Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Prakriya, Murali and Christopher J. Lingle. BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 µM nifedipine or Q-type Ca2+ channels by 2 µM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 µM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Elevations of cytosolic calcium
([Ca2+]i) mediated by influx through
voltage-dependent Ca2+ channels participate in a multitude
of cellular functions, including exocytosis, excitation-contraction
coupling, synaptic plasticity, ion channel gating, gene expression, and
the growth and death of neurons (Berridge 1998). An
unresolved question is how, if at all, specificity in signaling may
arise when multiple kinds of Ca2+ channels with comparable
activation properties may all contribute to the average elevation of
submembrane [Ca2+] within a cell. One possibility is that
specificity in function of different Ca2+ signals may be
encoded, in part, by the expression of different voltage-gated
Ca2+ channels, each selectively coupled to particular
Ca2+-dependent processes and each functionally optimized to
provide a localized pattern of Ca2+ influx that is
appropriate for a particular Ca2+-dependent process. As one
step in addressing this issue, here we examine whether activation of
large conductance Ca2+- and voltage-dependent
K+ channels (BK channels) in rat chromaffin cells may be
selectively coupled to particular subtypes of Ca2+ channels.
In rat chromaffin cells, 10-20% of BK channels appear to be
sufficiently tightly coupled to Ca2+ channels that the
activation of those BK channels during brief Ca2+ influx
steps is resistant to high concentrations of intracellular EGTA
(Prakriya et al. 1996
). During Ca2+ influx,
these BK channels appear to be driven to high open probabilities with
[Ca2+]i in the vicinity of those BK channels
reaching
60 µM. This coupling of a portion of the BK channels to
Ca2+ channels is essential to allow the BK channels to
participate in the rapid repolarization of membrane potential during
action potentials in the rat chromaffin cells (Solaro et al.
1995
). Because chromaffin cells express multiple subtypes of
Ca2+ channels (Artalejo et al. 1994
;
Gandia et al. 1995
; Hollins and Ikeda
1996
), the observed functional coupling of BK channels to Ca2+ channels raises the question of whether BK current
activation is dependent on Ca2+ influx through a particular
subtype of Ca2+ channel.
We address two main issues. First, we confirm the identities of the
various Ca2+ channel subtypes found in rat chromaffin cells
(Gandia et al. 1995; Hollins and Ikeda
1996
). Second, we examine their contribution in driving
activation of BK channels. The results indicate that, of at least four
classes of Ca2+ channels expressed in rat chromaffin cells,
L-, N-, P-, and Q-type channels, BK current activation during brief
Ca2+ influx steps characteristic of action potentials is
coupled to Ca2+ influx predominantly through the L- and,
when present, the Q-type channels. Because activation of BK current is
important for the rapid termination of action potentials in rat
chromaffin cells (Solaro et al. 1995
), these results
suggest that selective modulation of L- and Q-type Ca2+
channels by second messengers may affect the extent of BK current activation and hence cellular excitability.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chromaffin cell culture
Adrenal glands were removed from Sprague-Dawley rats and
enzymatically digested with an enzyme cocktail containing 3%
collagenase, 2.4% hyaluronidase, and 0.2% DNAse as described earlier
(Neely and Lingle 1992). Chromaffin cells were plated on
collagen-coated dishes and maintained at 37°C and 5% CO2
in a medium composed of DMEM supplemented with 10% fetal bovine serum
and 25 µl/ml ascorbic acid.
Electrophysiological methods
Whole cell recordings were performed at room temperature on
cells 2-14 days after plating. In perforated patch-clamped cells, amphotericin was employed as the permeabilizing agent
(Herrington et al. 1995). Whole cell currents
were recorded with an Axopatch-1C amplifier (Axon Instruments; Foster
City, CA) with a 500-M
feedback resistor. Whole cell voltage clamp
was controlled with the Clampex program in the pClamp software package
(Axon Instruments; Foster City, CA). Currents were obtained at a
digitization rate of 2 kHz. Previous experiments (Prakriya et
al. 1996
) indicated that changing the sampling rate from 2 to
20 kHz produces only small effects on the estimates of the
instantaneous BK current following the voltage step to +81 mV.
In some experiments involving Ca2+ channels, run-down of
the Ca2+ current was noted. To prevent errors arising from
run-down, only those cells were selected that satisfied the criteria
that 1) Ca2+ currents exhibited stable
responses during the control application of the Ba2+
solution, 2) the response to toxins displayed both a
clear onset of block and an approximate exponential convergence toward
a stable current level, and 3) recovery of response to
reversible toxins such as nifedipine or brief applications of -CnTx
MVIIC was complete.
Solutions
The standard extracellular solution contained (in mM) 140 NaCl, 5.4 KCl, 10 HEPES, 1.8 CaCl2, and 2.0 MgCl2 titrated to pH 7.4 with N-methylglucamine (NMG). In some experiments CaCl2 was excluded from the standard extracellular solution. The divalent concentration was kept constant by including 3.8 mM MgCl2 in this solution; 200 nM apamin was included in all experiments to block SK channels. For perforated patch-clamp experiments where both BK and Ba2+ currents were recorded from the same cell, the pipette saline contained (in mM) 120 K-aspartate, 30 KCl, 10 HEPES (H+), and 2 MgCl2 adjusted to pH 7.4 with NMG. To isolate IBa, the following extracellular solution was used: 10 BaCl2, 40 tetraethylammonium (TEA) chloride, 10 HEPES, and 90 NaCl with pH adjusted to 7.4 with NaOH. In experiments where only the Ba2+ currents were recorded, the composition of the internal saline was (in mM) 110 Cs-methane sulfonate, 14 phosphocreatine, 10 HEPES, 9 EGTA, 5 Mg-ATP, and 0.3 Tris-GTP. The pH was adjusted to 7.3 with CsOH. Osmolarity was measured by dew point (Wescor Osmometer) and adjusted between 290 and 310. Extracellular solution changes and drug applications were accomplished via a multibarrel perfusion system.
Drugs
Stock solutions of nifedipine (Sigma; St. Louis, MO) and Bay K
8644 (Bay K, Sigma) were prepared in ethanol at concentrations of 10 and 5 mM, respectively. -Conotoxin MVIIC (CnTx MVIIC),
-conotoxin
GVIA (CgTx GVIA) (both from RBI; Natick, MA),
-Agatoxin IVA (Aga
IVA, gift of Pfizer), and
-Agatoxin TK (Aga TK, Peptide Institute;
Japan) were dissolved in dH2O at stock solutions of 500, 500, 100, and 100 µM, respectively. Final concentrations are given in
the legends. In some experiments, L-type Ca2+ current was
isolated by blocking P-, N-, and/or Q-type Ca2+ currents by
preincubating chromaffin cells with a cocktail containing
-Aga IVA
(100 nM),
-CgTx GVIA (2 µM), and
-CnTx MVIIC (2 µM) for
15
min before electrophysiological recordings. P-type Ca2+
current and/or N-type Ca2+ was also preblocked in some
experiments by exposure to
-Aga IVA (100 nM) and/or
-CgTx GVIA (2 µM) for
15 ins before recording.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium current in rat chromaffin cells
Biophysical and pharmacological strategies were used to identify
the various Ca2+ currents expressed in cultured rat
chromaffin cells. Voltage-activated Ca2+ currents can be
broadly classified into high-voltage-activated (HVA) and
low-voltage-activated (LVA) currents De Waard et al. 1996). LVA currents display rapid voltage-dependent
inactivation, require relatively mild voltage stimuli for activation,
and completely inactivate at depolarized holding potentials in the
range of
60 to
50 mV. HVA currents in contrast lack rapid
voltage-dependent inactivation and require more positive potentials for
activation (Artalejo et al. 1991
; Scroggs and Fox
1992
). Therefore the presence of LVA current can be detected by
comparing the I-V relationship at negative
holding potentials to that at more depolarized holding potentials
(Artalejo et al. 1991
; Scroggs and Fox
1992
). Here Ca2+ current I-V
relationships were obtained at two holding potentials,
85 and
50
mV, and the difference current at each depolarizing step was
determined. A typical Ca2+ current
I-V relationship obtained at holding potential of
60 mV in a rat chromaffin cell is shown in Fig.
1A. In 10 mM Ba2+,
current activation occurred at voltages positive to
40 mV and peaked
around 0 mV. The I-V relationship was very
similar in 10 mM Ca2+ except for a slight (~5 mV) shift
to the right of the peak of the response (n = 4 cells).
As shown in Fig. 1B, when the holding potential was changed
from
50 to
85 mV, the I-V relationship was
unaltered at the more negative potentials, indicating minimal presence
of LVA current in these cells (6/6 cells). Thus calcium current in the
rat chromaffin cells studied here is predominantly of the HVA type.
|
HVA Ca2+ current can arise from several subtypes of
Ca2+ channels, including L-, N-, P-, Q-, and R-type
channels (De Waard et al. 1996). These can be
distinguished from one another based on differential selectivity to
various inhibitors. L-type channels are sensitive to dihydropyridines
(DHPs), N-type channels are irreversibly blocked by the snail toxin
-CgTx GVIA, and P-type channels are blocked by low concentrations of
-Aga IVA (for review see De Waard et al. 1996
).
Q-type channels can be blocked by high concentrations of
-Aga IVA or
by the snail toxin
-CnTx MVIIC (McDonough et al.
1996
; Randall and Tsien 1995
).
Figure 2A illustrates the
effects of sequential application of -CgTx GVIA,
-Aga IVA, and
nifedipine on the Ba2+ current (IBa)
evoked at 0 mV.
-CgTx GVIA (1-2 µM) and
-Aga IVA (0.1 µM)
produced irreversible inhibition of some IBa in
most cells, indicative of blockade of N- and P-type Ca2+
channels, respectively. Nifedipine (5 µM) produced a rapid (<20 s),
reversible block of IBa in all cells, indicating
the presence of L-type Ca2+ channels. There was substantial
variability in the extent of block produced by each drug. The mean
irreversible block produced by 1-2 µM
-CgTx GVIA applied for
90
s was 24 ± 3% (SE; n = 16 cells).
-Aga IVA
(0.1 µM) elicited only minor effects; the mean reduction of
IBa by 0.1 µM
-Aga IVA applied for
120 s
was only 12 ± 3% (n = 5 cells). Nifedipine
showed the most prominent reduction of IBa, with
a mean percentage block of 33 ± 3% (n = 13 cells). A substantial fraction (30-40%) of the total
IBa was unaffected by the combined actions of
nifedipine,
-CgTx GVIA, and 100 nM
-Aga IVA, indicating the
presence of other HVA currents.
|
-CnTx MVIIC is a calcium channel antagonist that blocks P-, Q-, and
N-type calcium currents (McDonough et al. 1996
;
Randall and Tsien 1995
). In CNS neurons, the onset of
block of P- and Q-type current by
-CnTx MVIIC is slow and only
slowly reversible, whereas block of N-type current is fast and quickly
reversible (McDonough et al. 1996
). Figure 2B
illustrates the effects of 2 µM
-CnTx MVIIC on
IBa elicited at 0 mV in 6 mM Ba2+.
Effects of
-CnTx MVIIC were evaluated in 6 mM Ba2+
rather than the 10 mM Ba2+ used in previous experiments
because of the observation that higher concentrations of
Ba2+ result in slower block of the P- or Q-type
Ca2+ current by this toxin (McDonough et al.
1996
). Two kinetically distinct components of blockade by
-CnTx MVIIC could be detected, 1) a fast component (mean
amplitude = 20 ± 3%; n = 14 cells) that was
mostly complete within 40 s and was fully reversible and
2) a very slow component (mean = 27 ± 4%;
n = 9 cells) that was largely irreversible over the
time course of a typical experiment (Fig. 2B). When
-CgTx
GVIA was applied, the fast reversible effect of
-CnTx MVIIC was
consistently eliminated (6/6 cells), which suggested that it was
mediated by N-type calcium channels. The slow component of
-CnTx
MVIIC block was similar to the block of P- and Q-type Ca2+
channels reported by McDonough et al. (1996)
. To examine if this arose
solely from P-type Ca2+ channels or whether Q-type channels
were also present, we next tested the efficacy of
-CnTx MVIIC after
preblocking N-type current by 1 µM
-CgTx GVIA and P-type current
by 100 nM
-Aga IVA (Fig. 2C). Under these conditions, in
10 of 15 cells examined,
-CnTx MVIIC produced an additional slowly
developing block of some current, consistent with the presence of
Q-type Ca2+ channels.
The presence of Q-type channels was also confirmed by -Aga IVA. This
toxin blocks both P- and Q-type Ca2+ channels, although the
relative affinities are widely different; P-type channels are maximally
blocked by 100 nM
-Aga IVA, whereas Q-type Ca2+ channels
are blocked only at micromolar concentrations of
-Aga IVA
(Randall and Tsien 1995
). Chromaffin cells were
preincubated in 100 nM
-Aga IVA for 15 min before
electrophysiological recording, and the effect of 2 µM
-Aga IVA
was examined on the remaining Ba2+ current. In 8 of 11 cells tested, 2 µM
-Aga IVA elicited a substantial block of the
remaining IBa, indicating the presence of Q-type Ca2+ channels in these cells (Fig. 2D).
In some cells, -CgTx GVIA produced reversible block of a component
of IBa (data not shown). The amplitude of this
current could be substantially reduced by simultaneously applying
nifedipine, suggesting that this was arising from L-type channels
sensitive to
-CgTx GVIA. We did not further characterize these
currents. However, overlap between
-CgTx GVIA and DHP inhibitors of
L-type Ca2+ channels was reported in some cells
(Kasai and Neher 1992
), and it was suggested that the
Ca2+ channels underlying this current may be L-type
channels arising from the
1D pore-forming subunit
(Williams et al. 1992
). A more recent study, however,
indicates that the L-type Ca2+ channels in sympathetic
neurons arising from the
1D subunit are not
-CgTx
GVIA sensitive (Lin et al. 1996
). Thus the identity of
this current remains to be elucidated.
In sum, the previous experiments demonstrate that rat chromaffin cells
express four major classes of HVA Ca2+ channels: L-, N-,
P-, and Q-type channels. The average contributions of each type are
summarized in Fig. 6. We also often observed a small residual
Ca2+ current even in the combined presence of all the
calcium antagonists mentioned previously (not shown). This current may
be similar to the R-type current reported in previous studies that is
insensitive to all the known Ca2+ channel toxins (De
Waard et al. 1996).
BK current in rat chromaffin cells
Rat chromaffin cells express inactivating (BKi) or
noninactivating (BKs) variants of BK current. Approximately
75% of the chromaffin cells express predominantly BKi
current, and the rest express either noninactivating or a mixture of
inactivating and noninactivating BK current (Ding et al.
1998; Solaro et al. 1995
). All the data shown in
this paper are from cells displaying BKi currents, although
we observed no difference between the two cell types in the nature of
BK-Ca2+ channel coupling.
Depolarizing steps that elicit Ca2+ influx through
Ca2+ channels can robustly activate BK current in rat
chromaffin cells (Solaro et al. 1995). Here BK current
activation was studied in perforated patch-clamped cells by stepping
the voltage to a potential that activates Ca2+ current
followed by a test pulse to +81 mV where BK current activation was
evaluated. Because minimal Ca2+ influx occurs at +81 mV,
the effects of conditioning steps that activate Ca2+ influx
can be easily distinguished from those that do not produce influx.
Figure 3A shows the effect of
stepping the voltage directly to +81 mV in the presence and absence of
1.8 mM [Ca2+]ext. Direct steps to +81 mV
would be expected to only activate Ca2+-independent
voltage-activated K+ currents (Solaro et al.
1995
). Consistent with this, the current activated by a direct
step to +81 mV in 1.8 mM [Ca2+]ext is
identical to that elicited in 0 [Ca2+]ext. In
contrast, a 5-ms step to 0 mV before the +81 mV test pulse produces
Ca2+ influx through voltage-gated Ca2+ channels
and results in substantial BK current activation at +81 mV (Fig.
3B).
|
A closer examination of the BK current at +81 mV reveals two current
components. First, there is an instantaneous current, which is the
ohmic increase in current resulting from the increased driving force on
K+ ions passing through BK channels open at 0 mV (Fig.
3B, arrow) (for additional information about the
instantaneous current see Prakriya et al. 1996). Second,
there is a slower time-dependent increase in current that results from
the activation of additional BK channels caused by the increased open
probability of BK channels at +81 mV. The amplitudes of both these
components of BK current increase as the duration of the influx step is
increased (Fig. 3C). High concentrations of EGTA
differentially affect these two components of BK current; the
instantaneous current is unaffected by EGTA up to concentrations of 5 mM, whereas the slow component of BK current is eliminated by EGTA
(Prakriya et al. 1996
). These effects were interpreted
to suggest the presence of two populations of BK channels at differing
distances from Ca2+ channels, the instantaneous current
possibly reflecting BK channels in close association with
Ca2+ channels and exposed to high
[Ca2+]i and the more slowly activating BK
current reflecting channels at greater distances from sites of
Ca2+ influx and exposed to the much lower bulk
[Ca2+]i. In this study, the contribution of
influx through various subtypes of Ca2+ channels found in
chromaffin cells to the activation of BK channels by brief influx steps
is examined. Ca2+ influx steps were confined to brief
duration (5 ms) because brief steps will favor elevation of the
[Ca2+]i in the immediate vicinity of the
active Ca2+ channels while less significantly altering the
global [Ca2+]i. Thus BK channels activated by
brief Ca2+ influx steps are more likely to be driven by
Ca2+ elevations in the vicinity of Ca2+ channels.
Selective blockade of L- but not N-type Ca2+ current affects BK current activation
The role of the various subtypes of Ca2+ channels
toward BK current activation was next examined with the previous
Ca2+ channel inhibitors. Figure
4 shows an example of the effects of 5 µM nifedipine and 2 µM -CnTx MVIIC on BK currents and
Ba2+ currents activated by 5-ms Ca2+ influx
steps. BK current was recorded in the standard external solution
containing 1.8 mM [Ca2+], and Ba2+ current
was recorded from the same cell by changing the standard external
solution to one containing 10 mM Ba2+. Nifedipine caused a
very substantial reduction in the amount of BK current activated by
calcium influx. In contrast, applications of
-CnTx MVIIC lasting
40-50 s resulted in only minor effects on BK current activation (Fig.
4). The average nifedipine-induced reduction in the amplitude of
instantaneous BK current activated by calcium influx was 77 ± 2% (mean ± SE; n = 15 cells), whereas the
nifedipine-sensitive Ba2+ current comprised 33 ± 3%
of the total IBa. In contrast,
-CnTx MVIIC reduced BK current activation by only 11 ± 3%
(n = 9 cells), although the
-CnTx
MVIIC-sensitive Ba2+ current in these cells comprised
21 ± 3% of the total IBa. Even in
cells with comparable amplitudes of nifedipine and CnTx MVIIC-sensitive Ba2+ currents, blockade of L-type Ca2+ channels
by nifedipine evoked dramatically larger reductions in BK current than
blockade of N-type Ca2+ channels by CnTx MVIIC. Thus these
results suggest that BK current activation during brief
Ca2+ influx steps primarily occurs via influx through L-
but not N-type Ca2+ channels.
|
We also determined the effects of the irreversible N-type blocker
-CgTx-GVIA.
-CgTx-GVIA (1-2 µM) inhibited BK current by 20 ± 4% (n = 15 cells) and
IBa by 24 ± 3% (n = 16 cells). These effects on BK current are larger than those elicited
by block of N-type channels by
-CnTx MVIIC. However, as pointed out
earlier,
-CgTx GVIA produced both irreversible and reversible
effects on IBa in some cells, and much of
the reversible effect could be blocked by nifedipine. Thus the larger
effect of
-CgTx GVIA on BK current relative to
-CnTx MVIIC may
result from the reversible block by
-CgTx GVIA of a rarely
occurring, novel Ca2+ channel variant that is susceptible
to nifedipine blockade.
In separate experiments, any direct effects of Ca2+ channel inhibitors on BK current were assessed by introducing 10 µM [Ca2+]i into cells via the patch pipette to directly activate BK current. These experiments did not indicate any direct effects of the inhibitors on BK channels.
Blockade of Q- but not P-type Ca2+ channels affects BK current activation
The contribution of P-type channels toward BK current activation
during calcium influx was evaluated with 100 nM -Aga IVA. Measurements of
-Aga IVA-mediated inhibition of BK current
activation and of the block of IBa were
determined in different cells because of the irreversible nature of
-Aga IVA block. Application of 100 nM
-Aga IVA produced a small
decrease in BK current activation in many cells (7/11 cells; no effect
in 4 cells; Fig. 5A). The mean
reductions in BK current and IBa were 14 ± 5% (n = 7 cells) and 12 ± 2% (n = 8 cells), respectively. These figures might be overestimates of the
true contribution of P-type current because 100 nM Aga IVA would be
expected to block Q-type Ca2+ current also. Thus these
results indicate that the contribution of P-type Ca2+
channels to BK current activation is minor.
|
In contrast, applications of 1.5-2 µM -Aga IVA produced
substantially greater effects on both IBa and on
BK current activation (Fig. 5B). After preblocking P-type
Ca2+ channels by incubating the cells in 100 nM
-Aga IVA
for 15 min before the beginning of a recording, application of 2 µM
-Aga IVA resulted in a mean reduction in BK current of 42 ± 4% (4/6 cells, no detectable effect in 2 cells). When
IBa was examined, 1-2 µM
-Aga IVA produced
a mean block of 23 ± 4% (8/11 cells, no effect in 3 cells) after
preblock with 100 nM
-Aga IVA. It is unlikely that these effects of
-Aga IVA result from a nonselective blockade of some L-type
Ca2+ channels because several cells with substantial L-type
currents were totally insensitive to 1-2 µM
-Aga IVA. The true
fraction of Q-type Ca2+ current and its contribution to BK
current activation might be somewhat higher than these numbers indicate
because preincubation with 100 nM
-Aga IVA may by itself produce
some block of Q-type Ca2+ current. Regardless of the exact
amount of Q-current the results argue that, when Q-type channels are
present, Ca2+ influx through these channels participates in
initiating activation of BK channels.
Figure 6 summarizes the effects of all
the Ca2+ channel antagonists used here on
IBa and BK currents. Selective blockade of L-type Ca2+ channels had the greatest effect on BK current
activation, whereas blockade of N- and P-type Ca2+ channels
caused only minor effects. Blockade of Q-type Ca2+ channels
by micromolar concentrations of -Aga IVA also reduced BK current
activation significantly.
|
Blockade of L- and Q-type Ca2+ channels inhibits BK current activation during action potentials
We next examined the role of Ca2+ influx during action potential waveforms on BK current activation in perforated patch-clamped chromaffin cells. Currents were activated by an action potential voltage-clamp waveform that was generated by digitizing a chromaffin cell action potential recorded in the current-clamp mode. The ability of the previous Ca2+ channel blockers to inhibit the action potential-induced BK current was estimated by comparing K+ currents obtained in 0 [Ca2+]ext, which should block all the BK current induced by the action potential. Figure 7 shows typical examples of the effects of removing [Ca2+]ext and of the various Ca2+ channel blockers in perforated patch-clamped cells. In the majority of cells, a prominent shoulder or "hump" in the outward K+ current during the falling phase of the action potential is observed. When the external solution is replaced with a 0 [Ca2+]ext solution, the hump disappears, indicating that it arises because of the activation of BK current driven by Ca2+ influx during the action potential. In addition, the 0 [Ca2+]ext solution also reduces the peak outward K+ current in many cells, suggesting that BK current is also activated to some extent during the rising phase of the action potential.
|
Analogous to results obtained from BK current measurements by 5-ms
influx steps, block of L-type Ca2+ channels by nifedipine
significantly reduced BK current activation during action potentials
(Fig. 7A). In all cells (7/7), the peak outward
K+ current was reduced, and the prominent hump in the
K+ current was completely eliminated by nifedipine. In
fact, the outward K+ current profile was very similar to
that elicited in 0 [Ca2+]ext, indicating that
nearly all BK current activation is prevented by the blockade of L-type
Ca2+ channels during the action potential. In contrast, the
same experiment done with transient (40-50 s) applications of -CnTx
MVIIC to block N-type Ca2+ channels showed little or no
effect in all cases (Fig. 7B). In these cells, the outward
K+ currents were virtually identical to the control
K+ currents activated in the normal
[Ca2+]ext solution. In four of the six cells
that were examined in this way, the amplitude of the N-type
Ca2+ current was then determined by 40-s applications of 2 µM
-CnTx MVIIC with 10 mM Ba2+ as the charge carrier.
The mean amplitude of the
-CnTx MVIIC-sensitive IBa was found to be 24% of the total
IBa in these cells. Thus the lack of an effect
by
-CnTx MVIIC on BK current activation in these cells was not
because of the lack of
-CnTx MVIIC-sensitive Ca2+ channels.
The role of P- and Q-type Ca2+ channels toward BK current
activation during action potentials was assayed by blocking these Ca2+ channels with Aga TK. Analogous to Aga IVA, low
concentrations of Aga TK selectively block P-type Ca2+
currents, whereas higher concentrations have been also shown to block
Q-type Ca2+ currents (Teramoto et al.
1995). We used 100 nM and 2 µM Aga TK to selectively block P-
and Q-type Ca2+ currents, respectively (Fig. 7,
C and D). Aga TK (100 nM) had little or no effect
on the outward K+ current in all cells tested (5/5 cells),
indicating that P-type Ca2+ currents do not contribute to
BK current activation during action potentials. In contrast, 2 µM Aga
TK elicited a substantial inhibition of the outward K+
current in four of five cells tested (Fig. 7D).
Collectively, these data are consistent with results obtained by 5-ms Ca2+ influx steps and show that blockade of L- and Q-type Ca2+ channels results in significant inhibition of BK current activation during action potentials, whereas selective blockade of N- and P-type Ca2+ channels produces little or no effect.
Bay K increases the BK current activated by calcium influx
The effects of the DHP agonist Bay K 8644 (Bay K), which is known
to increase the amplitude of the ensemble Ca2+ current by
favoring long-duration openings of L-type Ca2+ channels
(Kokubun and Reuter 1984; Nowycky et al.
1985
), were next examined on the depolarization-evoked
Ca2+ and BK currents.
Bay K elicited a significant increase in the amplitude of the peak
Ca2+ current (peak IBa increased
~143% of control; n = 10 cells). To examine if this
is a consequence of an increase in the number of L-type
Ca2+ channels or an increase in the
Popen of L-type Ca2+ channels, we
used noise analysis (Sigworth 1980) to estimate the
number of Ca2+ channels and their average
Popen (Fig. 8). In
these experiments, the contribution of non-L-type Ca2+
channels to the Ba2+ current was minimized by preincubating
chromaffin cells for
15 min with a cocktail of 100 nM
-Aga IVA, 1 µM
-CnTx GVIA, and 1 µM
-CnTx MVIIC. As shown in Table
1, the primary effect of Bay K was to
increase the peak mean current with no obvious effects on average
single-channel conductance or channel number. Thus this result argues
that the increase in peak Ca2+ current occurs mainly by an
increase in the Popen of L-type Ca2+
channels.
|
|
The consequences of the Bay K-mediated increase in L-type Ca2+ current on the BK current activated by brief influx steps were also examined. Bay K produced very large increases in the amplitude of BK current activated by brief Ca2+ influx steps (Fig. 9A). Although the maximal peak BK current activated by long-duration influx steps was the same in the presence or absence of Bay K (Fig. 9B), the instantaneous BK current activated by brief influx steps increased 2.2-fold on average (n = 5 cells). In these cells, IBa increased 1.38-fold on Bay K treatment. Thus this result indicates that, when L-type Ca2+ channel open probability is increased, the fraction of BK channels activated by Ca2+ influx during brief Ca2+ influx steps also increases significantly.
|
We estimated the fraction of BK channels that can be activated by brief
influx steps in the presence of Bay K by comparing the instantaneous BK
current activated by 5-ms Ca2+ influx steps in the
presence of Bay K with the total BK current present in the cell. For
each cell, total current was estimated by measuring the peak BK current
activated by 50 µM muscarine at +81 mV, which activates nearly all
the BK channels in the cell (Prakriya et al. 1996). This
procedure indicated that Bay K increased the fraction of BK channels
activated by brief influx steps twofold, from 17.4 ± 3% to
36 ± 5% (n = 5 cells). Thus
30-40% of the BK channels in rat chromaffin cells are positioned sufficiently close to
L-type Ca2+ channels so as to be influenced by
Ca2+ influx through these Ca2+ channels during
brief depolarizing steps.
Ca2+ influx through L- and Q-type Ca2+ channels is more effective than Ca2+ influx through other Ca2+ channels in driving BK current activation
The previous pharmacological experiments argue that BK channels are functionally associated with L- and Q-type but not the N- and P-type Ca2+ channels. How do these results compare with manipulations that do not discriminate among the various types of calcium channels but affect all of them? We examined this issue by lowering or raising [Ca2+]ext to determine the dependence of BK and Ca2+ current activation on the [Ca2+]ext (Fig. 10A). BK current at various [Ca2+]ext was then plotted against the Ca2+ current amplitude at the respective [Ca2+]ext (Fig. 10B). Note the sigmoidicity in the activation plot in Fig. 10B. This may represent the cooperative activation of BK channels by Ca2+.
|
We found that, although -CnTx MVIIC,
-CgTx-GVIA, and 100 nM
-Aga IVA produced measurable reductions in BK current activation, these effects were not obviously different from those obtained by simply lowering the [Ca2+]ext to produce
an equivalent reduction in the overall Ca2+ current.
However, the selective blockade of L-type Ca2+ channels by
nifedipine or Q-type Ca2+ channels by 1.5-2 µM
-Aga
IVA evoked reductions in BK current activation that were significantly
greater than what could be produced by simply lowering the
[Ca2+]ext to produce an equivalent reduction
in the overall Ca2+ current. Similarly, enhancing the
L-type Ca2+ current by Bay K produced an increase in BK
current amplitude that was far in excess of the increase that could be
produced by increasing the [Ca2+]ext to evoke
a similar overall Ca2+ current. Thus the larger role of
L-type Ca2+ current is not simply because this current
comprises the largest Ca2+ current component. Instead BK
current is more effectively activated when Ca2+ influx
occurs through L- and Q-type Ca2+ channels than when it
occurs through N- or P-type Ca2+ channels.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multiple types of Ca2+ channels are frequently expressed in the same cellular compartment, and all contribute to the elevation of the average [Ca2+]i. Under such conditions, an unresolved question is whether there is specificity in Ca2+ signaling triggered by influx through the various subtypes of Ca2+ channels. We addressed this question with the Ca2+- and voltage-dependent BK-type channel on rat adrenal chromaffin cells and asked whether BK channels are specifically coupled to a particular Ca2+ channel subtype. Our results indicate, in fact, that BK channels appear to be preferentially activated by Ca2+ influx through L- and Q-type Ca2+ channels.
We assumed that the proportion of Ca2+ influx that occurs in the solutions used to record BK current (with 2 mM [Ca2+]) is comparable with the Ca2+ current amplitudes we measured with 10 mM Ba2+. In trying to relate the amount of influx that would occur in 2 mM [Ca2+]ext from the measured influx amounts in 10 mM [Ba2+]ext, two factors must be considered, 1) the increased permeability of HVA Ca2+ channels to Ba2+ over Ca2+ and 2) the saturable increase in the conductance of HVA Ca2+ channels as the divalent concentration is increased. If these effects occur in roughly the same proportion in all the HVA Ca2+ channel subtypes, the proportion of influx occurring at 10 mM [Ba2+]ext through a channel subtype can be assumed to be the same as that occurring in 2 mM [Ca2+]ext.
Examination of the Ba2+-Ca2+ permeability
ratios of native L- and N-type channels from published studies show
that these two currents do not differ significantly in their relative
ability to conduct Ca2+ and Ba2+. The
permeability ratio is roughly two- to threefold
(Ba2+-Ca2+) in these channels (Fox et
al. 1987; Kasai and Neher 1992
; Smith et
al. 1993
). Moreover, the conductance dependence of native L- and N-type Ca2+ channels on the [divalent]ext
at subsaturating concentrations appear to be comparable and show a
roughly twofold increase as the divalent concentration is increased
from 2 to 10 mM (McNaughton and Randall 1997
;
Smith et al. 1993
; Zhou and Jones 1995
).
Detailed information on permeation and the divalent concentration
dependence of the current is unavailable for the P-, Q-, and R-type
Ca2+ channels. However, the
Ba2+-Ca2+ ratio of the whole cell P-type
current in Purkinje cells was reported to be ~2 (Regan
1991
). Further, Bourinet et al. (1996)
find that the
Ba2+-Ca2+ conductance ratios of the
cloned
1A,
1B, and
1C channels, which may form the
pore-forming subunits of the P-Q-, N-, and L-type Ca2+
channels (De Waards et al. 1996
), are ~1.5, 1.5, and
2, respectively. Their study also showed that the
[Ca2+]ext dependence of these channels is
more or less similar. Collectively, these observations suggest that the
relative amounts of influx through the L-, N-, P-, and Q-type
Ca2+ channels may be similar in 2 mM
[Ca2+]ext to that measured in 10 mM
[Ba2+]ext. Although these are indirect
arguments, there is no observation that requires us to reject this assumption.
Because blockade of L-type Ca2+ channels had the greatest effect on BK current activation, we also directly examined the fraction of Ca2+ current blocked by 5 µM nifedipine in 2 mM [Ca2+]ext. In two cells, ~30% of the Ca2+ current in 2 mM [Ca2+]ext was inhibited by nifedipine, which is virtually identical to the fraction of the DHP current in 10 mM Ba2+ (Fig. 6). On the basis of the previous arguments, this result suggests that the proportion of the remaining Ca2+ currents in 2 mM [Ca2+]ext may also be comparable with our measurements in 10 mM [Ba2+]ext. However, irrespective of the precise proportion of influx contributed by the different Ca2+ channels in 2 mM [Ca2+], it does not affect the central conclusion that BK current activation is affected more significantly by the blockade of L- and Q-type Ca2+ channels rather than N- and P-type Ca2+ channels.
Ca2+ currents in rat chromaffin cells
Our observations indicate that rat chromaffin cells express L-,
N-, P-, and Q-type Ca2+ channels. The presence of each of
these currents is generally consistent with previous reports
(Gandia et al. 1995; Hollins and Ikeda
1996
). L-type current was the largest component
(~33%) of total Ca2+ current, whereas N-type current
comprised ~20-25%. Q-type current was found in ~70% of rat
chromaffin cells, consistent with Gandia et al. (1995)
, but differing
from Hollins and Ikeda (1996)
. P-type current appears to be a minor
contributor to total ICa in rat chromaffin cells.
We obtained no evidence of LVA current, contrasting with Hollins and
Ikeda (1996), who found T-type current in ~25% of the cells they
examined. It is possible that we did not sample enough cells to
determine whether T-type currents are sometimes present. Alternatively,
the Ca2+ currents present in the different strains of rats
used in the two studies may differ.
Activation of BK current by L- and Q-type Ca2+ channels
Our results indicate that, although rat chromaffin cells express
multiple types of HVA Ca2+ channels, Ca2+
influx through L- and Q-type channels dominates the activation of BK
current. Although the precise physical mechanisms that mediate the
specificity of this functional coupling will have to await future
studies, in principle, it could arise if L- and Q-type Ca2+
channels are located much closer to BK channels than N- or P-type Ca2+ channels. Indeed the preferential targeting of ion
channels to particular cellular locations was described in many cell
types, and the underlying mechanisms may involve, at least in part,
selective protein-protein interactions between ion channels and
certain cytoskeletal proteins (Sheng and Wyszynski
1997). Analogous mechanisms may be at work in implementing the
functional specificity that we observe between the BK and
Ca2+ channels in rat chromaffin cells.
Selective functional association of Ca2+-activated
K+ channels with particular Ca2+ channel
subtypes was described in several types of neurons, and the specificity
of the coupling appears to differ among different cell types. For
example, in chick ciliary ganglion neurons, BK current activation has
been shown to occur via Ca2+ influx through a L-type
Ca2+ current but not the N-type Ca2+ current
(Wisgirda and Dryer 1994). In contrast, in chick
sympathetic ganglion neurons, the N-type current appears to dominate
activation of BK current (Wisgirda and Dryer 1994
). In
rat superior cervical ganglion neurons, Ca2+ influx through
L-type channels preferentially activates a BK-type current, whereas
influx through N-type Ca2+ channels activates an SK-type
current (Davies et al. 1996
). In hippocampal neurons, BK
channels are selectively coupled to N-type Ca2+ channels,
whereas SK channels are selectively coupled to L-type Ca2+
channels (Marrion and Tavalin 1998
). Finally, in rat
dorsal motor vagus nucleus neurons, N-type Ca2+ channels
are responsible for SK current activation, whereas BK current
activation appears to be dependent on Ca2+ influx through
-CgTx GVIA- and nifedipine-insensitive Ca2+ channels
(Sah 1995
).
In neurons, the complement of Ca2+ channels found in the
various cellular compartments can be often markedly different. For example, N- and P-type Ca2+ channels are primarily
localized to the dendrites and synaptic terminals (Westenbroek
et al. 1992), whereas L channels are primarily localized to the
soma (Hell et al. 1993
). Similar differences in
Ca2+ channel distributions occur in hippocampal neurons, in
neurons of the cerebral cortex, and in the olfactory bulb neurons
(Bischofberger and Schild 1995
; Hell et al.
1993
; Westenbroek et al. 1992
). A consequence of
this markedly different channel localization is that functional
specificity in the association between Ca2+ and
Ca2+-activated K+ channels may result, at least
in part, simply because of segregation of channels to different
membrane compartments. In contrast, rat chromaffin cells are spherical
cells without complex morphological features such as dendrites or
axons. BK channels can be detected in virtually all patches pulled from
chromaffin cells, suggesting that these channels are present, at least,
to some degree in all parts of the cell membrane (unpublished
observations). However, the failure of N-and P-type channels to
activate BK current during single action potentials implies that the
[Ca2+]i elevations resulting from influx
through these channels can be exquisitely localized even in simple
spherical cells. Chromaffin cells may thus prove to be of considerable
benefit for questions addressing the molecular basis for how
specificity in coupling between Ca2+ channels and target
proteins may occur.
Implications for cellular excitability
Ca2+ channels are known to be targets of numerous
kinases, phosphatases, and G-proteins that modulate their properties
and affect Ca2+ influx (Catterall 1997).
Because BK current is the primary outward current involved in action
potential repolarization in rat adrenal chromaffin cells (Solaro
et al. 1995
), an important implication of our results is that
the properties of L- and Q-type Ca2+ channels might have
important regulatory effects on action potential properties. Any second
messengers that modulate the properties of L- and Q-type
Ca2+ channels will also be able to dynamically regulate the
extent of BK current activation and produce profound effects on
cellular excitability. In fact, it is precisely because of the
existence of selective coupling of particular Ca2+ channel
variants to BK channel activation that modulation of Ca2+
current may be allowed to exert important effects on cellular excitability. If BK channel activation were not selectively coupled but
influenced by the cumulative Ca2+ arising from all
Ca2+ channel subtypes, regulation of any particular
Ca2+ channel subtype would have only minimal consequences
on both Ca2+ influx during action potentials and on any
Ca2+-dependent process. Thus selective coupling may provide
a key mechanism that enables specific modulation of certain
Ca2+ channel subtypes to exert more profound physiological
consequences. The advantage of employing multiple Ca2+
channel variants, in this case the L- and Q-type, which may be differentially modulated by differing signaling cascades, would be an
ability to regulate cellular excitability over a wide range of
environmental conditions.
Are the Ca2+ channels involved in BK current activation the same as those involved in secretion?
L- and Q-type Ca2+ channels, which we show here to be
involved in BK current activation, were also implicated in the control of exocytosis in bovine and cat chromaffin cells (Artalejo et al. 1994; Lopez et al. 1994a
,b
). This finding
raises the interesting possibility that the Ca2+ channels
involved in BK current activation may also be involved in the control
of secretion. In fact, in the presynaptic nerve terminals of the frog
neuromuscular junction and in auditory hair cells, it was reported that
Ca2+ and Ca2+-activated K+ channels
are colocalized in such a way that the same Ca2+ signal
that triggers transmitter release also activates BK channels (Roberts et al. 1990
; Robittaile et al.
1993
). Our finding that the Ca2+ channel subtypes
involved in BK current activation may be the same as those implicated
for catecholamine secretion raises the intriguing possibility that,
analogous to these examples, Ca2+ and
Ca2+-activated K+ channels may be colocalized
at release sites to control exocytosis.
![]() |
ACKNOWLEDGMENTS |
---|
We thank S. Swanson for the chromaffin cell preparations and Drs. S. Mennerick, J. P. Ding, and J. H. Steinbach for helpful discussions and comments.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-37671.
![]() |
FOOTNOTES |
---|
Address for reprint requests: C. J. Lingle, Dept. of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
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 12 November 1998; accepted in final form 10 February 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|