TGFbeta 1 Regulates the Gating Properties of Intermediate-Conductance KCa Channels in Developing Parasympathetic Neurons

Loic Lhuillier and Stuart E. Dryer

Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5513


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INTRODUCTION
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Lhuillier, Loic and Stuart E. Dryer. TGFbeta 1 Regulates the Gating Properties of Intermediate-Conductance KCa Channels in Developing Parasympathetic Neurons. J. Neurophysiol. 82: 1627-1631, 1999. The developmental expression of Ca2+-activated K+ channels (KCa) in chick ciliary ganglion (CG) neurons is regulated by a target-derived avian isoform of TGFbeta 1, which evokes a robust increase in the number of functional large-conductance (BK) KCa channels but which produces no change in their kinetics. However, CG neurons express multiple KCa channel subtypes. Here we show that TGFbeta 1 regulates the gating properties of intermediate-conductance (IK) KCa channels in developing CG neurons. IK channels in inside-out patches excised from control E9 CG neurons became active on exposure to 1-5 µM free Ca2+ but then remained active on return to Ca2+-free salines. In contrast, IK channels in TGFbeta 1-treated cells became active on exposure to 1-5 µM Ca2+, but became quiescent immediately on return to Ca2+-free salines. In contrast to its effects on BK channels, TGFbeta 1 had no effect on the mean number of IK channels detected in excised patches. IK channels were not activated in cell-attached patches on E9 neurons depolarized by bath application of 145 mM KCl in the presence of 5 mM external Ca2+. However, BK channels were activated immediately by this procedure and were detected at a higher density in TGFbeta 1-treated cells. In addition, analyses of macroscopic KCa fluctuations, and the voltage-dependence of KCa tail currents, suggest that IK channels do not contribute to voltage-evoked whole cell KCa. IK channels therefore may have some other function. These results indicate that the effects of TGFbeta 1 on CG neurons entail distinct actions on multiple KCa channel subtypes.


    INTRODUCTION
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INTRODUCTION
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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 TGFbeta 1 (Cameron et al. 1998). Thus application of TGFbeta 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 TGFbeta 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 TGFbeta 1 treatment also can regulate the functional expression of IK channels in chick CG neurons. We now report that TGFbeta 1 alters the ability of IK channels to deactivate when Ca2+ is removed from the cytoplasmic face of patch membranes. However, TGFbeta 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.


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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 TGFbeta 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 TGFbeta 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)/2pi 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).


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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. open circle , 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).

However, marked differences were observed when active patches were returned to Ca2+-free salines containing 10 mM EGTA. In cells treated with 1 nM TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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.

Results from many inside-out patches excised from E9 CG neurons are summarized in Table 1. Treatment of CG neurons with 1 nM TGFbeta 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 TGFbeta 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 TGFbeta 1 treatment had no effect on voltage-independent low-conductance (<20 pS) KCa channels observed in excised patches (data not shown).


                              
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Table 1. Effects of TGFbeta 1 treatment on functional properties of 45 and 115 pS channels in inside-out patches

Given that TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 1-treated cells). As with TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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.

To further address this question, Ca2+-dependent macroscopic current fluctuations were examined in TGFbeta 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 TGFbeta 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
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ABSTRACT
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We have previously shown that target-derived TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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 TGFbeta 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, TGFbeta 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 TGFbeta 1 entail distinct and complex actions on multiple KCa channel subtypes, which differ dramatically in their susceptibility to activation by voltage-evoked Ca2+ influx.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

Address reprint requests to: S. E. Dryer.

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 4 December 1998; accepted in final form 24 May 1999.


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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society