Coupling strength between localized Ca2+ transients and K+ channels is regulated by protein kinase C

Orline Bayguinov, Brian Hagen, James L. Kenyon, and Kenton M. Sanders

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Localized Ca2+ transients resulting from inositol trisphosphate (IP3)-dependent Ca2+ release couple to spontaneous transient outward currents (STOCs) in murine colonic myocytes. Confocal microscopy and whole cell patch-clamp techniques were used to investigate coupling between localized Ca2+ transients and STOCs. Colonic myocytes were loaded with fluo 3. Reduction in external Ca2+ ([Ca2+]o) reduced localized Ca2+ transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca2+ transients and STOCs showed increased coupling strength between Ca2+ transients and STOCs when [Ca2+]o was reduced. Gd3+ (10 µM) did not affect Ca2+ transients but increased STOC amplitude and frequency. Similarly, an inhibitor of Ca2+ influx, 1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C (PKC) inhibitor, GF-109203X, also increased the amplitude and frequency of STOCs but had no effect on Ca2+ transients. Phorbol 12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequency but did not affect Ca2+ transients. 4alpha -Phorbol (1 µM) had no effect on STOCs or Ca2+ transients. Single channel studies indicated that large-conductance Ca2+-activated K+ (BK) channels were inhibited by a Ca2+-dependent PKC. In summary 1) Ca2+ release from IP3 receptor-operated stores activates Ca2+-activated K+ channels; 2) Ca2+ influx through nonselective cation channels facilitates activation of PKC; and 3) PKC reduces the Ca2+ sensitivity of BK channels, reducing the coupling strength between localized Ca2+ transients and BK channels.

calcium puffs; spontaneous transient outward current; gastrointestinal motility; inositol 1,4,5-trisphosphate; sarcoplasmic reticulum


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LOCALIZED CA2+ transients are important in regulating membrane potential, excitability, and responses to agonists in several smooth muscles (e.g., see Refs. 1, 2, 7, 9, 11, 30, 36, and 42). These events result from spontaneous opening of ryanodine-sensitive Ca2+ release channels (Ca2+ sparks) or inositol 1,4,5-trisphosphate (IP3) receptor-operated channels (Ca2+ puffs) in the sarcoplasmic reticulum. In some cells, sparks or puffs can become regenerative, establishing Ca2+ waves that can spread locally around the initiation site or through the entire cell (1, 5, 11, 16). Localized Ca2+ release, in close proximity to the plasma membrane, generates a transient and spatially restricted rise in Ca2+ concentration estimated to be in excess of 1 µM (32).

The rise in Ca2+ opens Ca2+-activated ion channels in the plasma membrane that are close to sites of Ca2+ release. Specific cells might respond with either net inward (usually resulting from Ca2+-activated Cl- currents; see Ref. 42) and/or net outward currents (usually resulting from Ca2+-activated K+ currents; see Refs. 1, 30, and 42). Activation of membrane conductances is transient in nature, and the resulting currents have been referred to as spontaneous transient inward or outward (STOCs) currents (3, 30, 42). Studies of intact smooth muscle tissues have also demonstrated localized Ca2+ transients, and inhibition of these events or the ionic conductances activated by the Ca2+ transients have demonstrated changes in membrane excitability, membrane potential, and the contractile state of the muscles (7, 18).

A few studies have examined factors regulating localized Ca2+ transients and the membrane currents that they activate. In particular, cAMP and cGMP increase (33) and protein kinase C (PKC) inhibits Ca2+ sparks (6) in vascular myocytes. Furthermore, ZhuGe et al. (41) measured the amount of Ca2+ released during a spark and correlated this to spontaneous transient outward current (STOC) amplitude in smooth muscle myocytes. Interestingly, from spark to spark, there was a highly variable relationship between the amount of released Ca2+ and STOC amplitude that the authors attributed to different numbers of large-conductance Ca2+-activated K+ (BK) channels at the various sparking sites. An alternative possibility is that there could be variations in the Ca2+ sensitivity of the BK channels activated by a Ca2+ transient, i.e., the strength of the coupling between Ca2+ release and activation of the STOC might be subject to regulation. This suggestion is consistent with observations that muscarinic activation reduces the Ca2+ sensitivity of BK channels in colonic myocytes (8) and hippocampal neurons (29).

A recent study showed that coupling strength between Ca2+ transients and ion channels is an important determinant of physiological responses. In animals where expression of the beta 1-subunit of the Ca2+-activated K+ channel was knocked out, Ca2+ sparks were normal, but these events were poorly coupled to STOCs (7). The change in coupling strength between sparks and STOCs was attributed to the reduced Ca2+ sensitivity of BK channels conveyed by loss of beta 1-subunits (27).

We have shown that localized Ca2+ transients in murine colonic myocytes are the result of phospholipase C (PLC)-dependent activation of Ca2+ release via IP3 receptor-operated stores (1, 2). Both inhibitory (ATP) and excitatory (ACh) agonists interact with this pathway to affect responses in murine colonic myocytes. In our initial study of this phenomenon, we performed simultaneous measurements of Ca2+ transients and STOCs in colonic myocytes and found, in a limited population of cells, a relatively linear correlation between these events. In performing more extensive studies, we noted 1) not all Ca2+ transients of a given amplitude coupled to STOCs of equal amplitude; some puffing sites generated larger or smaller STOCs than others; and 2) blocking Ca2+ entry through nonselective cation channels increased the amplitude and frequency of STOCs without increasing Ca2+ transients. In the present study, we have investigated Ca2+ entry-dependent regulation of coupling strength between Ca2+ puffs and STOCs and explored the hypothesis that a Ca2+-dependent PKC regulates the coupling strength in murine colonic myocytes.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation. BALB/C mice (60-90 days old) of either sex were anesthetized with CO2 and killed by cervical dislocation and decapitation. Colons were excised and opened along the mesenteric border, and the luminal contents were removed with Krebs-Ringer bicarbonate buffer (KRB; see Solutions and drugs). Tissues were pinned to the base of a Sylgard-coated dish, and the mucosa and submucosa were dissected away.

Strips of colonic muscle were cut and equilibrated in Ca2+-free solution for 60 min. Next, the tissues were digested at 37°C for 16 min without agitation in an enzyme solution containing collagenase F (Sigma Chemical; see Ref. 1). At the end of the digestion period, the tissues were washed four times with Ca2+-free Hanks' solution to remove the enzyme. Next, the partially digested tissues were triturated with blunt pipettes of decreasing tip diameter to free single smooth muscle cells.

Confocal microscopy. Suspensions of cells were placed in a specially designed 0.5-ml chamber with a glass bottom. The cells were incubated for 35 min at room temperature in Ca2+-free buffer containing fluo 3-AM (10 µg/ml; Molecular Probes, Eugene, OR) and pluronic acid (2.5 µg/ml; Teflabs, Austin, TX). Cell loading was followed by a 25-min incubation in a solution containing 2 mM Ca2+ to restore the normal concentration of extracellular Ca2+ and to allow the cells to adhere tightly to the bottom of the chambers during deesterification of fluo 3. All measurements were made within 45 min after restoring extracellular Ca2+.

An Odyssey XL confocal laser-scanning head (Noran Instruments, Middleton, WI) connected to a Nikon Diaphot 300 microscope with a ×60 water immersion lens (numeric aperture = 1.2) was used to image the cells. The cells were scanned using INTERVISION software (Noran Instruments) running on an Indy workstation (Silicon Graphics, Mountain View, CA). Changes in the fluo 3 fluorescence (indicating fluctuations in cytosolic Ca2+) were recorded for 20-s test periods using T-series acquisition and a laser wavelength of 488 nm (excitation for FITC). Six hundred frames were acquired per test period (1 frame every 33 ms), creating 20-s movie files.

Ionic currents of single cells. Ionic currents were measured in isolated muscle cells using the whole cell, perforated-patch (amphotericin B) configuration of the patch-clamp technique. An Axopatch 200B amplifier with a CV 203BU head stage (Axon Instruments, Foster City, CA) was used to measure ionic currents. Membrane currents were recorded using pClamp software (version 7.0; Axon Instruments) while holding cells at -30 or -40 mV (after correction for a -11-mV junction potential). Currents were digitized at 1 kHz. In some experiments, patch-clamped cells were simultaneously scanned for fluorescence changes in cells preloaded with fluo 3 as described above. All experiments were performed at room temperature (22-25°C).

Unitary ionic currents resulting from openings of single channels were measured in cell-attached patches. Cells were bathed in solutions with high K+ (140 mM) and either 1 or 0.01 mM free Ca2+. High-resistance seals (> 10 GOmega ) were obtained using borosilicate electrodes (7-12 MOmega ). Recordings were made using an Axopatch 200B amplifier with a CV 203BU head stage (Axon Instruments). The data were digitized at 2 kHz and filtered at 1 kHz using pClamp Software (version 6.0; Axon Instruments). Voltage-ramp protocols were used in which the patch potential was ramped from +100 to -50 mV over 3 s every 10 s. Ramps were repeated at least six times before and after test compound application, and current responses were averaged, creating current-voltage (I-V) relationships. The effects of test treatments on the activation of BK channel currents were assessed by measuring the amplitude of the current at +80 mV, a potential where BK channel activity was observed under all conditions. All single channel recordings were conducted with 4-aminopyridine (5 mM) and nicardipine (1 µM) in the pipette and bath solutions to decrease contributions from the delayed-rectifier K+ and voltage-dependent Ca2+ channels expressed by these cells. Experiments were also performed with charybdotoxin (ChTX; 200 nM) in the pipette solution. Under this condition, I-V curves were linear and small in amplitude, suggesting that most of the current response to depolarization was the result of openings of BK channels during typical recording conditions.

Solutions and drugs. The standard KRB used in this study contained (in mM): 120 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 NaH2PO4, and 11.5 dextrose. This solution had a final pH of 7.3-7.4 after equilibration with 97% O2-3% CO2. The bathing solution used in confocal microscopy studies and whole cell patch-clamp studies contained (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4). The enzyme solution used to disperse cells contained 1.3 mg/ml collagenase F, 2 mg/ml papain, 1 mg/ml BSA, 0.154 mg/ml L-dithiothreitol, 134 mM NaCl, 6 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). The pipette solution used in whole cell patch-clamp experiments contained 110 mM potassium aspartate, 30 mM KCl, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 0.05 mM EGTA (pH 7.2), and 250 µg/ml amphotericin B. Bath and pipette solution with free Ca2+ equal to 1 mM used in the cell-attached single channel contained (in mM): 140 KCl, 1 MgCl2, 10 glucose, 1 CaCl2, and 10 HEPES. Solution with free Ca2+ at 0.01 mM was calculated using the Max Chelator, provided by Dr. Chris Patton (www.stanford.edu/~cpatton/maxc.html), to contain (in mM): 140 KCl, 1 MgCl2, 10 glucose, 0.755 CaCl2, 1 N-hydroxyethyl-ethylenediamine-triacetic acid, and 10 HEPES.

4-Aminopyridine, 4alpha -phorbol ester, ChTX, gadolinium (III) chloride, and nicardipine were obtained from Sigma. 1-2-(4- ethoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365 hydrochloride), GF-109203X, and phorbol 12-myristate 13-acetate (PMA) were obtained from Tocris Cookson. Concentrations of drugs used were determined from the literature or by empirical determinations of effective concentrations on murine colonic myocytes.

Analysis of data. Image analysis was performed using custom analysis programs using Interactive Data Language software (Research Systems, Boulder, CO), as previously described (1). Baseline fluorescence (F0) was determined by averaging 10 images (out of 600) with no activity. F0 from control experiments was used to determine the ratio of the records after drug treatments. Ratio images were then constructed and replayed for careful examination to detect active areas where sudden increases in the ratio of fluorescence (F) to F0 occurred. F/F0 vs. time traces were further analyzed in Microcal Origin (Microcal Software, Northampton, MA) and AcqKnowledge Software (Biopac Systems, Santa Barbara, CA) and represent the averaged F/F0 from a box region of 2.2 × 2.2 µm centered in the active area of interest to achieve the fastest and sharpest changes. The goal of the present work was to study the relationship between the size of spontaneous Ca2+ release events and the amplitudes of the STOCs that resulted. Because Ca2+ release events in colonic myocytes consist of single Ca2+ puffs, clusters of puffs, and Ca2+ waves (see Ref. 1), they are more variable with regard to kinetics and volume of distribution than are Ca2+ sparks observed in some smooth muscles (18, 30, 41). Accordingly, quantitative methods developed to measure Ca2+ sparks are not appropriate for our experiments, and we integrated the area under the F/F0 recordings of the spontaneous Ca2+ events as described previously (1). This measure was chosen because it incorporated both the amplitudes and durations of Ca2+ events, two properties likely to affect the activation of BK channels.

Statistical analysis. Results are expressed as means ± SE where applicable. Statistical analysis was made with SigmaStat 2.03 software (Jandel Scientific Software, San Rafael, CA). ANOVA on ranks test was used to compare average I-V relationships before and after the application of test compound. STOC amplitudes were measured using the Mini Analysis Program (Synaptosoft, Leonia, NJ) with a threshold for detection set at 15 pA. The distributions of STOC amplitude were strongly skewed, resembling those of single-channel dwell times or survival curves. Accordingly, we have shown changes in STOC amplitudes in control and test conditions as cumulative distributions where the y-axis is the fraction of STOCs of amplitude greater than the picoampere value on the x-axis (cf. Ref. 26). For statistical analysis of STOC amplitudes, we calculated the mean STOC amplitude observed in each cell in control and test conditions and compared the means from all cells tested in a paired t-test and with a log rank test, a powerful method for determining if one group has a tendency toward larger values than another group (26). Both tests gave similar results, and we report P values from the log rank tests, with n being the number of cells.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

As we previously reported (1), colonic myocytes loaded with fluo 3 generated spontaneous, transient elevations in intracellular Ca2+ concentration ([Ca2+]i) that occurred either as highly localized events (Ca2+ puffs) or more widely spreading Ca2+ waves. Imaging of cells under whole cell voltage-clamp conditions demonstrated that localized Ca2+ transients were associated with STOCs. In most cells, Ca2+ puffs originated from multiple sites. In examining the relationship between Ca2+ puffs and STOCs, we found that Ca2+ puffs of a given magnitude originating at different sites triggered STOCs of different amplitude within a given cell. An example of this activity is shown in Fig. 1, with selected puffs and the STOCs they activated marked at different locations. This observation might be explained by differing numbers of BK channels (that are responsible for the STOCs) in proximity to different puffing sites or by regulation of the coupling strength between Ca2+ puffs and STOCs. Additional experiments were performed to test the latter hypothesis.


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Fig. 1.   Relationship between spontaneous Ca2+ transients and spontaneous transient outward currents (STOCs). Cell shown in panels 1-3 displayed spontaneous Ca2+ transients at 3 discrete sites. The 3 images were selected from 600 images taken during a 20-s scan of the cell shown and depict Ca2+ transients at the 3 sites. Fluorescence records from sites 1-3 are shown in the 3 traces. Simultaneous recordings of whole cell currents were made from the same cell (bottom). Multiple Ca2+ transients were recorded from each site. Comparison of the amplitude of the Ca2+ transients shows that transients of similar amplitude at different sites did not yield STOCs of the same amplitude. Relationship between STOC amplitude vs. the ratio of fluorescence to baseline fluorescence (F/F0) is plotted for the 3 sites on bottom (color of trace corresponds to color of symbols).

To investigate the relationship between the amplitude of the Ca2+ puffs and STOCs, we examined the effect of reducing external Ca2+ concentration ([Ca2+]o) on the amplitudes of puffs and STOCs. Fluorescence measurements of Ca2+ found that lowering [Ca2+]o from 2 to 1 mM reduced the area of Ca2+ transients to 60.17 ± 6.8% of control (n = 8, P < 0.001). In contrast, as shown in Fig. 2, reducing [Ca2+]o significantly increased the amplitude and frequency of STOCs. In particular, STOC amplitude increased from 20.7 ± 2.2 to 33.2 ± 2.9 pA (n = 8, P < 0.01), and STOC frequency increased from 55.7 ± 12.2 to 96.3 ± 15.8/min (n = 8, P < 0.01). Similar changes were observed in simultaneous recordings of intracellular Ca2+ and membrane currents (Fig. 3). In these experiments, reduction of [Ca2+]o increased STOC amplitude from 25.5 ± 2.5 to 44.6 ± 1.9 pA (n = 5, P < 0.001), whereas Ca2+ puff amplitudes decreased from 2.2 ± 0.2 to 1.8 ± 0.04 pA (n = 5, P < 0.05). The increase in coupling between Ca2+ transients and STOCs was also manifest in the observation that Ca2+ transients at some sites failed to elicit STOCs when cells were bathed in 2 mM Ca2+ but did elicit STOCs after a reduction in [Ca2+]o (Fig. 4). This observation may explain the increase in STOC frequency described above.


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Fig. 2.   STOC amplitude increased when external Ca2+ was reduced. A: extended recording of STOCs from a cell held at -40 mV. During continuous recording, the external Ca2+ was reduced from 2.0 to 1.0 mM. This caused a significant increase in the amplitude and frequency of STOCs (B). C: graphic representation of the change in STOC amplitude before and after reduction in Ca2+. Graph plots fraction of STOCs > X in 2 mM Ca2+ (control) and in 1 mM Ca2+ vs. STOC amplitude. The continuous lines under each condition result from connecting data points from thousands of individual STOCs recorded during 8 experiments. The data show a significant increase in STOC amplitude after reduction of Ca2+ (P < 0.01).



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Fig. 3.   Simultaneous recordings of Ca2+ transients and STOCs before and after reduction in Ca2+. A: fluorescence traces (A, a-d) from 4 sites during 20-s scans. Ae: STOCs recorded simultaneously during the scans. B: fluorescence transients from the same sites as in A and STOCs during a 20-s scan after reduction of external Ca2+ to 1 mM. C and D: Ca2+ transients of the same amplitude before (black trace on left) and after (red trace on left) reduction in external Ca2+ evoked STOCs of different amplitudes (black and red traces on right). Expanded traces correspond to Ca2+ transients and STOCs marked with triangles (2 mM Ca2+) and circles (1 mM Ca2+ in panels A, c-e and B, c-e, respectively).



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Fig. 4.   Coupling between Ca2+ transients and STOCs increases after reduction in external Ca2+. A: example of a site producing regular Ca2+ transients (trace on top) but only a single transient (solid arrow) coupled to an STOC (trace on bottom). Open arrows denote Ca2+ transients that were not productive in eliciting STOCs. B: fluorescence trace (top) from the same site after reduction of external Ca2+ to 1 mM. Note that majority of Ca2+ transients (although slightly reduced in amplitude) coupled to STOCs (solid arrows), and only two events did not produce resolvable STOCs (open arrows). Increasing the efficacy of Ca2+ transients to generate STOCs explains the increase in STOC frequency when external Ca2+ was reduced.

Role of Ca2+ entry pathways in regulating coupling between Ca2+ puffs and STOCs. It is possible that the effects of changing [Ca2+]o on coupling between Ca2+ puffs and STOCs could be linked to specific Ca2+ entry pathways. As we found previously, nicardipine (1 µM), an effective blocker of L-type Ca2+ current in murine myocytes (1), did not significantly affect localized Ca2+ transients (see Ref. 1) but reduced STOCs (data not shown). Thus blocking Ca2+ entry via this pathway reduced coupling between Ca2+ puffs and STOCs.

In addition to L-type Ca2+ channels, Ca2+-permeant nonselective cation channels might influence coupling between Ca2+ transients and STOCs. Gd3+ (10 µM), a blocker of nonselective cation channels, had no effect on Ca+ transients, as we previously reported (2), but significantly increased the frequency (from 21.9 ± 7.8 to 65.1 ± 14.7/min, n = 8, P < 0.01) and amplitude (from 22.9 ± 1.03 to 38.7 ± 4.7 pA, n = 5, P < 0.005) of STOCs (Fig. 5, A-C). Simultaneous recordings of Ca2+ puffs and STOCs also demonstrated an increase in efficacy between Ca2+ transients and STOCs, similar to the effects of reducing [Ca2+]o (Fig. 5, D and E). In addition, 1 µM SKF-96365, an inhibitor of receptor-mediated Ca2+ influx channels previously shown to have no effect on spontaneous Ca2+ transients in murine myocytes (2), also caused a dramatic increase in STOC amplitude (from 29.1 ± 8.3 to 68.5 ± 8.6 pA, n = 5, P < 0.01) and frequency (from 48.3 ± 15.2 to 104 ± 25.4/min; Fig. 6, A-C). Simultaneous recordings of Ca2+ puffs and STOCs demonstrated an increase in the coupling between these events (Fig. 6, D and E).


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Fig. 5.   Effects of Gd3+ on STOC amplitude. A: control STOCs recorded in a cell held at -40 mV for an extended period of time. B: excerpt of a continuous recording from the same cell in A after addition of Gd3+ (10 µM). Note the increase in the amplitude of STOCs. C: graphic representation of the change in STOC amplitude before and after Gd3+ (P < 0.01). Graph plots fraction of STOCs > X in control conditions and after addition of Gd3+ (10 µM) vs. STOC amplitude. The data show a significant increase in STOC amplitude after Gd3+. D and E: simultaneous recordings of Ca2+ transients and STOCs before (D) and after (E) Gd3+ (10 µM). Ca2+ transients were not greatly affected by Gd3+, but the coupling between Ca2+ transients and STOCs was increased by Gd3+.



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Fig. 6.   Effects of SKF-9635 on STOCs and Ca2+ transients. A: STOCs recorded in a cell held at -40 mV. B: excerpt from a continuous recording from the same cell after addition of SKF-96365 (1 µM). Note the increase in the amplitude of STOCs. C: graphic representation of the change in STOC amplitude before and after SKF-96365 (P < 0.01). Graph plots fraction of STOCs > X in control conditions and after addition of SKF-96365 vs. STOC amplitude. The data show a significant increase in STOC amplitude after SKF-96365. D and E: simultaneous recordings of Ca2+ transients and STOCs before (D) and after (E) SKF-9635. Note that Ca2+ transients were not greatly affected, but the coupling between Ca2+ transients and STOCs was greatly increased by SKF-9635.

Second messenger pathway mediating the increase in coupling strength. It seemed paradoxical that reducing the gradient for Ca2+ entry or inhibiting Ca2+ influx via nonselective cation conductances would increase the amplitude of currents resulting from Ca2+-activated K+ conductances. These observations suggested that local Ca2+ entry might activate Ca2+-dependent enzymes that reduce the Ca2+ sensitivity of BK channels. A candidate enzyme expressed in gastrointestinal muscles is PKC (25, 37). Therefore, we tested an inhibitor of PKC, GF-109203X (1 µM; see Ref. 15). This compound had no effect on localized Ca2+ transients (i.e., 77.4 ± 4.7% of control area, n = 6, P = 0.331). In contrast, treatment of cells with GF-109203X mimicked the effects of reduced [Ca2+]o and the addition of Gd3+ or SKF-96365 and greatly increased the amplitude (from 26.9 ± 3.3 to 52.5 ± 8.4 pA, n = 8, P < 0.005) and frequency (from 26.4 ± 8.2 to 86.7 ± 23.3/min, n = 8, P < 0.01) of STOCs (Fig. 7, A, B, and D). In four experiments, cells treated with GF-109203X were subsequently exposed to reduced Ca2+ (1.0 mM). This caused no further enhancement in the amplitude (P = 0.8383) and frequency (P = 0.918) of STOCs (Fig. 7C).


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Fig. 7.   Effects of GF-109203X on STOCs and enhancement in STOCs caused by reduced extracellular Ca2+. A: whole cell currents recorded from a cell held at -40 mV. B: STOCs recorded from the same cell after treatment with GF-109203X (1 µM). Note the significant increase in amplitude and frequency of STOCS. C: after GF-109203X, the external Ca2+ was reduced to 1.0 mM, and STOC activity was recorded from the same cell. STOCs were not increased by reduced Ca2+ in the continued presence of GF-109203X. D: graphic representation of the change in STOC amplitude before and after GF-109203X. Graph plots fraction of STOCs > X in control conditions and after addition of GF-109203X vs. STOC amplitude. The data show a significant increase in STOC amplitude after GF-109203X (P < 0.01).

An opposite effect from that of GF-109203X was observed with the PKC activator PMA. PMA (1 µM) strongly reduced STOC amplitude (from 39.3 ± 7.7 to 17.8 ± 1.8 pA, n = 5, P < 0.01) and frequency (from 65.8 ± 12.9 to 21 ± 9.1/min, n = 5, P < 0.01; Fig. 8, A and C) but had no effect on Ca2+ transients (i.e., 106.3 ± 14.6% of control area, n = 6, P = 0.59). An inactive derivative of PMA, 4alpha -phorbol (1 µM), did not affect either STOC amplitude (from 41.2 ± 6.5 to 40.9 ± 6.4 pA, n = 5, = 0.2004) or frequency (n = 5, P = 0.308; Fig. 8, B and D) or Ca2+ transients (i.e., 87.14 ± 6.1% of control area, n = 8, P = 0.157).


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Fig. 8.   Effects of active and inactive forms of phorbol esters on STOCs. A: typical STOC activity from a cell held at -40 mV. Second trace in A shows part of a continuous recording from the same cell after exposure to phorbol 12-myristate 13-acetate (PMA; 1 µM). A drastic reduction in STOCs was noted. B: typical STOC activity from a different cell held at -40 mV. Second trace in B shows continued recording from the same cell after exposure to 4alpha -phorbol (1 µM). No significant reduction in STOCs was noted under these conditions. C and D: significant decrease in STOC amplitude after PMA (P < 0.01) but no significant effect after addition of 4alpha -phorbol (P > 0.5).

Which component of STOCs is regulated by PKC? We showed previously that the STOCs elicited by Ca2+ puffs in murine colonic myocytes consisted of currents via BK and small-conductance Ca2+-activated K+ (SK) channels (1). In the present study, we found that ChTX (200 nM; treatment for 15 min) reduced but did not abolish STOC amplitude (from 41.3 ± 0.5 to 24.1 ± 1.8 pA, P < 0.001, n = 5). In the presence of ChTX, reduced [Ca2+]o had no effect on STOCs (P = 0.861, n = 5), suggesting that primarily ChTX-sensitive channels (i.e., BK channels) are affected by the Ca2+-dependent mechanism described above (Fig. 9A). This was further tested by demonstrating that ChTX (200 nM) reduced STOCs to control levels after pretreatment with Gd3+ (from 96.6 ± 2.6 to 28.2 ± 1.6 pA, P < 0.001 n = 5) or GF-109203X (from 43.3 ± 2.1 to 24.3 ± 1.5 pA, P < 0.005, n = 5). Examples of these experiments are shown in Fig. 9, B and C.


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Fig. 9.   Effects of reduced Ca2+ or added Gd3+ or GF-109203X on STOCs were the result of effects on large-conductance Ca2+-activated K+ (BK) channels. A: first trace shows control STOCs (holding potential -40 mV). Second trace shows STOCs after exposure to charybdotoxin (ChTX; 200 nM) for 15 min. After ChTX, reduced Ca2+ (10-min exposure) had no effect on remaining STOCs (3rd trace). B: first trace shows control STOCs (holding potential -40 mV). Second trace shows STOCs after exposure to Gd3+ (200 nM) for 10 min. Next, ChTX was added (15-min exposure), and this reversed the increase in STOCs caused by Gd3+ (3rd trace). C: first trace shows control STOCs (holding potential -40 mV). Second trace shows STOCs after exposure to GF-109203X (1 µM) for 10 min. Next, ChTX was added (15-min exposure), and this reversed the increase in STOCs caused by GF-109203X (3rd trace).

Changes in BK channel activity as a result of regulation by PKC. Our experiments suggest that negative modulation of BK channels via a Ca2+-dependent PKC may provide an important means of regulating coupling strength between Ca2+ puffs and STOCs in murine colonic myocytes. To further test this idea, on-cell recordings of BK channel activity were performed. In the first experiments, cells were bathed with low [Ca2+]o (0.01 mM) to reduce the gradient for Ca2+ entry and activity of the PKC that appears to be activated by Ca2+ influx. On-cell patches were ramped from -50 to +100 mV, and current responses were recorded. Figure 10A shows a typical current response averaged from six ramps. Addition of GF-109203X (1 µM) had no effect on the voltage dependence of the activation of the BK channels, as measured by the amplitude of the current at +80 mV (n = 6, P > 0.5). However, raising Ca2+ to 1.0 mM, which is expected to increase Ca2+ influx, shifted the activation of BK channels to more positive potentials, reducing the outward current compared with that observed in 0.01 mM Ca2+ (Fig. 10B, n = 6, P < 0.005). In 1 mM Ca2+, addition of GF-109203X (1 µM) shifted the I-V curve to the left, significantly increasing the amplitude of the current at +80 mV (n = 6, P < 0.01; Fig. 10B). These data suggest that the open probability of BK channels is reduced by PKC.


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Fig. 10.   Effects of various conditions shown to regulate coupling between Ca2+ transients and STOCs on unitary currents because of BK channels. A: current (I)-voltage (V) curves obtained by ramping membrane patch from -50 to 100 mV 6 times and averaging current responses. External and patch solutions contained 0.01 mM Ca2+ (control responses in black and test responses in red in A-D). Addition of GF-109203X to the bath solution caused no change in the I-V relationship. B: similar experiment in which external and pipette solutions contained 1 mM Ca2+. Under these conditions, I-V curve was shifted to the right (vs. control I-V in A). Addition of GF-109203X in B caused a rightward shift in the I-V relationship. C: effects of PMA (1 µM) on I-V relationship. Experiment performed with 0.01 mM Ca2+ in external and pipette solutions. Addition of PMA caused a rightward shift in the I-V relationship. D: in similar experiments, 4alpha -phorbol caused no significant change in the I-V relationship. E and F: control experiments in which tests of PMA (E) and GF-109203X (F) were repeated on additional cells with ChTX (200 nM) in the pipette solution to block BK channels. Black I-V traces are responses to single ramps during the 1st min after obtaining giga seals. Note openings of channels at positive ramp potentials. The red traces show control responses after ChTX blocked BK channels. Green traces show that PMA and GF-109203X had no effect on I-V relationships after BK channels were blocked.

We also tested the effects of PMA and its inactive form (4alpha -phorbol). On-cell patches were ramped from -50 to +100 mV, and current responses were recorded with 0.01 mM Ca2+ in the bath solution. Figure 10C shows a current response averaged from six ramps. Addition of PMA (1 µM) caused a significant rightward shift in the current response (n = 6, P < 0.05). In contrast, in similar experiments, we found that 4alpha -phorbol (1 µM) had no effect on the I-V relationship (n = 6, P > 0.05). Additional control experiments were performed in which ChTX (200 nM) was added to the pipette solution. This resulted in block of most of the channels activated by the ramp protocols (Fig. 10, D and E). In the presence of ChTX, PMA (1 µM) and GF-109203X (1 µM) had no effect on current responses to the voltage ramps (e.g., ChTX vs. PMA: P > 0.5, n = 5 and ChTX vs. GF-109203X: P > 0.05, n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

It is well established that localized Ca2+ transients resulting from release of Ca2+ from internal stores are important regulators of membrane potential, excitability, and responses to agonists in smooth muscles (e.g., reviewed in Ref. 17, see also Refs. 1, 2, and 12). Localized Ca2+ transients may influence several Ca2+-dependent effectors in the vicinity of Ca2+ release sites. A major means of coupling localized Ca2+ transients to cellular responses is activation of ionic conductance in the plasma membrane. In principle, this coupling is subject to two mechanisms of regulation. First, the size and frequency of Ca2+ transients can be altered (6, 33, 41). Second, as we show here, the Ca2+ sensitivity of the membrane ionic conductances can be regulated. A recent study dramatically demonstrated how altering the Ca2+ sensitivity of membrane conductances can affect coupling strength in animals in which the beta 1-subunit of BK channels was knocked out (7). beta 1-Subunits greatly increase the Ca2+ sensitivity of BK channels (27), and the mutant animals showed diminished STOCs in response to Ca2+ sparks that were the same as in wild-type cells. Our observations in the present study suggest that the Ca2+ sensitivity of BK channels is regulated dynamically by a Ca2+-dependent isoform of PKC and that Ca2+ entry in myocytes via nonselective cation channels participates in the ongoing activity of a PKC close to BK channels. This pathway may provide regulation of the coupling strength between Ca2+ transients and Ca2+-activated K+ channels in colonic smooth muscle cells.

PKC isoforms comprise one of the major second messenger systems that regulate the behaviors of smooth muscle cells. There are several isozymes of PKC expressed in gastrointestinal smooth muscle cells. For example, immunoblot analysis demonstrated expression of eight isoforms (alpha , beta , gamma , delta , epsilon , zeta , iota , and lambda ) in rat antrum smooth muscle (25), and esophageal smooth muscles express a variety of Ca2+-dependent isoforms, including alpha -, beta 2-, and gamma -isozymes (37). Under physiological conditions, PKC isozymes can be activated by diacylglycerol, [Ca2+]i, or phospholipids (4, 10, 13, 14, 38). Cellular distribution of PKC isozymes varies, and some forms, including Ca2+-activated isoforms, are translocated from the cytosol to the membrane upon activation (19, 23, 37). Activation and translocation of the Ca2+-dependent isozyme PKC-alpha occurs at basal or near basal levels of [Ca2+]i in some smooth muscles (19), and analyses of cellular distributions have shown this isoform to be present in both the soluble (cytosolic) and particulate (membrane) fractions in unstimulated cells (31). Thus it is likely that Ca2+-dependent isoforms of PKC are positioned at or near the membrane to phosphorylate membrane targets in resting cells. Others have shown that PKC-alpha can be rapidly translocated to specific membrane domains by localized increases in [Ca2+]i (24). Thus localized Ca2+ transients may also affect the spatial and temporal targeting of Ca2+-dependent isoforms of PKC.

The role of PKC in regulating intracellular Ca2+ signaling and coupling to membrane conductances has been considered previously in studies of vascular smooth muscles. STOCs, which are the result of activation of Ca2+-activated K+ conductances by localized Ca2+ transients (see Introduction), are inhibited by PKC-mediated depletion of Ca2+ stores in rabbit portal vein (20). Bonev et al. (6) showed that PKC inhibits the activity of ryanodine-sensitive Ca2+ channels in rat cerebral arteries. Our data suggest that PKC activation does not significantly affect the occurrence of IP3 receptor-mediated Ca2+ transients in colonic myocytes. Instead the data suggest downstream regulation of the coupling between Ca2+ transients and STOCs. Bonev et al. (6) also reported that activators of PKC slightly reduced STOC amplitude and suggested that this might have been the result of direct effects of PKC on BK channels. Others have provided direct evidence of regulation of BK currents by PKC. Whole cell currents in rat tail artery smooth muscle cells resulting from Ca2+-activated K+ channels were reduced by PKC (34), and the open probabilities of single BK channels in porcine coronary artery cells were reduced by PKC (28). Similar results have been reported in studies of the BK conductance in rat anterior pituitary cells (35). Phorbol esters reduced whole cell currents and caused an eightfold reduction in the open probability of BK channels. We found that the I-V relationship of currents resulting from opening of BK channels shifted rightward along the voltage axis when the solution bathing cells was increased from 0.01 to 1 mM Ca2+ and that this effect was blocked by an inhibitor of PKC. A similar effect on currents resulting from BK channels was observed with phorbol ester. We have previously demonstrated that PKC does not affect the open probability of SK channels in murine colonic myocytes (21). Taken together, these data suggest that a Ca2+-dependent PKC regulates the open probability of BK channels, and this action may explain the role of PKC in regulating the coupling between Ca2+ puffs and STOCs.

In addition to the increase in amplitude of STOCs noted upon inhibition of PKC, we observed an increase in frequency. It is possible that the increase in frequency was a result of the increase in STOC amplitude. Under control conditions, there may have been many subthreshold STOCs that could not be detected above the noise. By inhibiting PKC and increasing the coupling between Ca2+ transients and STOCs, these events may have increased in amplitude to a point where they could be detected as STOCs.

Previous studies of BK channels in gastrointestinal muscles have demonstrated that agonists that are typically coupled to activation of PKC suppress the open probability of BK channels. For example, stimulation of canine colonic myocytes with ACh, via muscarinic receptor activation, shifted the voltage dependence of activation in a manner consistent with decreasing the sensitivity of BK channels to Ca2+ (8). This action was also shown to be G protein mediated (8), suggesting that it was likely to be linked to production of diacylgycerol, a primary activator of PKC. The muscarinic effect on BK channels was highly specific, and application of ACh outside on-cell patches was ineffective. This suggests tight coupling between muscarinic receptors and the intracellular pathways linked to regulation of BK channels.

We have shown in a previous study that ATP, acting through P2Y purinoreceptors and PLC, increased the occurrence of Ca2+ puffs and promoted the development of Ca2+ waves (1). These events led to substantially increased STOC activity that resulted from activation of BK and SK channels (Fig. 11 and see also Ref. 22). The increase in Ca2+ puffs in murine colonic myocytes in response to ATP may have been the result of additional production of IP3. In addition to production of IP3, diacylglycerol is also a byproduct of the action of PLC on its substrate, phosphatidylinositol bisphosphate. Diacylglycerol is a potent physiological activator of PKC (39). Therefore, it is possible that the pathway that we have identified in the present study (i.e., desensitization of BK channels to localized Ca2+ release) may provide negative feedback to purinergic responses or to selectively increase the relative contribution of SK channels vs. BK channels in response to ATP.


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Fig. 11.   Coupling of Ca2+ puffs to Ca2+-activated K+ channels in murine colonic myocytes. Summary of major findings made with respect to localized [inositol trisphosphate receptor (IP3R) mediated] Ca2+ transients. I: ATP increases Ca2+ puffs and STOCs via a P2Y receptor/phospholipase C (PLC)/IP3-dependent pathway. STOCs in these cells are the result of openings of BK and small-conductance Ca2+-activated K+ (SK) channels. PIP2, phosphatidylinositol bisphosphate. II: Ca2+ entry via a pathway blocked by Gd3+ or SKF-96365 activates a Ca2+-dependent protein kinase C (PKC) that inhibits BK channels and reduces coupling between Ca2+ puffs and STOCs. This pathway may also be activated by diacylglycerol (DAG) that is produced by PLC. When the Ca2+-PKC is blocked (either by blocking the Ca2+ entry pathway or by blocking PKC), Ca2+ puffs generate STOCs of greater amplitude. III: like ATP, ACh has a tendency to increase Ca2+ puffs and STOCs via a muscarinic/PLC/IP3-dependent pathway. This would tend to increase K+ channel openings, generate hyperpolarization, and reduce the effectiveness of ACh as an excitatory agonist, but this tendency is inhibited by a tonic rise in intracellular Ca2+ concentration that occurs by M2- and/or M3-mediated activation of a nonselective cation current and shuts off Ca2+ puffs. It is unlikely that the Ca2+ entry pathway coupled to PKC is the same NSCC activated by muscarinic stimulation because this conductance is inactive in the absence of receptor occupation. NSCC, nonselective cation channel; SR, sarcoplasmic reticulum.

Regulation of K+ channels by Ca2+ entry into a submembranous compartment and activation of Ca2+-dependent PKC has also been reported in studies of cultured dorsal root ganglion neurons (40). These authors found suppression of delayed-rectifier K+ current by phorbol ester and blockade of this effect by an inhibitor of PKC. In the case of neurons, the submembranous [Ca2+]i and PKC were supplied by an N-type Ca2+ conductance. By dialyzing cells with Ca2+ buffers with different rate constants, these investigators deduced that the Ca2+-dependent PKC, N-type Ca2+ channels, and the K+ channels that were regulated by PKC were closely organized into a compartment within 40 nm to 1 µm of the plasma membrane.

Our current concepts regarding the regulation of Ca2+ puffs in murine colonic myocytes are summarized in Fig. 11. In the present study, we have added a new facet to this pathway by showing that the coupling strength between Ca2+ puffs and STOCs is regulated by a Ca2+-dependent PKC. This form of regulation suggests specialized organization between BK channels, Ca2+-permeable nonselective cation channels, and PKC. The findings of the present study and previous work (Refs. 1 and 2 and Fig. 11) suggest the following. Ongoing PLC activity generates IP3 and diacylgylcerol. IP3 levels are high enough in unstimulated cells to initiate spontaneous Ca2+ transients. This could mean that diacylglycerol levels are also high enough to produce basal activation of a Ca2+-dependent PKC that resides near the plasma membrane. Basal activity of PKC is facilitated by Ca2+ influx via a pathway blocked by Gd3+ and SKF-96365. The influx of Ca2+ is small and localized enough that it does not affect global Ca2+. PKC reduces the Ca2+ sensitivity of BK channels, thus regulating the coupling strength between Ca2+ puffs and STOCs. This mechanism may provide negative feedback to limit the influence of Ca2+ puffs acting through BK channels on resting potentials and excitability in resting cells and to limit the activation of Ca2+-activated outward current during excitatory stimulation.


    ACKNOWLEDGEMENTS

We are grateful for many valuable discussions with Dr. Adrian Bonev.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315.

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology/MS 352, Univ. of Nevada School of Medicine, Reno, NE 89557-0046 (E-mail: kent{at}physio.unr.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. Section 1734 solely to indicate this fact.

Received 8 March 2001; accepted in final form 19 June 2001.


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DISCUSSION
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