Regulation of Ca2+-activated K+ channels by multifunctional Ca2+/calmodulin-dependent protein kinase

Steven C. Sansom, Rong Ma, Pamela K. Carmines, and David A. Hall

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Activation of mesangial cells by ANG II provokes release of intracellular Ca2+ stores and subsequent Ca2+ influx through voltage-gated channels, events that are reflected by a large transient increase in intracellular concentration [Ca2+]i followed by a modest sustained elevation in [Ca2+]i. These ANG II-induced alterations in [Ca2+]i elicit activation of large Ca2+-activated K+ channels (BKCa) in a negative-feedback manner. The mechanism of this BKCa feedback response may involve the direct effect of intracellular Ca2+ on the channel and/or channel activation by regulatory enzymes. The present study utilized patch-clamp and fura 2 fluorescence techniques to assess the involvement of multifunctional calcium calmodulin kinase II (CAMKII) in the BKCa feedback response. In cell-attached patches, KN62 (specific inhibitor of CAMKII) either abolished or reduced to near zero the ANG II-induced BKCa feedback response. This phenomenon did not reflect direct effects of KN62 on the BKCa channel, because this agent alone did not significantly alter BKCa channel activity in inside-out patches. KN62 also failed to alter either the transient peak or sustained plateau phases of the [Ca2+]i response to ANG II. In inside-out patches (1 µM Ca2+ in bath), calmodulin plus ATP activated BKCa channels in the presence but not the absence of CAMKII. These observations are consistent with the postulate that CAMKII is involved in the BKCa feedback response of mesangial cells, acting to potentiate the influence of increased [Ca2+]i on the BKCa channel or a closely associated regulator of the channel. An additional effect of CAMKII to activate a voltage-gated Ca2+ channel cannot be ruled out by these experiments.

mesangial cells; angiotensin II; patch clamp; fura 2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

IN GLOMERULAR MESANGIAL CELLS (GMC) (28), brain (26) and vascular smooth muscle (4), large Ca2+-activated K+ channels (BKCa) are activated and repolarize the cell membrane as a feedback response to agonist-induced elevations in [Ca2+]i and membrane potential (4, 28). GMC have proven to be excellent models for studying the signaling pathways leading to the feedback activation of BKCa channels. These cells express abundant BKCa channels, the activity of which can be relatively easily monitored at the single channel level in the cell-attached mode. The contractile properties of GMC are identical to vascular smooth muscle for at least ten generations in culture.

The exact mechanism underlying the BKCa feedback response to contractile agonists remains unclear. Contractile agonists such as ANG II induce the release of intracellularly sequestered Ca2+, which leads to the activation of Ca2+-sensitive Cl- channels (15, 23), subsequent membrane depolarization and activation of voltage-operated Ca2+ channels (VOCC). Although the BKCa feedback response is regulated by VOCC-dependent Ca2+ influx in many vascular smooth muscle beds (6, 17, 21), agonist-induced elevations in GMC [Ca2+]i may not be large enough to directly influence BKCa channels, which require an [Ca2+]i greater than 500 nM for significant activation at membrane potentials greater than 0 mV.

As in other cell signaling systems, regulation of GMC BKCa channels by Ca2+ may involve Ca2+-activated enzymes such as protein kinase C or multifunctional calcium calmodulin kinase (CAMKII). CAMKII could have a particularly interesting role because it can be converted to a Ca2+-independent, autophosphorylated form after membrane depolarization and entry of Ca2+ into the cell (1, 2, 9). CAMKII has been linked recently to the phosphorylation and regulation of several plasmalemmal ion channels, including L-type Ca2+ channels in cardiac myocytes (32), Ca2+-activated Cl- channels in tracheal smooth muscle (31), nonselective cation channels in T84 cells (3), and K+ channels in a variety of cell types (16, 24, 27). In the present study, we tested the hypothesis that CAMKII, abundantly expressed in GMCs (14, 20), is an intermediary in the agonist-induced feedback activation of BKCa channels. In this scenario, CAMKII could amplify and/or convert spatially and temporally fluctuating Ca2+ signals to a controlled and reversible phosphorylation reaction, thus permitting BKCa channels to more efficiently govern the tone of contractile cells.


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mesangial cell cultures. Cultured human GMCs were subpassaged from generation five to ten in DMEM supplemented with 10 mM N-2-hydroxyethylpiperazine-n-2-ethanesulfonic acid (HEPES; pH=7.4), 2.0 mM glutamine, 0.66 U/ml insulin, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin and 20% fetal bovine serum. On reaching confluency, cells were studied within 56 h of passage onto 22 × 22 mm cover slips (Fisher; Pittsburgh, PA), which served as the floor of the perfusion chamber (Warner RC-2OH, 23°C) used in either patch-clamp or fura 2 experiments.

Patch-clamp procedure. Patch-clamp experiments were performed either with the pipette attached to the membrane (cell-attached) or excised from the cell (inside-out). For cell-attached patches, the physiological saline (PSS) bathing solution contained (in mM) 135 NaCl, 5 KCl, 10 HEPES, 2 MgCl2, and 1 CaCl2 (pH 7.4). For inside-out patches, the bath solution contained (in mM) 140 KCl, 10 HEPES, and 1 CaCl2 (pH 7.4). For all patch-clamp experiments, the pipette solution contained 140 mM KCl, 1 µM CaCl2, and 10 mM HEPES (pH 7.4). In some experiments, the free Ca2+ concentration of the bath was adjusted to 1.0 µM by buffering with 1.08 mM EGTA, according to the calcium concentration program by MTK Software.

Single channel analysis was performed at 23°C by using standard patch-clamp techniques (12, 28). The patch pipette, partially filled with solution, was in contact with a Ag-AgCl wire on a polycarbonate holder connected to the head stage of a patch clamp (501A, Warner Instrument, Hamden CT). The pipette was lowered onto the cell membrane and suction was applied to obtain a high resistance (> 5 GOmega ) seal. The unitary current (i), defined as zero for the closed state (C), was determined as the mean of the best-fit Gaussian distribution of the amplitude histograms. Channels were considered to exist in an open state (S) when the total current (I) was >(n - 1)I and <(n + 1)I, where n is the maximum number of current levels observed. The open probability (NPo) was defined as the time spent in an open state divided by the total time of the analyzed record. When multiple channels occupied a patch, channel activity was calculated as NPo = Sigma  nPn, where Pn is the probability of finding n channels open. The Axoscope and pCLAMP program set 6.02 (Axon Instruments; Foster City, CA) was used to record and analyze currents.

Measurement of [Ca2+]i. [Ca2+]i was monitored in GMC by using fura 2 and dual excitation wavelength fluorescence microscopy, as previously described (5, 10). In brief, cells were loaded with fura 2 by incubation for 70-90 min (20°C) in PSS (135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES) containing 7 µM fura 2 AM, 0.09 g/dl DMSO and 0.018 g/dl Pluronic F-127 (Molecular Probes, Eugene, OR). After loading with fura 2, GMC were placed in a perfusion chamber mounted on the stage of a Nikon Diaphot-300 inverted microscope. With light provided by a DeltaScan dual monochromator system (Photon Technology International, Monmouth Junction, NJ), the cells were illuminated alternately at excitation wavelengths of 340 and 380 nm (bandwidth = 3 nm). To limit detection of emitted fluorescence (510 nm; 20-nm bandpass) to that emitted from a single cell, an adjustable optical sampling window was positioned within the light path upstream from the photon-counting photomultiplier. Background-corrected data were collected at a rate of 5 points/s, stored, and analyzed by using the FeliX software package (Photon Technologies). Calibration of the fura 2 signal was performed according to established methods previously described (5, 10). In all fura 2 experiments, the initial bathing solution contained (in mM): 135 NaCl, 5 KCl, 10 HEPES, and 1 CaCl2.

Reagents and statistical analyses. ANG II, CAMKII, KN62, and KN93 were obtained from Calbiochem (San Diego, CA). Differences among groups of data were determined by using the one-way ANOVA plus Student-Newman-Keuls test. P values <0.05 were considered statistically significant. Data are reported as means ± SE, n = number of cells.


    RESULTS
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INTRODUCTION
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DISCUSSION
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Effects of CAMKII blockers on feedback response. BKCa channels were identified in patches by their characteristic large amplitudes and voltage dependence as previously described in GMC (28). The BKCa feedback response to ANG II involves an elevation in [Ca2+]i that activates BKCa channels, as evidenced by an increase in the open probability (NPo). Figure 1A illustrates a typical BKCa feedback response to 1 µM ANG II in a cell-attached patch. In this example, NPo increased within 10 s from a basal value of 0.01 to a peak value of 0.69, and subsequently declined back toward baseline after 2-3 min. Figure 1B illustrates the BKCa response to ANG II in the presence of 10 µM KN62, a CAMKII inhibitor that prevents the interaction between calmodulin and CAMKII (30). Because KN62 does not inhibit the active form of CAMKII, KN62 was always applied before adding ANG II. In this example, ANG II only increased NPo from the baseline value of 0.01 to a peak value of 0.05 (10 s after ANG II exposure), returning to baseline levels 50 s after ANG II exposure. Figure 1C summarizes the effects of KN62 on the peak BKCa response. Baseline NPo values did not differ significantly between control cells (untreated; 0.0018 ± 0.0004, n = 5) and KN62-treated cells (0.005 ± 0.006, n = 7); however, the peak NPo value achieved on ANG II exposure was diminished by ~90% in the presence of KN62 (Control, 0.27 ± 0.05; KN62, 0.036 ± 0.014; P < 0.05).


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Fig. 1.   Effects of the specific inhibitor of CAMKII (KN62) on the large Ca2+-activated K+ channels (BKCa) feedback response to ANG II. A: single BKCa currents (I) in a cell-attached patch showing the response to addition of 1 µM ANG II to the bathing solution. B: effect of 1 µM ANG II on BKCa currents during exposure to 10 µM KN62. C: bar graph summary of effects of 10 µM KN62 on the ANG II-evoked peak Po of BKCa channels. *P < 0.05 vs. Control.

Experiments addressing the possibility that KN62 acts by directly inhibiting BKCa channels were performed by using inside-out patches. In the typical tracings of Fig. 2A (holding potential = 40 mV), the NPo of BKCa channels was 0.52 before (upper tracing) and 0.53 after (lower tracing) the addition of 10 µM KN62 to the bathing solution. Figure 2B provides a summary of six inside-out experiments performed at potentials (-Vp) of either -40 or 40 mV. By using a paired t-test, NPo values before KN62 (0.09 ± 0.03 at -40 mV) did not differ significantly from those measured in the same patches during KN62 exposure (0.11 ± 0.02 at -40 mV).


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Fig. 2.   Effects of KN62 on the Po of BKCa channels in inside-out patches. The bath solution contained 140 mM KCl and 1 µM CaCl2. A: typical tracing showing BKCa currents at a holding potential of 40 mV before (top) and during (bottom) exposure to 10 µM KN62. Arrows indicate closed state. B: results of 6 individual experiments showing the effects of KN62 on the Po of BKCa channels in inside-out patches studied at holding potentials of either 40 mV () or -40 mV (open circle ).

The intracellular Ca2+ indicator, fura 2, was utilized to determine whether the effect of KN62 on the BKCa feedback response could be attributed to an alteration in the ANG II-evoked increase in [Ca2+]i. In Control (untreated) cells, 1 µM ANG II evokes changes in [Ca2+]i that are typified by Fig. 3A. In this example, ANG II exposure caused a rapid increase in [Ca2+]i from the baseline value of 55 nM to a peak value of 1100 nM, followed by a return toward baseline that achieved a plateau at 90 nM. We previously demonstrated that the transient peak response reflects release of intracellular Ca2+ stores whereas the sustained elevation in [Ca2+]i involves Ca2+ influx via L-type voltage-gated channels (11). In the example provided in Fig. 3B, 10 µM KN62 did not alter baseline [Ca2+]i (62 nM) or the response to ANG II (peak, 1200 nM; plateau 105 nM). As shown in the summary bar graph of Fig. 3C, ANG II provoked a rapid transient increase in [Ca2+]i to a level that averaged 1,300 ± 270 nM in Control cells (n = 7) and 1,118 ± 224 nM in the presence of KN62 (n = 7). During the subsequent sustained phase of the ANG II response, [Ca2+]i was maintained at levels averaging 21 ± 4 and 29 ± 9% above baseline in Control and KN62-treated cells, respectively (not significant). These results confirm that KN62 affects neither the release of Ca2+ from intracellular stores nor the entry of Ca2+ through voltage-gated Ca2+ channels of the plasmalemmal membrane.


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Fig. 3.   Effect of KN62 on the intracellular Ca2+ concentration ([Ca2+]i) response to ANG II. A: typical response of a Control (untreated) cell, to addition of 1 µM ANG II to the bathing solution. B: [Ca2+]i response to ANG II in the presence of 10 µM KN62. C: bar graph summary of the effects of KN62 on the peak and sustained responses to ANG II. triangle , Change.

Thus the CAMKII inhibitor KN62 blocked the ANG II-induced BKCa feedback response but did not exert direct effects on BKCa channels or on the [Ca2+]i response to ANG II. These observations suggest that CAMKII participates in the BKCa feedback response by amplifying the effects of [Ca2+]i to activate BKCa channels or an associated regulator of BKCa channels. To test this postulate, additional experiments were performed to determine whether the NPo of the BKCa channel could be directly influenced by the enzymatic activity of CAMKII. Figure 4 shows a representative current tracing of BKCa channel activity in an inside-out patch at a holding potential of -40 mV. The NPo was 0.008 under basal conditions (1 µM Ca2+ in the bath), and remained unchanged after addition of 10 µM calmodulin and 10 µM ATP to the bathing solution. Subsequent addition of 100 ng/ml CAMKII to the bathing solution increased the NPo to 0.19 in this example. This behavior was confirmed in 3 out of 3 patches, where the NPo of BKCa channels increased on addition of CAMKII and calmodulin plus ATP to the bathing solution. However, the magnitude of the effect of CAMKII was variable. In the other patches, NPo increased from 0.002 to 0.022 and from 0.008 to 0.170. Thus in the presence of Ca2+, calmodulin and ATP, CAMKII increased BKCa channel activity in inside-out patches.


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Fig. 4.   Effects of CAMKII on BKCa channels in an inside-out patch. The bathing solution contained 140 mM KCl, 10 mM HEPES, and 1 µM Ca2+. Tracings illustrate channel activity under Control conditions (top), after addition of 10 µM calmodulin and 10 µM ATP to the bathing solution (middle), and on subsequent addition of 100 ng/ml CAMKII to the bath.


    DISCUSSION
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INTRODUCTION
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DISCUSSION
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The results of this study showed that pharmacological blockers of the interaction between calmodulin and CAMKII inhibited the ANG II-induced BKCa feedback response. These data suggest that endogenous CAMKII is necessary to elicit the BKCa feedback response, apparently acting to potentiate the influence of agonist-induced [Ca2+]i responses on BKCa channel activity.

The BKCa feedback response is thought to involve an agonist-induced elevation in [Ca2+]i which activates BKCa channels. The resultant hyperpolarization of membrane potential reduces the influx of Ca2+ and attenuates the contractile response. In arterial smooth muscle cells, spatially localized Ca2+ sparks of 200-300 nM magnitude and 400 ms duration are responsible for activating spontaneous transient outward currents that likely reflect BKCa channel activity (21). It is not known whether the localized increase in [Ca2+]i induced by ANG II is also in this range in GMC; however, at Ca2+ concentrations less than 500 nM and at a membrane potential of 0 mV, BKCa channels in GMC are activated to an NPo of no greater than 0.05 (29). This value is in the range of the peak NPo response of BKCa channels to ANG II observed in the presence of KN62 in the present study (Fig. 1B). On the basis of this information, it is reasonable to postulate that BKCa channels are not activated directly by an agonist-induced rise in [Ca2+]i but, rather, are activated by an intermediary such as a Ca2+-sensitive kinase that amplifies the Ca2+ signal.

CAMKII inhibition failed to alter the [Ca2+]i response to ANG II and did not exert a direct effect on BKCa channel activity. However, inside-out patch experiments revealed the ability of CAMKII to directly activate BKCa channels (or a membrane-associated regulatory protein) in the presence of Ca2+, calmodulin, and ATP. Viewed en masse, these observations suggest that the site of CAMKII involvement in the BKCa feedback response lies between the elevated [Ca2+]i and the BKCa channel. However, the Ca2+ signal to other channels could also affect the BKCa response. For example, CAMKII mediates the inactivation of Ca2+-activated Cl- channels in tracheal smooth muscle (31). In these cells, as well as in mesangial cells, Cl- channels are implicated in the development of agonist-induced membrane depolarization. If CAMKII exerts its effects on mesangial cells via Cl- channel inactivation, KN62 treatment should allow prolonged or augmented membrane depolarization that, in turn, would increase Ca2+ influx through voltage-gated channels. This series of events would be evident as an increase in the sustained [Ca2+]i response to ANG II, a phenomenon that was not observed in our experiments. CAMKII has also been reported to increase current through cardiac L-type Ca2+ channels via phosphorylation of the channel or other events that occur in close proximity to the channel (32). If CAMKII exerts a similar effect on mesangial cells to promote Ca2+ influx through L-type channels, KN62 treatment should reduce the sustained [Ca2+]i response to ANG II. This phenomenon was not evident in our experiments. However, it remains possible that CAMKII participation in the mesangial cell response to ANG II involves opposing effects on the L-type Ca2+ channel and the BKCa channel. The rationale behind this suggestion lies in the prediction that the CAMKII-dependent activation of the BKCa channel (demonstrated in inside-out patch experiments) should limit the membrane depolarization and Ca2+ influx response evoked by ANG II. This scenario should be evident as an increased sustained [Ca2+]i response to ANG II in the presence of KN62, a phenomenon which was not observed in our studies. We propose that an effect of KN62 to inhibit L-type Ca2+ channels might counteract the influence of this compound on BKCa channels, resulting in no discernible alteration in the [Ca2+]i response to ANG II.

In the cell-attached patch, BKCa channels were activated by ANG II to a larger NPo (0.69) compared with the activation of these channels by CAMKII in the inside-out patch (to an NPo of 0.19). These results suggest that an additional intracellular intermediary is involved. It has been shown that BKCa channels are activated by cGMP-activated kinase (PKG) and inactivated by protein phosphatase 2A (28). It is possible that CAMKII affects the phosphorylation state of BKCa channels by either stimulating PKG or inhibiting a protein phosphatase. Whether or not CAMKII also activates an L-type Ca2+ channel and/or an intermediary activator of BKCa channels, the major role of CAMKII appears to be to amplify the Ca2+ influence on the BKCa channel after the cell has depolarized.

Activation of BKCa channels by CAMKII is not a novel concept. BKCa channels in pig cochlear outer hair cells are activated by CAMKII (19). In these cells, opening of K+ channels facilitates cell exit of K+ with a consequent cell shortening due to a loss of cell volume. Moreover, calmodulin is involved in the activation of Ca2+-activated K+ channels from a variety of cell types, including mouse fibroblasts (22), adipocytes (25), and the colonic cell line, HT29-19A (7). Deficits in the genetic sequence encoding calmodulin results in the mutant pantophobiac A (pntA), characterized in Paramecium tetraurelia (13) by a loss of the Ca2+-dependent K+ current. In snail neurons, activation of the Ca2+-activated K+ current by Ca2+/calmodulin involves channel phosphorylation, suggesting a Ca2+/calmodulin-dependent protein kinase (24).

In cerebellar granule cells (8), rat pancreatic acini (29) and nerve terminals (9), transient increases in [Ca2+]i can convert CAMKII to the calcium-independent, autophosphorylated form. In this configuration, CAMKII is directly sensitive to the frequency of [Ca2+]i oscillations (18) and is necessary for amplification and leveling of the oscillatory Ca2+ signal. Thus small, spatially oscillating Ca2+ concentrations near the membrane surface are capable of converting CAMKII to the autophosphorylated form. The results of the present study suggest that the role of CAMKII in the BKCa feedback response may be to sense the frequencies and amplitudes of the spatially and temporally irregular Ca2+ sparks that accompany activation by contractile agonists, ultimately acting to refine the signal into a uniform BKCa response.


    ACKNOWLEDGEMENTS

We are grateful to Hanna Abboud of the University of Texas Health Science Center at San Antonio for providing us with cultures of human mesangial cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49561 (to S. C. Sansom). R. Ma was supported by a fellowship grant from the American Heart Association (Heartland Affiliate).

Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Physiology and Biophysics, Univ. of Nebraska Medical Center, 984575 Nebraska Medical Center, Omaha, NE 68198-4575 (E-mail: ssansom{at}unmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 4 November 1999; accepted in final form 29 February 2000.


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RESULTS
DISCUSSION
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