1 Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085; and 2 Department of Physiology, University of Melbourne, Parkville, Australia 3052
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ABSTRACT |
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These experiments were performed to determine the effects of
reducing Ca2+ influx
(Cain) on
K+ currents
(IK) in
myocytes from rat small mesenteric arteries by
1) adding external
Cd2+ or
2) lowering external
Ca2+ to 0.2 mM. When measured from
a holding potential (HP) of 20 mV
(IK20),
decreasing Cain decreased
IK at voltages
where it was active (>0 mV). When measured from a HP of
60 mV
(IK60),
decreasing Cain increased
IK at voltages
between
30 and +20 mV but decreased IK at voltages
above +40 mV. Difference currents
(
IK) were
determined by digital subtraction of currents recorded under control
conditions from those obtained when
Cain was decreased. At test
voltages up to 0 mV,
IK60 exhibited
kinetics similar to control
IK60, with rapid
activation to a peak followed by slow inactivation. At 0 mV, peak
IK60 averaged
75 ± 13 pA (n = 8) with
Cd2+ and 120 ± 20 pA
(n = 9) with low
Ca2+ concentration. At test
voltages from 0 to +60 mV,
IK60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20
mV. At +60 mV, the initial peak
IK60 averaged
115 ± 18 pA with Cd2+ and 187 ± 34 pA with low Ca2+. With 10 mM pipette BAPTA, Cd2+ produced a
small inhibition of
IK20 but still
increased IK60 between
30 and +10 mV. In
Ca2+-free external solution,
Cd2+ only decreased both
IK20 and
IK60. In the
presence of iberiotoxin (100 nM) to inhibit
Ca2+-activated
K+ channels
(KCa),
Cd2+ increased
IK60 at all
voltages positive to
30 mV while BAY K 8644 (1 µM) decreased
IK60. These
results suggest that Cain, through L-type Ca2+ channels and perhaps
other pathways, increases KCa
(i.e., IK20) and decreases voltage-dependent K+
currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.
L-type calcium channels; vascular smooth muscle; mesenteric arteries; electrophysiology; patch clamp
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INTRODUCTION |
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AGONIST ACTIVATION of smooth muscle initiates a complex sequence of excitation-contraction coupling events (36). These include coupling of cell surface receptor occupancy to the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate by a specific phospholipase C, with the resultant production of inositol trisphosphate (IP3) (2), the release of intracellular Ca2+ stores (26), myosin light chain phosphorylation (20), and rapid force development (11). Additional mechanisms come into play to sustain contraction, despite the fact that the increases in IP3, cytosolic free Ca2+, and myosin light chain phosphorylation are largely transient (2, 11, 26). The mechanisms responsible for force maintenance are less well understood than those responsible for force development but have been suggested to involve protein kinase C activation (19, 21) and/or an increase in Ca2+ sensitivity of the contractile proteins (5, 32).
Force maintenance has been shown to have a critical dependence on extracellular Ca2+ influx as well as a close coupling to membrane potential (17, 27, 37). In the absence of extracellular Ca2+, contractile responses to agonists cannot be maintained (9, 34). Agonist activation causes membrane depolarization, with subsequent activation of voltage-dependent Ca2+ influx (29), and also indirectly activates nonselective cation channels (33, 41).
Mechanisms contributing to the depolarization of the membrane potential
by agonists have only recently been identified.
Ca2+ released from the
sarcoplasmic reticulum (SR) by agonists activates Ca2+-sensitive
Cl channels
(ClCa) (41) and inhibits
voltage-dependent K+ channels
(Kv) (13). Both of these effects
are thought to contribute to the membrane depolarization. There is
direct evidence that membrane depolarization is sustained for the
duration of agonist exposure on the basis of membrane potential
measurements (27). Furthermore, there is indirect evidence based on the
observation that organic Ca2+
channel blockers can inhibit sustained agonist-induced contractions in
arterial smooth muscle (31), especially in resistance arteries (4) and
in arteries from hypertensive subjects (1).
Considering the fact that SR Ca2+
release is transient (26) and ClCa
currents inactivate with sustained increases in cytosolic Ca2+ concentration
([Ca2+]) (43), the
question arises as to how the membrane depolarization associated with
agonist activation is sustained. Recently, it was demonstrated that
L-type Ca2+ channels
(CaL) exhibit measurable open
probability under steady-state conditions at voltages between 40
and
20 mV in arterial smooth muscle (35). It has
also been shown that a voltage "window" exists for
CaL activation, which produces a
similar voltage window of sustained, elevated cytoplasmic
[Ca2+] (12). These
results suggest that sustained
Ca2+ influx through
CaL associated with membrane
depolarization may also provide a mechanism to inhibit
Kv. It was the purpose of the
experiments reported in this study to test the hypothesis that
Ca2+ influx can inhibit
Kv and provide a mechanism to
produce sustained membrane depolarization during agonist activation,
thereby contributing to force maintenance.
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METHODS |
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Cells were isolated from rat small mesenteric artery branches using techniques previously described in detail (6, 8). Briefly, individual branches were cut open longitudinally and incubated at 37°C for 60 min in a Ca2+-free buffer. The segment was then cut into small pieces and incubated in buffer with collagenase (250 U/ml) and elastase (25 U/ml) for ~30 min without agitation at 37°C. The tissue was gently aspirated with a Pasteur pipette, and released cells were separated by filtration through 210-µm nylon mesh.
Electrophysiology. An aliquot of cells
was placed in a perfusion chamber mounted on an inverted microscope
(Nikon Diaphot), allowed to adhere to the glass bottom, then superfused
with a HEPES-buffered solution. The external
[Ca2+] was slowly
increased to 2 mM to avoid damaging the cells. Cells were sealed to
pipettes with gentle suction, and the membranes were ruptured by
negative pressure at a holding potential of 40 mV. Membrane
currents were recorded using whole cell or perforated patch-clamp
techniques (18) with a voltage-clamp amplifier (model 8900; Dagan).
Fire-polished micropipettes (2-3 M
resistance) were fashioned
from capillary tubing (Kwik-fil; WPI) using a micropipette puller
(model P-80/PC; Sutter Instruments). Series resistance and stray
capacitance compensation were employed using the amplifier circuitry.
For perforated-patch conditions, junction potential was compensated
before measurements. Current and voltage signals were converted from
analog to digital form at a sampling rate of 2 kHz (Labmaster A/D
board) and stored in a computer (Gateway 2000 486DX) for subsequent analysis.
Protocols. Whole cell currents were
measured using both voltage-ramp and voltage-step protocols from
holding potentials of 60 and
20 mV. With the ramp
protocol, voltage was increased at 1 mV/ms from either holding
potential to a maximum value of +60 mV. With the step protocol, 1-s
voltage steps were applied from
60 to +60 mV from both values of
holding potential in 10-mV increments at intervals of 15 s. For
statistical analysis and comparisons, peak currents and currents
averaged over the last 100 or 200 ms of each voltage-clamp step were
determined. For most conditions, current responses were recorded before
and during an intervention. Difference currents were determined between
such conditions by subtracting digital current records obtained under control conditions from those obtained during an intervention at each
value of test voltage. Current records were analyzed using pCLAMP
software (version 5.5.1; Axon Instruments).
Chemicals and solutions. Collagenase
was purchased from Worthington Biochemical, Freehold, NJ (type CLS3),
and elastase was from ICN Nutritional Biochemicals, Cleveland, OH (hog
pancreas). All other chemicals were obtained from Sigma Chemical, St.
Louis, MO. Water (>18 M) was obtained from a Barnstead
purification system with an organic final filter. The solution used for
myocyte isolation contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 10 dextrose
at pH 7.4 (with NaOH). The external perfusion solution was the same,
with 2 mM Ca2+ added. The pipette
solution for whole cell studies contained (in mM) 140 KCl, 5 NaCl, 5 MgATP, 10 HEPES, and 0.2 or 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) at pH 7.2 (with KOH) and had a resistance of 2.5-3.5 M
. HEPES was increased with the 0.2 mM BAPTA internal solution to
maintain osmolarity constant at ~300 mosM. The pipette solution for
perforated-patch studies contained (in mM) 110 potassium gluconate, 30 KCl, 5 NaCl, 0.1 CaCl2, and 10 HEPES with 150 µg/ml amphotericin at pH 7.2 (with KOH).
Statistical analysis. Statistical comparisons of membrane currents were performed using a two-way ANOVA with repeated measures for paired data using the STATWORKS application on a Power Macintosh computer. Probability values of <0.05 were considered to be significant.
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RESULTS |
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In the first series of experiments, the effects of 0.1 mM
Cd2+ (to inhibit
CaL) on
K+ current
(IK) were
determined. Figure 1 shows current
responses to voltage ramps from each holding potential before and
during the addition of 0.1 mM Cd2+
to the perfusate. When
IK was recorded
from a holding potential of 60 mV
(IK60),
Cd2+ increased net outward current
at voltages just above the activation threshold (Fig.
1A). At more positive voltages up
to +60 mV, however, net outward current was decreased with
Cd2+. When recorded from a holding
potential of
20 mV
(IK20),
Cd2+ only decreased net outward
current at voltages >0 mV (Fig.
1B). Difference currents, obtained
by digital subtraction of current ramps before and during
Cd2+, also shown in Fig. 1,
confirm this description. We initially assumed that, under the
conditions of these experiments,
K+ currents dominate these
responses. Whole cell K+ currents
have been shown in many smooth muscle cells to be composed primarily of
Kv and
Ca2+-activated
K+ channel
(KCa) components (22, 29).
Because Kv currents are expected
to be inactivated (decreased availability) at a holding potential of
20 mV (22, 46), the
IK20 data should
primarily represent the KCa
component. The above
IK20 results
suggest that inhibiting CaL with
Cd2+ reduced
KCa current. Because both
Kv and
KCa currents are expected to
contribute to
IK60, and since
Cd2+ inhibits
IK20 (i.e.,
KCa) only at voltages >0 mV,
one interpretation of the
IK60 responses at
voltages <0 mV is that Kv
currents are augmented in the presence of
Cd2+. The presence of both
Kv and
KCa components of
IK60 were
observed in every cell studied.
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Current responses to voltage ramps are only qualitative at best, since
voltage-dependent activation and inactivation kinetics may obscure the
details of currents recorded by this method. To study the effects of
Cd2+ on
IK
quantitatively, measurements of
IK were made in
response to voltage steps from holding potentials of 60 and
20 mV with 2 mM external
[Ca2+] and 0.2 mM
pipette BAPTA in the absence then the presence of 0.1 mM
Cd2+. Figure
2 shows representative results of whole
cell currents recorded at two values of test potential (0 and +60 mV)
from the two values of holding potential (
60 and
20 mV).
The addition of Cd2+ to the
perfusion solution consistently increased
IK60 at a test voltage of 0 mV (Fig. 2A,
top) but decreased
IK60 at a test
voltage of
60 mV (Fig. 2C,
top) compared with control
conditions. Inspection of the current responses suggests that the
kinetics of the current was also altered by
Cd2+. Therefore, difference
currents
[
IK60(Cd)]
were calculated between the two conditions. At 0 mV test voltage,
IK60(Cd)
increased rapidly to a peak and subsequently declined to a steady level that was maintained for the duration of the voltage step (Fig. 2A,
bottom). The peak value of
IK60(Cd)
averaged 75 ± 13 pA, whereas the "steady" level (last 100 ms
of the clamp step) averaged 31 ± 9 pA
(n = 8). Although the majority of
IK60(Cd)
recorded at the +60-mV test voltage was negative (smaller net outward
current), there was an early positive phase for the difference current
(arrow in Fig. 2C,
bottom). The peak value of this
early current averaged 115 ± 18 pA, whereas the steady level
averaged
148 ± 52 pA (n = 8) at +60 mV. When recorded at a test voltage of 0 mV from a holding
potential of
20 mV, the currents were very small (Fig. 2B,
top) and the difference current
[
IK20(Cd)]
was essentially zero. When measured at a test potential of +60 mV (Fig.
2D,
bottom),
IK20(Cd) was
consistently decreased in the presence of
Cd2+ and never exhibited an early
positive peak as found in
IK60(Cd). The
steady level of
IK20(Cd)
averaged over the last 200 ms of the voltage-clamp step was
288 ± 48 pA (n = 8).
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To expand on these observations, measurements were repeated at several
test potentials from both values of holding potential (i.e.,
IK20 and
IK60). Examples
of difference currents determined from currents measured before and
during Cd2+ at various values of
test potential are shown in Fig. 3. Over a
test potential range from 20 to +20 mV (Fig.
3A),
IK60(Cd) rose
to an initial peak that increased with test voltage then appeared to
"inactivate" with time. This secondary decline increased with
increasing test voltage and was also associated with an increase in
current "noise." With further increases in test potential >20 mV, the initial peak became shorter in duration and was followed by a
net negative outward current.
IK60(Cd)
always exhibited an initial positive phase at all test potentials, but
values during the sustained phase of the voltage-clamp step exhibited a
transition from positive values at negative test potentials to negative
values at positive test potentials. This behavior was different from that recorded from a holding potential of
20 mV (Fig.
3B). Values of
IK20(Cd) were
zero at all voltages up to 0 mV (Fig.
3B). At test potentials >0 mV,
values of
IK20(Cd) were
only negative (decreased net outward current or increased net inward
current) over the entire duration of the voltage-clamp step.
IK20(Cd) never
demonstrated an early positive value.
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Peak values and steady values of
IK60(Cd)
averaged over the last 100 ms of the voltage-clamp step were determined
at different test potentials, and average results are shown in Fig.
4A. Peak values of
IK60(Cd)
"activated" at about
30 mV and increased with voltage up
to +60 mV. Steady values of
IK60(Cd) also
activated at about
30 mV, increased to a maximum value at about
+10 mV, then decreased with increasing test potential, becoming
negative at about +40 mV and higher. Steady values of
IK20(Cd) were
determined over the last 200 ms of the voltage-clamp step, and results
are summarized in Fig. 4B. Steady
values of
IK20(Cd) were
significantly different from zero only at voltages >0 mV, where they
were always negative (smaller net outward current). Steady values of
IK20(Cd) were
significantly larger (i.e., more negative) than steady values of
IK60(Cd) at
all voltages >0 mV. These results are qualitatively similar to those
presented in Fig. 1 for ramp current responses.
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The current responses at the two holding potentials have been
interpreted above based on the assumption that only
KCa currents contribute to
IK20, whereas
both Kv and
KCa contribute to
IK60. The
validity of the conclusions concerning the effects of
Cd2+ on
IK components
rests in part on the validity of this assumption. Therefore,
experiments were performed to determine the effects of blockers of
Kv (4-aminopyridine) and
KCa (iberiotoxin) on
IK20 and
IK60 to test this
assumption. As shown in Fig. 5, 100 nM
iberiotoxin completely inhibited
IK20, whereas it
had a relatively small effect on
IK60 (Fig.
5B), including reducing the current
noise. In the presence of iberiotoxin, prominent tail currents remain
in IK20, representing the recovery of Kv
from inactivation following clamp steps to voltages negative to the
20-mV holding potential. When 1 mM 4-aminopyridine was added
with iberiotoxin,
IK60 was reduced substantially, as was the tail current in
IK20 (Fig.
5C). Similar results were obtained
in six cells. These results confirm the assumption that
IK20 represents
primarily KCa currents, whereas IK60 represents
both Kv and
KCa currents.
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If the responses shown in Figs. 1-4 are the result of inhibiting
Ca2+ influx on
IK components by
Cd2+ as suggested, then it would
be expected that decreasing Ca2+
influx by lowering external
[Ca2+] should have
similar effects. The effects of decreasing external [Ca2+] from 2 to 0.2 mM on IK were
determined to test this expectation. External
[Mg2+] was
simultaneously increased to 2.8 mM to maintain divalent ion
concentration constant. The results are summarized in Fig. 6. At a test potential of 0 mV, decreasing
external Ca2+ increased
IK60, and the
difference current,
IK60(Ca),
increased rapidly to a peak then declined slowly for the duration of
the clamp step (Fig. 6A). The peak
value of
IK60(Ca) at 0 mV test voltage averaged 120 ± 20 pA while the steady value
averaged over the last 100 ms of the clamp step was 59 ± 19 pA
(n = 9). As with Cd2+,
IK60(Ca)
recorded at a +60 mV test voltage exhibited an early positive phase
(arrow in Fig. 6C) followed by a
sustained negative phase. The peak value of this early current phase
averaged 187 ± 34 pA while the steady level averaged
296 ± 44 pA (n = 9). When recorded
from a holding potential of
20 mV, however,
IK20 was only
decreased when external Ca2+ was
decreased, and only negative difference currents (smaller net outward
currents) were observed over the entire duration of the voltage-clamp
step. At a test potential of 0 mV, the difference currents were also
essentially zero (Fig. 6B), but at
+60 mV (Fig. 6D)
IK20(Ca)
averaged
455 ± 78 pA. In contrast to
IK60(Ca),
IK20(Ca) never
exhibited an early positive phase at a test potential of +60 mV (Fig.
6D). When the voltage dependence of
the peak and late values of difference currents were determined (Fig.
6, E and
F), they exhibited characteristics
similar to those found with Cd2+
(Fig. 4) but with larger magnitudes.
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The positive difference currents produced by decreasing external
Ca2+ could be due (in part) to a
shift in the voltage dependence of IK availability
as a result of altered masking of surface charge even with the
compensatory increase in external
[Mg2+] (15). Figure
7 shows a comparison of the effects of
external [Ca2+] on
steady-state inactivation of
IK. There was no
significant difference in the voltage dependence of this relationship
when external [Ca2+]
was reduced to 0.2 mM and external
[Mg2+] increased to
2.8 mM. The addition of 0.1 mM external
Cd2+ also had no significant
effect on IK
availability (data not shown). It is unlikely, therefore, that a shift
in the voltage dependence of
IK contributed to
the effects of decreasing external
Ca2+ on whole cell current
described above.
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If these current responses are the result of an effect of
Ca2+ influx on global
intracellular [Ca2+],
then IK responses
to Cd2+ should be decreased in
cells dialyzed against a pipette solution containing 10 mM BAPTA to
buffer intracellular
[Ca2+]
(45). As shown in Fig. 8, responses to the
addition of 0.1 mM Cd2+ to the
external perfusate with 10 mM pipette BAPTA were smaller but showed some similarities to those obtained with 0.2 mM pipette BAPTA (Fig. 4). Peak values of
IK60(Cd)
measured with 10 mM pipette BAPTA exhibited a bell-shaped voltage
dependence with a peak near 0 mV test voltage (Fig.
8A).
IK60(Cd) at 0 mV test voltage exhibited an early positive value (increased net
outward current) that averaged 68 ± 11 pA
(n = 7) and inactivated more
completely toward baseline over the course of the 1-s voltage-clamp
step (Fig. 8A). Values of
IK60(Cd) with
10 mM BAPTA measured at a +60-mV test voltage did not exhibit an early
positive peak, but a significantly negative difference current
developed during the sustained voltage-clamp step (Fig.
8C). The late value of
IK60(Cd)
averaged
59 ± 23 pA at a +60-mV test voltage with 10 mM
pipette BAPTA. Values of IK20 recorded
with 10 mM pipette BAPTA were generally smaller than those recorded
with 0.2 mM pipette BAPTA as expected and were reduced by the addition
of external Cd2+. These results
suggest that, when cytosolic Ca2+
is dialyzed to low (nM) levels by 10 mM pipette BAPTA, blocking Ca2+ influx with external
Cd2+ still increased
IK60 (smaller
effect compared with 0.2 mM BAPTA), but to a smaller extent and over a
smaller voltage range.
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The direct effects of Cd2+ on
IK were
determined by recording responses in the absence of external
Ca2+. These results are summarized
in Fig. 9.
IK was again
measured from holding potentials of 20
(IK20) and
60 mV
(IK60) with 0 mM external Ca2+ (replaced by
Mg2+) and 0.2 mM pipette BAPTA.
Currents measured from a holding potential of
20 mV were
significantly reduced at voltages above a test voltage of +40 mV by
Cd2+ (Fig.
9F), but the effect was much smaller
than noted under previous conditions. At test voltages above
20
mV, Cd2+ significantly reduced
currents recorded from the
60-mV holding potential (Fig.
9E).
Cd2+ never increased
IK under
conditions of Ca2+-free perfusion.
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To directly test the interpretation of the previous results,
experiments were performed after blocking
KCa currents with 100 nM
iberiotoxin. In addition,
IK was recorded
using the perforated-patch configuration (amphotericin) to provide more
physiological conditions. The effects of inhibiting
Ca2+ influx with 0.1 mM
Cd2+ under these conditions are
summarized in Fig. 10. When measured in
the presence of iberiotoxin, the increase in
IK60 (i.e.,
Kv) associated with the addition
of Cd2+ now occurred over the
entire voltage, where
IK60 was active
(from 30 to +60 mV). The maximum increase in
IK60 averaged 100 ± 6 pA (n = 4) at a test voltage
of +60 mV.
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To further test the interpretation of the previous results, experiments
were performed to determine the effects of increasing Ca2+ influx on
IK60.
IK was recorded
with the perforated-patch configuration in the presence of 100 nM
iberiotoxin to block KCa, and
CaL current was increased by
adding 1 µM BAY K 8644 to the perfusate. As shown in Fig.
11,
IK60 was
decreased following the addition of BAY K 8644, and the effect was
reversible. The decrease in
IK60 was significant at all voltages above 30 mV, where
Kv was active, and had a maximum
value of
97 ± 7 pA (n = 4)
at a test voltage of +60 mV.
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DISCUSSION |
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The results of these studies demonstrate that reducing
Ca2+ influx either by
1) decreasing external
Ca2+ or
2) adding external
Cd2+ increased steady values of
IK60 at test
voltages between about 40 and +10 mV but decreased
IK60 at test
voltages above +30 mV and decreased
IK20 at test
voltages above +10 mV. We have interpreted these results to indicate
that decreasing Ca2+ influx
relieves inhibition ("disinhibits") of
Kv and decreases the activation of
KCa in this tissue.
This interpretation of our experimental results based on whole cell
current measurements relies on two assumptions:
1) the contribution of
Kv and
KCa currents to
IK20 and
IK60 and
2) the relative contribution of
outward (primarily K+) and
inward currents (primarily
Cl and
Ca2+) to the net current
responses. Regarding the first assumption, IK20 but not
IK60 is nearly
completely inhibited by iberiotoxin, a selective inhibitor of
KCa, suggesting that
IK20 is primarily KCa current.
IK60 is only
partially inhibited by iberiotoxin but is further inhibited by
4-aminopyridine, suggesting that both Kv and
KCa contribute to
IK60. This latter
conclusion is consistent with the known steady-state availability of
Kv in vascular myocytes, which
have a voltage for 50% availability between
50 and
40 mV
(22, 46), as well as with the known properties of
KCa (10). Thus the first
assumption appears to be valid.
Regarding the second assumption, the increase in difference currents
associated with adding Cd2+ or
reducing external
[Ca2+] could be due to
an increase in outward current, a decrease in inward current, or a
combination of both. We have suggested that our results primarily
represent changes in outward currents. However, several candidate
inward currents could contribute to these responses, including
Cl,
Ca2+, and nonselective cation
currents (29, 33, 41, 44). Under the conditions of our experiments,
both Cl
(symmetrical
[Cl
])
and nonselective cation currents would be expected to have reversal
potentials near 0 mV (41, 42). The results shown in Figs. 2 and 6
demonstrate that reducing Ca2+
influx by either method increased whole cell current at a test potential of 0 mV (as well as others). It is unlikely that inhibition of either Cl
or
nonselective cation currents contributes significantly to the changes
in IK60 at 0 mV.
Furthermore, if changes in inward currents did contribute significantly
to these current responses, it would be expected that the holding
current at
60 mV would be increased (i.e., more positive)
following inhibition of Ca2+
influx, but it was not (Fig. 1). Furthermore, the difference currents
shown in Figs. 3A and
5A remain positive over the entire 1-s
voltage-clamp step. It is unlikely that
CaL are directly responsible for
this effect, since they are likely to be small under the conditions of
these experiments and they inactivate rapidly to small steady-state values (35). No voltage-gated Na+
currents or T-type Ca2+ currents
are present in this tissue (8, 24). Although a minor contribution from
inward currents may occur at negative test voltages, it is likely that
IK60 and
IK20 primarily
represent contributions from Kv
and KCa.
On the basis of this discussion, we have interpreted the results of
these experiments to indicate that decreasing
Ca2+ influx in rat small
mesenteric artery myocytes augments
Kv currents and inhibits
KCa currents. When recorded from a
holding potential of 20 mV where the dominant current is carried
by KCa, decreasing Ca2+ influx decreases
IK20 over the
voltage range where KCa currents are active (i.e., >0 mV). When recorded from a
60 mV holding potential where both Kv and
KCa currents contribute to
IK60, a mixed
result is predicted. Over the voltage range where
Kv current dominates (negative to
+10 mV), the effect of decreasing
Ca2+ influx is to increase
IK60. Over the
voltage range where KCa current
dominates (positive to +30 mV), the effect of decreasing Ca2+ influx is to decrease
IK60. The results
obtained in the presence of iberiotoxin confirm this interpretation.
When KCa currents are inhibited,
decreasing Ca2+ influx (with
Cd2+) increases
IK60 over the
entire voltage range from
30 to +60 mV. Furthermore, increasing
Ca2+ influx (with BAY K 8644)
decreases IK60,
as would be predicted based on our interpretation of these results.
These latter two sets of experiments provide strong evidence in support
of our interpretation of the results obtained in the absence of
K+ channel blockers.
In apparent conflict with this simple explanation was the observation
that, at positive test potentials, an initial rapid, transient positive
phase of the difference current was found in IK60 (Figs.
2C and
6C) but not
IK20 (Fig.
2D and
6D). This initial peak of
IK60 was
followed by a secondary decrease to negative net
IK60 values.
This biphasic response can be explained on the basis of a faster rate
of activation of Kv compared with
KCa currents (30, 40). At positive
test potentials, the effects of
Ca2+ influx on
Kv and
KCa contribute algebraically to
the overall response. The rapid, initial transient phase represents the
"disinhibition" of the more rapidly activating
Kv current, but the larger
inhibition of the more slowly activating
KCa current subsequently dominates the Kv contribution, and the
difference current declines to its final net negative value. Consistent
with this explanation, when difference currents between
20 and
+60 mV are inspected, a progressive transition in
IK60 is seen
over this voltage range (Fig. 3). Although
KCa dominate steady-state
responses at positive test potentials, the contribution of
Kv disinhibition can be seen in the initial transient current phase (Fig.
3A). However, since Kv are not available at a holding
potential of
20 mV, the inhibition of
KCa by decreased
Ca2+ influx completely explains
the observed responses of
IK20 (Fig. 3B).
To test the interpretation of these results, we developed
a simple model representation. We assumed that
IK could be
represented by the sum of Kv and
KCa components. The
Kv component was represented by an
activation function consisting of a single exponential function to the
second power
(n2) plus an
inactivation function consisting of two exponential components plus a
noninactivating component (40). The
KCa component was represented by a
similar activation function. Values for the amplitude and time
constants of the various functions were determined for
Kv and
KCa components from
IK60 data
obtained in the presence of iberiotoxin and from
IK20 data,
respectively, using the curve-fitting functions of SigmaPlot. Figure
12 shows some of the results predicted by
this model at a test voltage of +60 mV. First, we assumed that reducing
Ca2+ influx increased the
amplitude of Kv current by 30%
and decreased the amplitude of KCa
current by 50%. The net effect of these changes on
IK is shown in
Fig. 12F. The model predicts an early,
transient positive current response followed by a more slowly
developing net negative current response. If only a change in
KCa current is allowed to occur,
the response in Fig. 12G is obtained,
and there is no early positive peak, only a net negative current. This
simple model accurately represents the results shown above (Figs. 2, 3,
and 6) and gives support to the explanations provided. Thus the early
net positive outward current peak in the composite whole cell
IK response is
the result of the more rapid activation kinetics of
Kv compared with
KCa currents.
|
How does Ca2+ influx contribute to
these effects on
IK components?
The results obtained with 10 mM pipette BAPTA suggest that CaL current activated by
voltage-clamp steps directly contributes to these responses. The
primary evidence to support this conclusion comes from the voltage
dependence of the peak value of
IK60(Cd) recorded under these conditions, which mirror the voltage dependence of
CaL currents (7, 8). However, the
voltage dependence of peak
IK60(Cd) or
IK60(Ca)
recorded with 0.2 mM pipette BAPTA does not exhibit a similar voltage
dependence. Instead of exhibiting a bell-shaped variation with voltage
peaking near 0 mV, these responses extend to positive test potentials,
where they achieve their largest values. This is particularly apparent
in the results obtained in the presence of
KCa blocked by iberiotoxin (Fig.
10). This suggests that the time-dependent
CaL is not the primary determinant of the IK
responses to decreased Ca2+ influx
under more normal conditions (0.2 mM pipette BAPTA). It is likely,
therefore, that a finite open probability of
CaL contributes significantly to
global intracellular Ca2+ at a
holding potential of
60 mV (35). This conclusion is supported by
the observation that augmentation of
CaL by BAY K 8644 inhibits Kv over the entire test voltage
range up to +60 mV. The greater effect of time-dependent
CaL in the presence of 10 mM BAPTA
is likely due to the much larger current amplitude that is expected when global intracellular Ca2+ is
reduced, resulting in the loss of
Ca2+-induced inactivation of
CaL (15)
We chose to use Cd2+ to inhibit CaL in these experiments, because in initial studies we found that 100 nM nisoldipine inhibited IK60 with 0.2 mM external Ca2+ and 0.2 mM pipette BAPTA. Under these conditions, it is expected that only a small CaL would exist, so that the inhibition of IK by nisoldipine probably represents a direct inhibitory effect on Kv currents. Data in the literature demonstrate that heterologously expressed channels of the Shaker family are inhibited by dihydropyridines (16). A similar effect of dihydropyridines has been reported in rabbit coronary artery myocytes (22).
The effect of Cd2+ on
IK60 in the
voltage range from 20 to +10 mV was similar with 0.2 or 10 mM
pipette BAPTA (Figs. 2, 4, and 8). With the higher pipette BAPTA it
would be expected that global intracellular
Ca2+ would be much lower but that
the Ca2+ current would be larger
because of a reduction in
Ca2+-induced inactivation of
CaL (3). These results could be
interpreted to suggest that CaL
and Kv may be colocalized in the
plasma membrane in a region not readily accessible to BAPTA or in which
free diffusion of Ca2+ may be
restricted. The difference current recorded with 10 mM BAPTA exhibited
a continuous decrease with time, consistent with slow diffusion and/or
buffering of local Ca2+. Similar
suggestions have been made concerning the colocalization of functional
elements involved in smooth muscle excitation-contraction coupling,
including SR Ca2+-release channels
and plasma membrane KCa (28),
plasma membrane Na+ pump and
Na+/Ca2+
exchanger, and calsequestrin-containing regions of the SR (25).
The effects of Ca2+ influx on whole cell IK are similar to those demonstrated for the IK response to SR Ca2+ release mediated by either agonists or caffeine (13, 14). These investigators further demonstrated that Ca2+ added at micromolar levels to the cytoplasmic surface of inside-out patches inhibited the open probability of single-channel events by decreasing mean open time. Similar but more complicated results have been described for the effects of Ca2+ on inside-out patches of Xenopus oocytes expressing colonic smooth muscle Kv1.5 channels (39).
It has been known for some time that agonist activation of smooth muscle produces membrane depolarization (17, 27, 37). Ca2+ influx is then increased as a result of an increase in open probability of CaL associated with the membrane depolarization (29) and as a direct result of the effect of the agonist on CaL (23). Activation of ClCa (33) and inhibition of Kv (13) by SR Ca2+ release have both been suggested to participate in this response. This has been suggested to be a positive-feedback mechanism to maintain Ca2+ influx and elevated cytosolic Ca2+ (13). However, it is known that agonists, in addition to promoting SR Ca2+ release, maintain the release channels in the open configuration, preventing subsequent SR Ca2+ accumulation and release, thereby allowing Ca2+ influx to sustain contraction (38). The results of the present experiments suggest that Ca2+ influx can also inhibit Kv, thereby sustaining the membrane depolarization and the increased open probability of CaL. ClCa have been shown to inactivate during sustained increases in cytosolic Ca2+ (44) by a Ca2+/calmodulin kinase II-mediated process (43). This suggests that, while an action of SR Ca2+ release on ClCa and Kv may be involved in the initial phase of force development, sustained membrane depolarization and CaL influx may be maintained by an action of Ca2+ influx on Kv, contributing to force maintenance by a positive-feedback mechanism.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-28476.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: R. H. Cox, Dept. of Physiology, Univ. of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085 (E-mail: rcox{at}mail.med.upenn.edu).
Received 3 February 1999; accepted in final form 29 March 1999.
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