Charybdotoxin block of
Ca2+-activated
K+ channels in colonic muscle
depends on membrane potential dynamics
Bret W.
Frey1,
Andreas
Carl2, and
Nelson G.
Publicover1
1 Department of Physiology and
Cell Biology, University of Nevada School of Medicine, Reno, Nevada
89557; and 2 Department of
Pathology, University of Colorado Health Sciences Center, Denver,
Colorado 80262
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ABSTRACT |
Charybdotoxin
(ChTX) is a specific blocker of
Ca2+-activated
K+ channels. The voltage- and
time-dependent dynamics of ChTX block were investigated using canine
colonic myocytes and the whole cell patch-clamp technique with step and
ramp depolarization protocols. During prolonged step depolarizations,
K+ current slowly increased in the
continued presence of ChTX (100 nM). The rate of increase depended on
membrane potential with an e-fold
change for every 60 mV. During ramp depolarizations, the effectiveness
of ChTX block depended significantly on the rate of the ramp (50% at
0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanism
in which ChTX slowly "unbinds" in a voltage-dependent manner. A
simple kinetic model was developed in which ChTX binds to both open and
closed states. Slow unbinding is consistent with ChTX having little
effect on electrical slow waves recorded from circular muscle while
causing depolarization and contraction of longitudinal muscle, which
displays more rapid "spikes." Resting membrane potential and
membrane potential dynamics are important determinants of ChTX action.
voltage dependence; calcium-activated potassium channels; colonic
motility; model; maxi-potassium channels
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INTRODUCTION |
CALCIUM-ACTIVATED POTASSIUM (BK or
maxi-K+) channels are abundantly
expressed in a wide variety of smooth muscles, but their physiological
role in these tissues continues to be controversial. The development of
a number of specific blockers of BK channels such as charybdotoxin
(ChTX; Ref. 22) and iberiotoxin (IbTX; Ref. 8) has allowed a more
rigorous examination of the role of these channels in intact tissues.
For example, Suarez-Kurtz et al. (28) have tested the effects of ChTX
on smooth muscles from several sources. They report that depolarization
and contraction are stimulated by ChTX in guinea pig bladder, taenia
coli, and aorta but not in portal vein, uterus, or trachea. Because all of these tissues generously express BK channels, reasons for these differences in ChTX sensitivity remain unclear.
In a previous study (3), we investigated the effects of
Leiurus quinquestriatus scorpion venom
ChTX on electromechanical activity in intact muscle strips of isolated
longitudinal and circular layers of canine colon to evaluate the
physiological roles of these channels. The effects of ChTX in tissues
were compared with effects on whole cell outward currents in
enzymatically isolated myocytes from both muscle layers. In the
circular layer, ChTX does not affect in vitro basal electrical activity
but alters responses to excitatory agonists. The lack of an effect on
basal activity has been used to argue that BK channels do not play a role in the repolarization phase of the slow wave (15). In contrast, in
longitudinal muscle strips, ChTX depolarizes membrane potential and
increases burst duration as well as spiking frequency, resulting in
increases in the force of spontaneous contractions. Despite these
observations, no differences have been found in current density (pA/pF)
as well as in ChTX, voltage, or
Ca2+ sensitivity of BK channels in
myocytes isolated separately from the two muscle layers (3).
In the present study, we investigated the voltage- and time-dependent
kinetics of ChTX block of BK channels in colonic myocytes to determine
whether the apparent difference in ChTX sensitivity in longitudinal and
circular muscle strips may be due to the type of electrical activity
displayed by these cells [spiking vs. nonspiking and resting
membrane potential (RMP)]. The aim was to compare the ChTX
sensitivity of Ca2+-activated
K+ currents
[IK(Ca)]
elicited by different voltage protocols and to develop a quantitative
model of ChTX binding to BK channels that describes these kinetics.
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METHODS |
Preparation of cells.
Dogs of either sex were killed with pentobarbital sodium (100 mg/kg),
and the colon was removed. Circular and longitudinal muscle strips were
dissected from the proximal colon, viewed under a dissecting
microscope. Muscle strips were separated into small pieces and placed
in a Ca2+-free Hanks' solution
containing (in mM) 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4,
0.44 KH2PO4,
10 glucose, 2.9 sucrose, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) and 0.11 mg/ml ATP, 1 mg/ml collagenase type III
(Worthington), 2 mg/ml trypsin inhibitor (Sigma), 0.1 mg/ml protease
type XIV (Sigma), and 1 mg/ml bovine serum albumin (Sigma) at pH 7.4 (adjusted with KOH at 37°C). After a 20- to 30-min incubation
period, strips were rinsed in
Ca2+-free solution and briefly
agitated using a vortex mixer to disperse cells. Isolated cells were
collected and added to minimum essential medium Eagle
(Sigma) supplemented with (in mM) 0.5 CaCl2, 5 MgCl2, 4.17 NaHCO3, and 10 HEPES. Cells were
stored at 4°C and used within 1-8 h. Fully relaxed myocytes
with smooth-appearing membranes adhered to the glass bottom of a
recording chamber (volume of ~300 µl) mounted on the stage of a
Nikon TMS inverted microscope.
Whole cell patch-clamp recordings.
Currents were recorded using the cell-attached or whole cell mode of
the patch-clamp technique (10). High-resistance seals (>5 G
) were
formed using borosilicate electrodes (1.5-3 M
). An Axopatch 1D
amplifier (Axon Instruments) was used for current recordings. Data were
digitized at sampling rates of up to 10 kHz and filtered at 500 Hz.
Capacitance was compensated for, and residual capacitance current was
digitally removed. Current from hyperpolarizing pulses (represented by
x, in mV) was multiplied by (test
potential
holding potential)/x
and subtracted from currents recorded during test pulses. Series
resistance (between 4 and 8 M
) was not compensated for. Current
amplitudes were below 1,500 pA, resulting in a voltage error of <12
mV. Data are corrected for a
10-mV liquid junction potential.
Solutions.
The bath solution initially contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1.2 MgCl2, 10 dextrose, 10 HEPES, and
5 tris(hydroxymethyl)aminomethane at pH 7.4. In most experiments,
CaCl2 was replaced by equimolar MnCl2 to remove
Ca2+ inward currents. The standard
pipette solution contained (in mM) 20 KCl, 110 potassium gluconate, 5 MgCl2, 2.5 K2ATP, 0.1 Na2GTP, 2.5 Na2 creatine phosphate, 5 HEPES,
and 1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid at pH 7.0. All patch-clamp experiments were carried out at room
temperature (24°C).
ChTX was obtained from Peptides International (Louisville, KY). During
all procedures involving ChTX, the drug (100 nM, unless otherwise
noted) was added at least 2 min before voltage-clamp protocols. During
this time, cells were held at
50 mV.
Modeling and data analysis.
The model illustrated in Fig. 4 can be represented as a series of four
simultaneous, coupled differential equations. To solve these equations,
numerical integration was performed with the Gear method (12). The
fraction of channels in the open state (O in Fig. 4) was converted to a
current using the Goldman-Hodgkin-Katz equation (13) to account for the
effects of driving force, with intracellular
K+ concentration = 140 mM and
extracellular K+ concentration = 5 mM.
For acquisition and analysis of patch-clamp data, pCLAMP software (Axon
Instruments, version 5.5.1) was used. Data are expressed as means ± SE. Levels of significance were calculated using the Student's
t-test where appropriate.
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RESULTS |
Block of BK channels by ChTX: voltage ramps vs. steps.
To determine the effectiveness of ChTX block under conditions
comparable to components of electrical activity in vivo, ChTX block of
large-conductance BK channels was assessed using both step and ramp
depolarizations. Myocytes were isolated from the circular layer of
canine proximal colon. Inward Ca2+
currents were suppressed by replacement of bath
Ca2+ with 2 mM
Mn2+. Cells were either held at
50 mV and then step depolarized for 3 s to test potentials of 0 to +100 mV in 10-mV increments (Fig. 1,
A and
B,
top) or held at
50 mV and then ramped from
100 to +100 mV within 4 s
[ramp rate (dV/dt) = 0.05 V/s;
Fig. 1, A and B,
bottom]. Because ChTX blocks BK
channels with slow kinetics (11), one might expect that ChTX might be
more potent in blocking current elicited by step depolarizations than
current elicited by ramp depolarizations. However, our experiments
showed that ChTX was significantly more potent when 0.05 V/s ramp
depolarizations were used (Fig. 1).

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Fig. 1.
Block of Ca2+-activated
K+ current
[IK(Ca)]
by charybdotoxin (ChTX). Outward currents were recorded from myocytes
isolated from the circular layer of canine colon. With the use of a
step depolarization protocol from a holding potential (HP) of 50
mV to test potentials of 0 to +100 mV in 10-mV increments, both delayed
rectifier K+ current
(IdK) and
IK(Ca) were
apparent (A and
B, top
traces). Over the same range of test potentials,
IdK largely
inactivated when cells were held at 0 mV for >2 min, leaving
primarily IK(Ca)
(A and
B, middle
traces). Voltage ramps ( 100 to +100 mV) were
performed with a ramp rate (dV/dt) = 0.05 V/s (A and
B, bottom
traces). Compared with control
(A),
IK(Ca) in the
presence of ChTX (100 nM, B) was
reduced by ~75% during voltage ramps. Current reduction was less
(~65%) when elicited by 3-s step depolarizations.
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The outward currents shown in Fig. 1
(A and
B,
top) are composed of delayed
rectifier K+ currents
(IdK) as well
as IK(Ca). The
activation curve of BK channels is shifted toward positive potentials,
allowing current attributable to
IdK to be
partially distinguished from
IK(Ca). To reduce
the amount of IdK
through inactivation (3), cells were held at 0 mV for at least 2 min
before step depolarization to test potentials of 0 to +100 mV. In Fig.
1 (A and
B,
middle), reduced
IdK and total
current are demonstrated, especially at lower test potentials. By
comparing currents elicited from the
50-mV holding potential
with those from the 0-mV holding potential, it can be seen that ChTX
continued to suppress the noisy
IK(Ca), particularly at more positive potentials. The continued presence of
IdK shown in Fig.
1B
(top) suggests that ChTX does not
significantly affect
IdK.
In Fig. 1 (A and
B,
bottom), the degree of ChTX block
depends on the specific protocols used to measure binding. At +80 mV, ChTX (100 nM) blocked 61 ± 1% (n = 5 cells) of current elicited by 3-s step depolarizations. At the same
test potential, following a ramp depolarization at a rate
(dV/dt) of 0.05 V/s, ChTX blocked 73 ± 2% of current elicited by ramps
(n = 6 cells). At this ramp rate, the
degree of ChTX block differed compared with step depolarizations (P < 0.03). However, the
effectiveness of ChTX suppression of currents elicited by voltage ramps
was found to depend significantly on the rate of ramp depolarization.
Figure 2 shows some examples of the degree
of block of whole cell current at different ramp rates. ChTX (100 nM)
was less effective in suppressing current during slow ramps (Fig.
2A) than current during faster ramps
(Fig. 2, B and
C). The degree of block was
proportional to the concentration of ChTX, suggesting that availability
of drug was not a limiting factor (3). These data suggest that ChTX may
be less effective during slow ramps due to voltage-dependent
"unbinding."

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Fig. 2.
ChTX block depends on ramp rate. Reduction of
IK(Ca) by ChTX
was weaker during slow ramps compared with faster ramps (0 mV holding
potentials). At a ramp rate (dV/dt)
of 0.01 V/s (A), current was reduced
by 50% in the presence of ChTX (100 nM). At a rate of 0.05 V/s
(B), current was reduced by 68%,
and at 0.5 V/s (C) current was
reduced by 78%. All current traces were recorded from the same myocyte
isolated from the circular layer.
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To determine if the dependence of ChTX block on ramp rate might be due
to such a mechanism, currents were measured during prolonged
(30-60 s) step depolarizations. Figure
3 shows an example of current traces
recorded from a colonic myocyte during 60-s depolarizations to +80 mV.
Under control conditions (i.e., in the absence of ChTX), current was
sustained at a relatively constant level throughout the 60-s period. In
the presence of 100 nM ChTX, current was initially suppressed by
>80% compared with control. The current amplitude relaxed to 60% at
250 ms following depolarization and then declined to a steady-state
level with little apparent block remaining in this experiment (Fig. 3).
In four experiments, current suppression averaged 68 ± 7% when measured at 250 ms compared with 22 ± 10% once steady
state was achieved (P < 0.01). This current relaxation appeared to be due to a slow unbinding of ChTX during the sustained depolarization. The unbinding resulted in a
current relaxation that was well fit by a single exponential function.
The average time constant of relaxation in seven myocytes isolated from
the circular muscle layer at +80 mV was 8.7 ± 2.1 s. With
the use of the same protocol, a similar time constant for unbinding was
observed in cells isolated from the longitudinal layer (9 s;
n = 2).

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Fig. 3.
ChTX block weakens during prolonged depolarization. ChTX binding was
determined during sustained step depolarizations from a holding
potential of 0 to +80 mV. Compared with control
(A), relaxation of block was
observed in the presence of 100 nM ChTX
(B). When curve fit with a single
exponential function, the time constant of this current relaxation was
8.7 s.
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Model of ChTX block of BK channels.
These findings prompted the development a kinetic model of ChTX block
to test if results were consistent with a simple unbinding mechanism
and to quantitatively describe the dependence of ChTX block on ramp
speed. The model also allows the efficacy of ChTX block to be predicted
under any membrane potential conditions, including in vivo electrical
activity. The gating of BK channels in skeletal muscle cells (myotubes)
has been investigated in great detail by Magleby and co-workers (Refs.
17-20). They found that distributions of open- and closed-time
intervals were best described by the sum of three to four and six to
eight exponential components, respectively, suggesting the existence of
at least three open and six closed states (19). The kinetics of
open-open and closed-closed transitions are rapid compared with ChTX
binding. Therefore, for the present study, we have used a simplified
model (Fig. 4), which considers only one
open-close transition and assumes that ChTX blocks to closed and open
states with the same affinity.

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Fig. 4.
Model of ChTX block of
Ca2+-activated
K+ (BK) channels. C and O
represent the closed (nonconducting) and open states, respectively.
Cb and
Ob similarly correspond to closed
and open bound nonconducting states, where ChTX binds to both open and
closed states with the same affinity. Rate constants
k1,
k 1,
k2, and
k 2 were
determined as described in the text.
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As shown in Fig. 5, the activation time
constant of
IK(Ca) in the
range of +50 to +90 mV was largely independent of voltage. However,
channel activity (open channel probability as a function of
potential) follows a Boltzmann relationship (5).
Therefore, k1 was
set to the average rate at all voltages (30.9 ms
1) and
k
1 was
assigned a voltage dependence according to k
1 = c[1 + e(V1/2
V)/K],
where the voltage at half-maximal activation
(V1/2) is 110 mV and the slope factor (K) is 17 mV
(5). The constant, c, related to the
number of channels per cell, is a simple scale factor that controls the
magnitude of the response and the potential where detectable current
can be observed. To match experimental results,
c was set to 1.0 s
1.

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Fig. 5.
Time constant of BK channel activation is independent of test
potential. Rates of activation during 2-s step depolarizations were
measured from a holding potential of 0 mV to a range of test potentials
(+50 to +90 mV in 10-mV increments) in 11 colonic myocytes. Time
constants ( ) were determined from curve fits of a single exponential
function using pCLAMP software. SE bars represent at least 5 measurements. No significant voltage dependence of was found.
Average time constant of activation (all cells at all potentials) was
30.9 ± 2.2 ms (n = 41 measurements).
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The on-rate
(k2) for ChTX
binding was determined using a "recovery from unbinding" protocol
illustrated in Fig.
6A. In the presence of 100 nM ChTX, cells were initially stepped from a holding potential of 0 to +80 mV for 30 s to assess the total range of block
and to achieve a steady-state "unbound" condition. This was
followed by a recovery period (
t)
at 0 mV ranging from 3 to 120 s. Cells were then returned by step
depolarization to +80 mV when the degree of ChTX block was once again
determined. The difference in current at the beginning of the second
depolarization compared with steady-state current was normalized to the
total range of block in current measured during the initial
depolarization. The profile of recovery from unbinding followed an
exponential decline and is illustrated in Fig.
6B. From these data,
k2 was determined
to be 18.1 s
1.

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Fig. 6.
On-rate of ChTX block at 0 mV. A:
on-rate of ChTX block was estimated using initial step depolarization
(30 s) to +80 mV that was followed by recovery to 0 mV for varying
durations [recovery period
( t) = 3-120 s] and a
second step depolarization to +80 mV.
B: degree of ChTX block (i.e.,
difference current) at the beginning of the second step depolarization
was normalized to difference in current during the initial 30-s step
depolarization. When fitted by a single exponential, time constant for
recovery at 0 mV was 18.1 s.
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Rates of unbinding were measured using 30-s depolarizations over a
range of potentials up to +120 mV. The degree of block was difficult to
curve fit at potentials below +60 mV. However, between +60 and +120 mV
(illustrated in Fig. 7, see also Fig. 3),
results of ChTX unbinding of currents were well fit by single exponential functions. Rates were found to be voltage dependent, ranging from an average of 13 s at +60 mV to 5 s at +120 mV. The voltage dependence of off-rates could be described by a function of the
form k
2 = 0.027eV/60
mV s
1. In
other words, an e-fold change in
off-rate occurred for every 60 mV.

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Fig. 7.
Voltage dependence of unbinding. Colonic myocytes were stepped from a
holding potential of 0 mV to test potentials of +120 mV
(A) and +60 mV
(B) in the presence of 100 nM ChTX.
Rates of unbinding were found to be voltage dependent. Curve fits
(smooth traces) represent rates of 4.9 s at +120 mV and 15.5 s at +60
mV.
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Simulation of ChTX block during step and ramp depolarizations.
With the use of the parameters described above, simulations were
performed over a range of membrane potentials and voltage protocols. An
example of a response to a voltage step from a holding potential of
50 mV to a test potential of +80 mV is shown in Fig.
8. During the 30-s step depolarization, the
model demonstrates a current relaxation, in good agreement with
empirical data shown in Fig. 3. The current relaxation is due to a
decline in the fraction of channels in the blocked (either
closed-blocked or open-blocked) states, and the magnitude of initial
block depends on initial conditions (holding potential, corresponding
in tissues to RMP).

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Fig. 8.
Simulation of responses to a voltage-clamp step. A simulation of
responses to a voltage step from 50 to +80 mV was performed
using numerical techniques. Fraction of channels in the open state was
converted to current using the Goldman-Hodgkin-Katz equation.
Simulation of a 30-s step depolarization mimics the relaxation of ChTX
block observed in myocytes (as illustrated in Fig. 3). This relaxation
is due to a decline in the fraction of channels in the blocked states
and depends on initial conditions (i.e., fraction of channels starting
off in Cb and
Ob states).
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The model was also used to compute the effects of ChTX on current
elicited by voltage ramps (Fig. 9). We
found that (in agreement with experimental data) ChTX block was
significantly dependent on dV/dt. With
the use of slower ramps (Fig. 9A),
ChTX was less effective than during faster ramps (Fig. 9,
B and
C). The decreased effectiveness of ChTX during slow ramps was primarily due to a significant decline in the fraction of channels in the closed-bound (Cb) state during the voltage
ramp (Fig.
10A).
On the basis of simulations, there was also a progressive decline in
the fraction of channels in the closed state and even a decline in the
fraction of channels in the open-bound
(Ob) during the later part of
the slowest voltage ramp (see Fig.
10A). This is due to the flux of channels in transition from the Cb
state through the Ob to the open
state. During more rapid voltage ramps, more channels remain in bound
states (Fig. 10, B and
C).

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Fig. 9.
Simulation of currents in response to voltage ramps. Simulations of
responses to voltage ramps from 100 to +100 mV over a range of
rates from 0.01 to 0.5 V/s were performed using numerical techniques.
Normalized currents in absence (control) and presence of ChTX were
computed using the Goldman-Hodgkin-Katz equation. In agreement with
experimental observations (Fig. 2), ChTX was less effective in
suppressing current elicited by slow ramps
(A, 0.01 V/s) compared with current
elicited by faster ramps (B and
C, 0.05 and 0.5 V/s, respectively).
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Fig. 10.
Simulation of channel activity in response to voltage ramps. Fractions
of channels occupying each state (see model in Fig. 4) in the presence
of 100 nM ChTX under the same ramp protocols as Fig. 9 are shown.
During slow ramps (A, 0.01 V/s), there
is a leveling off and even a decline in the fraction of channels in the
Ob state at positive potentials.
During faster ramps (B and
C, 0.05 and 0.5 V/s, respectively),
more channels remain in blocked states.
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DISCUSSION |
The electrical activities of circular and longitudinal muscle layers of
the canine proximal colon differ significantly (5). The RMP of cells
through the thickness of the circular layer varies as a function of
distance from the submucosal border (RMP of about
80 mV) to the
myenteric layer (RMP of about
40 mV; Ref. 26). Cells near the
submucosal border of the circular layer generate slow waves consisting
of an upstroke to about
25 to
35 mV (rate of rise <1
V/s), a partial repolarization to about
30 to
40 mV, and
a sustained plateau potential that persists for several seconds before
repolarization to the RMP (26, 27). Cells in the longitudinal muscle
layer of the canine colon have a more positive resting potential (about
60 mV) and exhibit low-amplitude oscillations in membrane
potential (myenteric potential oscillations), with individual
Ca2+ action potentials
superimposed (7, 14, 27). These spikelike action potentials (SLAPs;
Ref. 16) have a much more rapid rate of rise (1-4 V/s), reach a
peak at about
10 to +10 mV, and repolarize rapidly with a total
duration <50 ms (29).
Although both tissues express BK channels with identical properties
(i.e., voltage sensitivity, Ca2+
sensitivity, ChTX sensitivity) and similar current density (pA/pF), the
effect of ChTX on these tissues is strikingly different (3). Bath-applied ChTX (100 nM) has no effect on basal (i.e., unstimulated by agonist) electrical slow waves in the circular muscle layer but
increases both spiking and contraction generated by the longitudinal muscle layer (3). Data in the present study suggest that part of this
difference may be due to the type of electrical activity (spiking vs.
nonspiking) as well as RMPs in the two muscle layers.
To measure binding/unbinding rates, it was necessary during patch-clamp
studies to use a positive range of membrane potentials compared with
physiological conditions. During patch-clamp protocols, there may be
additional differences compared with recordings from tissues, including
intracellular Ca2+ concentrations
and the effects of temperature. Despite these differences, it is
evident that unbinding may occur, in which simulations using the model
presented in Fig. 4 suggest that there is >50% unbinding during the
time course of a colonic slow-wave event (average duration of 6.3 s)
compared with <1% unbinding during SLAPs with durations of <50 ms
(see Figs. 3 and 8). These results suggest that experiments using
"slow blockers" (11) such as ChTX or IbTX during spontaneous
electrical activity in intact tissues need to be interpreted with
caution.
In circular muscle, BK channels likely contribute to the process of
slow-wave repolarization (4). However, the relative contribution of BK
channels compared with other voltage-dependent K+ channels and inactivation of
Ca2+ currents remains
controversial (for review, see Ref. 23). Part of the reason for this
controversy is the lack of effect of BK blockers such as ChTX and IbTX
on basal slow-wave activity. The lack of effect argues in favor of
little or no contribution to the repolarization process by BK channels
(e.g., Ref. 15). However, as shown in the present study, substantial
unbinding may occur during a slow wave, since repolarization follows a
prolonged depolarization.
Previous studies (3) have also shown that, although ChTX does not alter
basal slow-wave activity in circular muscle strips, it can alter
responses to excitatory agonists. In the presence of
10
6 M acetylcholine (ACh),
ChTX (100 nM) increased slow-wave duration by 0.6 s and slow-wave
amplitude by 4.4 mV, resulting in a doubling of the force of phasic
contractions. ACh alone causes both membrane depolarization (2) and a
sustained elevation in intracellular Ca2+ concentration
([Ca2+]i)
(25). Both factors might contribute to the apparent increase in ChTX
effectiveness. The Ca2+-channel
opener BAY K 8644 caused an increase in
[Ca2+]i
as well as an RMP of 8.5 mV over control. The further addition of ChTX
(100 nM) caused further depolarization (2.5 mV). Therefore, agonist-induced membrane depolarization as well as changes in [Ca2+]i
may alter the dynamics of ChTX block of BK channels.
Differences in the ability to buffer intracellular
Ca2+ might result in dramatically
different magnitudes and time courses of submembrane
Ca2+ transients and subsequent
activation of BK channels. Under conditions of low-intracellular
Ca2+ buffering (whole cell
voltage-clamp conditions utilizing 0.1 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid in the pipette solution), smooth muscle cells from the
longitudinal layer of canine colon often display spontaneous transient
outward currents (STOCs), whereas cells from the circular muscle layer do not display STOCs (Carl, unpublished observations). It is possible that longitudinal muscle cells reach higher transient submembrane Ca2+ levels than circular cells.
This would result in a larger degree of activation of BK channels in
the longitudinal layer that would result in a larger ChTX sensitivity
of this muscle layer, even if all other channel properties were the
same.
Suarez-Kurtz and colleagues (28) have pointed out that it is difficult
to relate the effects of ChTX and IbTX from one tissue or species to
another. However, some of the variability they reported may be
attributed to the electrical activities in each tissue. Spontaneous
contractions in tissues that were not affected by ChTX (portal vein and
uterus) were prolonged (up to 1 min) compared with tissues stimulated
by ChTX (bladder, taenia coli, and aorta). Prolonged depolarizations
(associated with contractions) may allow time for relaxation of ChTX
block. Guinea pig trachea showed no significant response to ChTX;
however, this tissue showed no spontaneous activity (in the presence of
indomethacin) and may possess an excess of
K+ channels to remain in a
hyperpolarized state.
Voltage ramps are widely used as an experimental protocol to rapidly
assess current-voltage relationships in a variety of cell types (e.g.,
Ref. 6). Ramp rates (dV/dt) are
selected to be slow compared with channel activation kinetics but rapid compared with inactivation to directly observe current-voltage relationships. When characterizing BK channels that do not inactivate (at room temperature), these criteria are easy to meet. On the other
hand, these criteria are difficult or impossible to meet when studying,
for example, IdK
that rapidly inactivate. When applying voltage ramps in the presence of
an agonist or antagonist, in addition to considering activation and
inactivation rates, rates of binding and unbinding must also be
considered (as illustrated in Fig. 2). Because ChTX binding/unbinding
is relatively slow and voltage dependent, responses vary depending on
ramp rate, even when well below the rates of channel opening.
The model presented in Fig. 4 is a simplified view of BK channel
kinetics and ChTX binding. McManus and Magleby (19) have reported that
there are at least three open states and five to six closed states that
contribute to current dynamics. Similar results have been obtained from
channels purified from aortic smooth muscle and reconstituted in planar
lipid bilayers (9). However, the open-to-open and closed-to-closed
transitions occur rapidly and are not apparent over the time frame used
to observe ChTX binding/unbinding kinetics. In our model, ChTX binds
with equal affinity to both the open and closed state of BK channels where BK channels continue to cycle through closed-blocked and open-blocked states. Alternatively, the voltage dependence of ChTX
block may be due to different affinities of ChTX to closed and open
channel states. For example, in BK channels incorporated into planar
lipid bilayers, Anderson and colleagues (1) found an off-rate of ChTX
block independent of channel state but a sevenfold faster on-rate for
the open state compared with the closed state. Our results are not
contradictory to these findings but rather indicate that the
macroscopic behavior of
IK(Ca) is well
described by a simplified model using identical rate constants. The
mechanism of ChTX block involves a simple 1:1 binding near the external mouth of the channel, thereby occluding ion flux through the channel. Although direct evidence for closed-blocked to open-blocked transitions was not given by Anderson et al. (1), these authors speculate that the
channel indeed may cycle through the blocked states on the basis of
identical off-rates. This is analogous with
Ba2+ block of BK channels, and
earlier work had clearly shown such interconversions (21). The voltage
dependence of the off-rate (k
2)
with an e-fold change every 60 mV is
less than that reported by Anderson and colleagues (1), in which they
report an e-fold change in rate every
28 mV. This, in part, may be due to the positive range of potentials
used in our studies in which clear unbinding could be measured. The
simple model (Fig. 4) can be used to generate simulated responses to a
variety of protocols that are in excellent agreement with experimental
responses, as well as to provide a basis to analyze and interpret
experimental results.
In summary, the dependence of ChTX block on the dynamics of membrane
potential changes might be the basis for explaining a wide variety of
apparently contradictory results in the literature: 1) The ability of ChTX to alter
basal electrical activity not only depends on the abundance of BK
channels but also on the RMP and dynamics (e.g., spiking vs.
nonspiking) of electrical events. 2)
Agonist-induced responses may alter the ability of ChTX to bind to BK
channels by modifying membrane potential (e.g., resting potential or
the dynamics of active events) via BK channels or any other conducting
channel present in the membrane. 3)
The apparent discrepancies between ChTX binding using patch-clamp
techniques on isolated cells and channels vs. effects on tissues may
depend on the dynamics of spontaneous electrical activity and specific voltage-clamp protocols. 4) In the
presence of ChTX and other slow blockers, protocols designed to measure
current-voltage relationships (e.g., voltage ramps) must take into
account activation/inactivation kinetics as well as voltage-dependent
binding/unbinding rates. The relatively simple model presented in Fig.
4 provides a useful tool to estimate the efficacy and dynamics of ChTX
binding.
 |
ACKNOWLEDGEMENTS |
We thank Nancy Horowitz for the cell preparations and Drs. J. L. Kenyon
and K. M. Sanders for their continuous advice throughout this study.
 |
FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-32176 and program project grant
DK-41315.
Address reprint requests to N. G. Publicover.
Received 5 September 1997; accepted in final form 21 November
1997.
 |
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