibutyryl cyclic GMP plus MgATP activated BK_Ca from 0.029 to 0.332, and the subsequent addition of PP2A inactivated BK_Ca to 0.098. However, there was no effect of PP1 on cGMP-activated BK_Ca (not shown). These data are summarized in the bar graph of Fig. 4B. In each experiment, after activation of BK_Ca by Bt₂cGMP plus MgATP in either the absence or presence of cGMP-activated protein kinase, PP2A (n = 5) decreased the P_o from 0.57 ± 0.11 to 0.40 ± 0.14 (p < 0.025, paired t test). However, the P_o was 0.66 ± 0.11 and 0.70 ± 0.13 (n = 3; not significant) before and after the addition of PP1.

DISCUSSION

This study further defined the signal transduction pathways for regulating BK_Ca channels in a contractile cell. Three phosphatase inhibitors, in concentrations that inhibit both PP1 and PP2A, caused a larger and more sustained increase in open probability of BK_Ca in response to Bt₂cGMP, the second messenger mediator for relaxation by nitric oxide and atrial natriuretic peptide. Basal open probability of BK_Ca was increased by okadaic acid in concentrations specific for PP2A but not by calyculin A, a more potent inhibitor of PP1, indicating that endogenous PP2A but not PP1 was maintaining BK_Ca in a dephosphorylated quiescent state. It was shown that exogenous PP2A, but not PP1, applied to the cytosolic side of BK_Ca in inside-out patches can specifically inhibit the activation of BK_Ca by cGMP-dependent protein kinase.

In both smooth muscle and mesangial cells, BK_Ca are activated by cGMP-dependent protein kinase (7, 9). However, after activation by Bt₂cGMP in cell-attached patches, the open probability of the mesangial BK_Ca rapidly runs down to base-line levels. The present study shows that the run-down phase is due to the presence of PP2A, which would dephosphorylate BK_Ca. Phosphatase-induced channel rundown has been more commonly described for channels in excised patches. Kubokawa et al. (21) found that renal K_d(ATP) channels run down in excised patches due primarily to the presence of PP2A. However, phosphatase-induced rundown is not only found in excised patches; it was also shown that okadaic acid prevents rundown of Ba²⁺ current (whole cell) in dissociated helix neurons (22).

It is not understood why the effects of cGMP-dependent kinase are transient and ultimately overcome by a phosphatase that presumably dephosphorylates and inactivates BK_Ca despite the continued presence of Bt₂cGMP. However, several mechanisms could be involved in the temporary inhibition and then activation of a protein phosphatase to initiate the run-down phase. A similar type of biphasic activation was demonstrated for Ca²⁺/calmodulin-dependent protein kinase II, also a substrate for PP2A (23). An increase in intracellular Ca²⁺ in the rat brain is accompanied by a sequential autophosphorylated increase and then decrease in phosphorylation level of Ca²⁺/calmodulin-dependent protein kinase II. The decrease in phosphorylation was blocked by 1 nM okadaic acid. It was suggested by these authors that an increase in intracellular calcium autophosphorylated a serine/threonine protein kinase (described by Guo et al. (24)) that would temporarily phosphorylate and inhibit PP2A. In time, PP2A would autodephosphorylate and inactivate Ca²⁺/calmodulin-dependent protein kinase II. A similar mechanism may be involved whereby cGMP temporarily activates an inhibitor of PP2A. Although a recent study described inhibition of PP1 by cGMP-dependent protein kinase (25), a cGMP-activated inhibitor of PP2A has not been described.

Although several studies have described regulation of ion-selective channels by cAMP- and cGMP-dependent protein kinases, the reversal of channel phosphorylation by phosphoprotein phosphatases has been investigated only recently (26-31). The present study is one of a few that have now implicated PP2A as a physiological regulator of BK_Ca channels (12, 13, 32). However, for cGMP-activated protein kinase-activated BK_Ca, at least two previous studies using three different cell types (12, 13) have shown either that PP2A activates BK_Ca or cGMP-activated protein kinase does not activate BK_Ca in the presence of inhibitors of PP2A. These results contrast with our study which showed that BK_Ca was inactivated by PP2A and activated by either cGMP-activated protein kinase or inhibitors of PP2A.

Our disparate results may be explained by another study by Reinhart et al. (32) who have shown that BK_Ca from the brain expresses two types of channels in planar bilayers with respect to PP2A regulation. Type 1 channels are activated by cAMP-activated protein kinase and inactivated by PP2A. Type 2 BK_Ca channels are inactivated by cAMP-activated protein kinase and activated by PP2A. Although this was a protein kinase A and not a cGMP-activated protein kinase-activated mechanism, it is possible that there are two types of BK_Ca with respect to regulation of phosphorylation and dephosphorylation by cGMP-activated kinase. However, these type 2 BK_Ca channels have not been observed in mesangial cells.² When the properties of the PP2A-activated (type 2) channels in tracheal smooth muscle (12) are compared with the mesangial BK_Ca of this study, we find that the reported single channel conductance (in symmetrical 140 mM KCl) of the tracheal BK_Ca (257 ± 13 picosiemens) is somewhat larger than the mesangial BK_Ca (206 ± 18 picosiemens, see Ref. 5). However, voltage-gated activation response in 1 µM Ca²⁺ is similar (see Refs. 5 and 12). It remains to be determined if these are distinct isoforms of BK_Ca.

The dominant type of BK_Ca in smooth muscle could be determined by the contractile response to low concentrations of okadaic acid. Since PP1 dephosphorylates myosin light chain kinase (33), higher concentrations of okadaic acid would be expected to induce contractions. If type 1 channels predominate, it would be expected that okadaic acid in concentrations of 1-10 nM would inhibit smooth muscle contraction. That range of concentrations of okadaic acid would induce smooth muscle contraction if type 2 channels predominate. In support of type 1 channels, at least two studies have shown that low concentrations of okadaic acid relax vascular smooth muscle (34, 35). Although the kinase specific for activation of the mesangial BK_Ca appears identical to smooth muscle, the effects of okadaic acid on mesangial contraction have not been determined.

It would not be surprising if BK_Ca is regulated by different mechanisms in different cell types. Tseng-Crank et al. (36) found that HSLO, the human gene encoding the calcium-activated potassium channel, contains multiple splice variants in the brain. It was recently found that the mesangial BK_Ca contains at least two of these variants of HSLO (37). It is therefore feasible that the differential expression of these vari-ants would confer different sites for regulation by kinases and phosphatases.

In summary, we have demonstrated that the mesangial BK_Ca is not regulated by an all-or-none phosphorylation or dephosphorylation mechanism but rather by a dynamic enzymatic balance between cGMP-activated protein kinase and protein phosphatase 2A, which specifically activate and inactivate BK_Ca, respectively. It remains to be established, however, if there are multiple molecular variants of BK_Ca that are differentially modulated by kinase/phosphatase signal transduction mechanisms.