Department of Biology and Biochemistry, University of Houston,
Houston, Texas 77204-5513
 |
INTRODUCTION |
The developmental expression of macroscopic
Ca2+-activated K+ currents
(KCa) in chick ciliary ganglion (CG) neurons is
regulated by interactions with target tissues in the eye. CG neurons
that develop in vitro (Dourado and Dryer 1992
) or in
vivo (Dourado et al. 1994
) in the absence of normal
target tissues fail to express whole cell KCa at
normal levels. We recently have presented evidence indicating that the
target-derived factor required for normal functional expression of
KCa is an avian form of TGF
1 (Cameron et al. 1998
). Thus application of TGF
1 to E9 CG neurons
developing in vitro or in vivo evokes a robust stimulation of whole
cell KCa expression, and normal developmental
expression of these currents is blocked by TGF
neutralizing antisera
in vivo and in vitro (Cameron et al. 1998
).
Mature (E13) chick CG neurons express three biophysically distinct
KCa channels, two of which can be detected at
high density in excised inside-out patches (Dryer 1998
;
Dryer et al. 1991). One of these is a large-conductance
(BK) KCa channel, similar to those observed in
many other neuronal populations. The gating of this channel is quite
voltage sensitive (Cameron et al. 1998
; Dryer et
al. 1991). The second KCa channel is an
intermediate-conductance (IK) channel with a unitary conductance of 110 pS with [K]o = 75 mM and
[K]i = 150 mM, and 45 pS when
[K]o = 37.5 mM and [K]i = 150 mM (Dryer 1998
; Dryer et al. 1991).
The gating of this channel is more Ca2+ sensitive
than the BK KCa channel. It is occasionally
possible to detect a third type of KCa channel
with a unitary conductance of <20 pS when [K]o = 37.5 mM and [K]i = 150 mM in excised
inside-out patches from E13 CG neurons. The extent to which the
different KCa channels contribute to
voltage-evoked macroscopic currents is unknown, an issue addressed in
this paper. An additional purpose of the present study was to determine
if TGF
1 treatment also can regulate the functional expression of IK
channels in chick CG neurons. We now report that TGF
1 alters the
ability of IK channels to deactivate when Ca2+ is
removed from the cytoplasmic face of patch membranes. However, TGF
1
has no effect on the number of IK channels detected in developing CG
neurons. Importantly, IK channels do not appear to contribute to
macroscopic KCa in intact cells, and
voltage-evoked KCa can be attributed entirely or
predominantly to activation of BK channels.
 |
METHODS |
E9 CG neurons were dissociated and cultured as described
previously (Cameron et al. 1998
; Dourado and
Dryer 1992
; Subramony et al. 1996
) in the
presence or absence of 1 nM recombinant human TGF
1 (R&D Systems,
Minneapolis, MN). After 12 h in vitro, the properties of
intermediate-conductance KCa channels were
examined in excised inside-out patches as described previously
(Cameron et al. 1998
). Briefly, inside-out patches were
excised into a Ca2+-free saline consisting of (in
mM) 150 KCl, 10 EGTA, and 5 HEPES-KOH, pH 7.2. The pipette solution
consisted of (in mM) 112.5 NaCl, 37.5 KCl, 10 EGTA, and 10 HEPES-NaOH,
pH 7.4. Patches were quiescent immediately after patch excision.
Channel activity was evoked by bath application of salines containing 1 µM or 5 µM free Ca2+. The composition of
Ca2+/EGTA buffers was calculated using software
written by Dr. R. A. Steinhardt of the University of California,
Berkeley, and the equilibrium constants reported by Steinhardt,
Zucker, and Schatten (1977)
. The reversal potential of the
unitary currents was determined by application of ramp voltage-commands
(
90 to +40 mV at 0.6 V/s) and/or by recording unitary currents at a
series of constant holding potentials. Data were stored on FM magnetic
tape for off-line analysis using PClamp version 6.04 (Axon Instruments,
Foster City, CA). For cell-attached patch recordings, pipette solutions
consisted of (in mM) 112.5 NaCl, 37.5 KCl, 5 CaCl2, and 10 HEPES-NaOH, pH 7.4. Bath salines
initially consisted of (in mM) 150 NaCl, 5.3 KCl, 5.4 CaCl2, 0.8 MgCl2, and 10 HEPES-NaOH, pH 7.4. Once a stable cell-attached patch was obtained, the
bath was switched to a solution consisting of (in mM) 145 KCl, 5 CaCl2, and 10 HEPES-NaOH, pH 7.4, which served to
depolarize the neurons and thereby activate voltage-dependent
Ca2+ channels throughout the cell (Schmidt
and Kater 1995
; Schmidt et al. 1996
). During
these experiments, the patch membrane was depolarized by an additional
25 mV from the cell membrane potential. Whole cell recordings of
KCa were performed as described previously (Cameron et al. 1998
; Dourado and Dryer
1992
; Dourado et al. 1994
; Dryer et al.
1991; Subramony et al. 1996
) in TGF
1-treated
E9 neurons. For fluctuation analysis, whole cell
KCa was evoked by a series of 150-ms steps to 0 mV from a holding potential of
40 mV. Data were digitized at 10 kHz
and power spectra were calculated for currents evoked in the presence
and absence of external Ca2+ (Axograph software,
Axon Instruments). Mean power spectra in each cell were obtained by
averaging spectra obtained from eight sweeps of steady-state current at
0 mV. The difference between the mean power spectra (normal saline
minus Ca2+-free saline) then was obtained by
digital subtraction, smoothed by averaging of adjacent points, and
fitted with a single Lorentzian curve of the form
S(f) = S(0)/2
fc, where S(f)
is power as a function of frequency, S(0) is the maximal
power, and fc is the half-power
frequency (Anderson and Stevens 1973
). Fitting of power spectra was performed in Axograph using a Simplex least-squares algorithm. Tail-current analyses were performed as described previously (Dryer et al. 1991). Briefly, currents were evoked by a
25-ms pulse to 0 mV from a holding potential of
40 mV, and the cell then was stepped through a series of test potentials (
20 to
50 mV).
This protocol was repeated six times in the presence and absence of
external Ca2+, and net
Ca2+-dependent currents were obtained by digital
subtraction and then averaged. The resulting mean
Ca2+-dependent tail-currents were fitted with a
single-exponential curve and the decay time-constants plotted against
the test potential. Statistical analysis was carried out using
Statistica software (StatSoft, Tulsa, OK).
 |
RESULTS |
IK (45-pS) KCa channels in E9 CG neurons
were quiescent on patch excision into Ca2+-free
salines containing 10 mM EGTA, regardless of the conditions under which
the neurons developed. Bath application of 1-5 µM free
Ca2+ caused robust activation of these channels
in all test groups, and the unitary currents reversed close to the
calculated EK (
35 mV; Fig.
1). Robust gating of these channels
occurred at membrane potentials between
90 and +40 mV (Fig.
1B). This behavior differs from BK (115-pS)
KCa channels of CG neurons, which are much more voltage dependent and show little activity at potentials negative to
10 mV (Cameron et al. 1998
). Voltage-independent
gating of the 45-pS IK channels was observed in the majority of E9 CG
neurons, regardless of culture conditions.

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Fig. 1.
Gating of 45-pS KCa channels at different membrane
potentials. A: channel activity in an inside-out patch
excised from an E9 ciliary ganglion (CG) neuron in the presence of 5 µm free Ca2+. This free Ca2+ concentration
ensured activation of 45- and 115-pS channels, both of which were
present in this patch. Patch potentials are indicated above each trace.
The 45-pS channel was active at all membrane potentials, whereas the
115-pS channel was only active at more depolarized potentials.
B: all-points histogram constructed from the same patch
shown in A at a holding potential of +25 mV.
C: currents evoked by voltage ramps ( 90 to +40 mV at
0.6 V/s) obtained from a different inside-out patch containing a single
45-pS channel in the presence of 1 µm free Ca2+. Six
superimposed traces are shown. , unitary current
amplitudes measured in the same patch at steady holding potentials.
This KCa channel reversed close to the calculated
EK ( 35 mV) and exhibited robust activity
at membrane potentials positive and negative to the reversal potential.
This patch was quiescent in the absence of free Ca2+ (not
shown).
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However, marked differences were observed when active patches were
returned to Ca2+-free salines containing 10 mM
EGTA. In cells treated with 1 nM TGF
1 for 12 h, the IK channels
became quiescent immediately on switching the bath back to
Ca2+-free salines (Fig.
2). This behavior is identical to that
observed in CG neurons isolated acutely at E13 (Dryer
1998
; Dryer et al. 1991) and also was observed
in E9 CG neurons cultured with iris extracts (data not shown). In
contrast, IK channels in control E9 CG neurons (cultured in the
absence of TGF
1) typically remained active for tens of
minutes after switching the bath to Ca2+-free
salines (Fig. 2).

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Fig. 2.
Effects of TGF 1 on reversibility of Ca2+-activation of
45-pS KCa channels. A: in control neurons,
patches were quiescent in Ca2+-free saline and became
active in the presence of 1 µm free Ca2+. These channels
remained active for up to 30 min after the patch was returned to
Ca2+-free solution containing 10 mM EGTA. Patches were not
usually monitored for longer than that. B: in patches
excised from TGF 1-treated cells, the 45-pS channels were well
behaved. Thus the channels were quiescent after excision into
Ca2+-free saline and became active in 1 µm free
Ca2+. Importantly, these channels quickly became
inactive on return to Ca2+-free saline.
Left: 1 s of typical raw data.
Right: probablity of channel opening
(Po) for representative 20-s periods before,
during, and after Ca2+ application.
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Results from many inside-out patches excised from E9 CG neurons are
summarized in Table 1. Treatment of CG
neurons with 1 nM TGF
1 did not affect the mean number of IK channels
detected per patch. Moreover, the mean open-times of the channels were not different in TGF
1-treated cells compared with control neurons (data not shown). For comparison, data are also presented for the BK
channels. It is worth noting that TGF
1 treatment had no effect on
voltage-independent low-conductance (<20 pS) KCa
channels observed in excised patches (data not shown).
Given that TGF
1 produces different effects on IK (45-pS) and BK
(115-pS) KCa channels, we next determined the
extent to which each subtype contributes to voltage-evoked current. In
one set of experiments, cell-attached patches were made onto E9 CG
neurons cultured for 12 h in the presence or absence of 1 nM
TGF
1 (Fig. 3A). The patch
pipette contained a solution similar to that used in inside-out patch
recordings except that it also contained 5 mM
CaCl2. The patch was depolarized by 25 mV from
the resting potential. The cells then were perfused with external
saline containing 145 mM KCl and 5 mM CaCl2 to
induce depolarization and activation of voltage-dependent
Ca2+ current throughout the cell. In
TGF
1-treated E9 cells this depolarization caused activation of BK
channels in 8 out of 9 patches, with a mean of 1.89 ± 0.39 channels per patch. However, this procedure caused activation of an IK
channel in only one of nine patches. Similar results were obtained in
acutely isolated E13 neurons (data not shown). In control E9 neurons,
this procedure caused activation of BK channels, but at much lower
levels; BK channels were observed in four of nine patches, with a mean
of 0.44 ± 0.18 channels per patch (mean ± SE;
P < 0.005 compared with TGF
1-treated cells). As
with TGF
1-treated cells, an IK channel was again observed in only
one of nine patches from control neurons. These results suggest that IK
channels do not contribute significantly to voltage-evoked KCa in intact CG neurons, but they confirm TGF
1
stimulation of functional BK channels (Cameron et al.
1998
).

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Fig. 3.
Activation of large-conductance (BK) but not
intermediate-conductance (IK) KCa channels by voltage
pulses in intact ciliary ganglion neurons. A:
cell-attached patch recordings from E9 CG neurons cultured for 12 h in the absence (top) and presence
(bottom) of 1 nM TGF 1. Throughout, the patch membrane
was depolarized by 25 mV from the cell resting potential.
Representitive traces are shown for patches in normal external saline
(containing 5.3 mM KCl) and thirty seconds after depolarization of the
cell by bath application of 145 mM KCl in the presence of 5 mM
CaCl2, as indicated. Note that BK (115-pS) channels are
evoked by cell depolarization in the TGF 1-treated neuron, but not in
the control neuron. IK (45-pS) channels were not detected in either
cell. B: characteristics of macroscopic KCa
fluctuations in a TGF 1-treated E9 neuron. Records are AC-coupled
traces of whole cell currents evoked by depolarizing step to 0 mV from
a holding potential of 40 mV in the presence and absence of external
Ca2+, as indicated. In this cell, mean macroscopic
Ca2+-dependent outward current was 1,100 pA.
Bottom: mean power spectrum of
Ca2+-dependent outward current from the same cell fitted
with a single Lorentzian curve with a corner frequency of 35 Hz. Note
that this is a double-log plot. Excellent fit to a single Lorentzian
suggests activation of a kinetically homogeneous population of
KCa channels. C: deactivation of macroscopic
KCa in TGF 1-treated E9 CG neurons. Left:
mean Ca2+-dependent currents obtained by digital
subtraction with voltage-clamp protocol shown above the
current traces. Averaged tail-currents (6 pulses per trace) are shown
fitted with a superimposed single-exponential curve indicating
kineticially homogeneous behavior at all test pulses.
Right: mean tail-current decay time-constants as a
function of test potential in 4 CG neurons. Note voltage-dependence of
KCa tail-current deactivation.
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To further address this question, Ca2+-dependent
macroscopic current fluctuations were examined in TGF
1-treated E9
neurons (Fig. 3B). Whole cell currents were evoked by a
series of 150-ms depolarizing pulses to 0 mV from a holding potential
of
40 mV in the presence and absence of external Ca2+.
Power spectra were calculated for currents evoked in the presence and
absence of Ca2+. The resulting KCa difference
spectra (obtained by digital subtraction) could be very fitted with a
single Lorentzian curve with a half-power frequency
(fc) of ~35 Hz. Deviations from the single
Lorentzian account for <1% of the total KCa current
variance, indicating that this macroscopic current is carried by a
population of channels with essentially uniform kinetic behavior. This
is supported by analyses of macroscopic KCa tail-current
deactivation. In these experiments, whole cell KCa was
evoked by a 35-ms pulse to 0 mV from a holding potential of
40 mV. At
the end of the activation pulse, the current was allowed to deactivate
over a range of different test potentials (
20 to
50 mV). This
protocol was repeated in the presence and absence of external
Ca2+, and macroscopic Ca2+-dependent currents
were obtained by digital subtraction. At all test potentials, the time
course of the resulting KCa tail currents could be fitted
with a single exponential (Fig. 3C). Moreover, the time
constants were markedly voltage dependent. This indicates that the
underlying KCa channels in TGF
1-treated cells are
kinetically homogeneous and voltage dependent. Because the voltage
dependence of intermediate and large-conductance KCa
channel gating is quite different, this suggests that most or all of
the voltage-evoked macroscopic current is carried by large-conductance
KCa channels, which exhibit markedly voltage-dependent gating.
 |
DISCUSSION |
We have previously shown that target-derived TGF
is required
for the normal developmental expression of macroscopic voltage-evoked KCa in chick CG neurons developing in vivo and in
vitro (Cameron et al. 1998
). This effect is due at least
in part to an increase in the number of functional BK (115-pS)
KCa channels in the plasma membrane. Treatment
with TGF
1 does not appear to alter the gating behavior of those
channels. In the present study, we have confirmed this observation in
intact cells (i.e., in cell-attached patches). In addition, we have
observed that 12-h treatment with TGF
1 also affects IK (45-pS)
KCa channels in CG neurons. However, the nature of the action is different and is associated with changes in the reversibility of Ca2+ activation rather than the
number of functional channels in the plasma membrane. Finally, we have
shown that macroscopic KCa is associated
primarily with activation of BK channels. IK channels are not activated
to any appreciable extent by voltage-evoked Ca2+-influx, although they can be activated
readily in excised patches when the entire cytosolic face of the patch
membrane is exposed to micromolar free Ca2+.
The mechanism of TGF
1 effects on IK channels is not known but may
entail changes in the association of auxiliary subunits or other
proteins with the KCa channels. One possibility
is that TGF
1 regulates the association of
Ca2+-dependent kinases or phosphatases with IK
channels. The fact that IK channels are not activated by voltage-pulses
in intact neurons suggests that they may not be closely associated with voltage-activated Ca2+ channels in the plasma
membrane. It is possible that IK channels become active under different
conditions as a result of more generalized, as opposed to spatially
discrete, changes in intracellular free Ca2+.
In summary, we have shown that 12 h exposure to TGF
1 alters the
reversibility of Ca2+ activation of IK
KCa channels in developing chick CG neurons but
does not affect the number of IK channels detected in excised patches.
In contrast, TGF
1 treatment causes a large increase in the number of
BK channels in developing CG neurons but does not change their gating
properties. These results show that the effects of TGF
1 entail
distinct and complex actions on multiple KCa
channel subtypes, which differ dramatically in their susceptibility to
activation by voltage-evoked Ca2+ influx.
We are grateful to J. Cameron for helpful discussions and for
participating in some of these experiments.
This work was supported entirely by National Institute of Neurological
Disorders and Stroke Grant NS-32748.
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.