Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046
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ABSTRACT |
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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. 4-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
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INTRODUCTION |
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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
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
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.
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METHODS |
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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).
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, 4Analysis 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.
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RESULTS |
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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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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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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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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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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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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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DISCUSSION |
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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 1-subunit of BK channels was knocked out
(7).
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
(,
,
,
,
,
,
, and
) in rat antrum smooth muscle (25), and esophageal smooth muscles express a
variety of Ca2+-dependent isoforms, including
-,
2-, and
-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-
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-
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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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.
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ACKNOWLEDGEMENTS |
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We are grateful for many valuable discussions with Dr. Adrian Bonev.
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FOOTNOTES |
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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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