BK-Type KCa Channels in Two Parasympathetic Cell Types: Differences in Kinetic Properties and Developmental Expression

Jill S. Cameron and Stuart E. Dryer

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cameron, Jill S. and Stuart E. Dryer. BK-Type KCa Channels in Two Parasympathetic Cell Types: Differences in Kinetic Properties and Developmental Expression. J. Neurophysiol. 84: 2767-2776, 2000. The intrinsic electrical properties of identified choroid and ciliary neurons of the chick ciliary ganglion were examined by patch-clamp recording methods. These neurons are derived from a common pool of mesencephalic neural crest precursor cells but innervate different target tissues and have markedly different action potential waveforms and intrinsic patterns of repetitive spike discharge. Therefore it is important to determine whether these cell types express different types of plasma membrane ionic channels, and to ascertain the developmental stages at which these cell types begin to diverge. This study has focused on large-conductance Ca2+-activated K+ channels (KCa), which are known to regulate spike waveform and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin immunoreactivity, express a robust macroscopic KCa carried by a kinetically homogeneous population of large-conductance (BK-type) KCa channels. However, the kinetic properties of these channels are different in the two cell types. Steady-state fluctuation analyses of macroscopic KCa produced power spectra that could be fitted with a single Lorentzian curve in both cell types. However, the resulting corner frequency was significantly lower in choroid neurons than in ciliary neurons, suggesting that the underlying KCa channels have a longer mean open-time in choroid neurons. Consistent with fluctuation analyses, significantly slower gating of KCa channels in choroid neurons was also observed during macroscopic activation and deactivation at membrane potentials positive to -30 mV. Differences in the kinetic properties of KCa channels could also be observed directly in single-channel recordings from identified embryonic day 13 choroid and ciliary neurons. The mean open-time of large-conductance KCa channels was significantly greater in choroid neurons than in ciliary neurons in excised inside-out patches. The developmental expression of functional KCa channels appears to be regulated differently in the two cell types. Although both cell types acquire functional KCa at the same developmental stages (embryonic days 9-13), functional expression of these channels in ciliary neurons requires target-derived trophic factors. In contrast, expression of functional KCa channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain KCa stimulatory activity. However, KCa stimulatory activity cannot be detected in extracts of the smooth muscle targets of choroid neurons.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The electrophysiological properties of neurons are typically tuned for a specific physiological function. However, the factors controlling the developmental expression of a specialized electrophysiological phenotype are not well understood. The chick ciliary ganglion (CG) is a useful model system in which to study this process, as it contains two populations of mesencephalic neural crest-derived neurons that innervate different target tissues in the eye (Marwitt et al. 1971). Mature ciliary neurons innervate striated muscle cells in the iris and ciliary body, whereas choroid neurons innervate smooth muscle cells in the choroid layer. Both cell types are cholinergic, but choroid neurons also express somatostatin whereas ciliary neurons do not, and choroid neurons tend to be smaller than ciliary neurons (Darland and Nishi 1998; De Stefano et al. 1993; Epstein et al. 1988). These differences provide convenient criteria for identification of these neurons in dissociated preparations.

Choroid and ciliary neurons in the intact CG have markedly different electrophysiological properties (Dryer 1994). Ciliary neurons fire spikes at a constant high frequency (>150 Hz) in response to sustained injection of depolarizing current. The intrinsic firing properties of ciliary neurons are well matched to those of their striated muscle targets, which exhibit tetanic contraction only when stimulated at 100-150 Hz (Pilar and Vaughan 1969). In contrast, choroid neurons exhibit a maximum firing frequency of ~35 Hz and show substantial spike-frequency adaptation. Equally notably, the spike duration at half-amplitude in choroid neurons is about twice that of ciliary neurons (Dryer 1994).

We have previously shown that the differentiation of excitability in CG neurons is regulated by cell-cell interactions. Large CG neurons that develop in vivo in the absence of normal target tissues fail to express functional Ca2+-activated K+ channels (KCa) (Dourado et al. 1994). Moreover, large CG neurons developing in vitro fail to express KCa unless iris extracts or iris-derived factors are included in the culture media (Cameron et al. 1998, 1999; Dourado and Dryer 1992; Subramony et al. 1996). The essential iris-derived factor appears to be an avian ortholog of TGFbeta 1. Thus application of TGFbeta 1 can markedly accelerate the functional expression of KCa in vivo or in vitro, and a TGFbeta neutralizing antiserum blocks the normal in vivo expression of KCa as well as the in vitro actions of iris extracts (Cameron et al. 1998). Macroscopic KCa in CG neurons is mediated by large-conductance (BK-type) channels (Cameron et al. 1998; Dryer et al. 1991; Lhuillier and Dryer 1999) encoded by the slo gene (Dryer 1998; Subramony et al. 1996). Although other types of KCa channels with lower unitary conductance can be detected in inside-out patches excised from CG neurons, these lower conductance KCa channels cannot be detected on depolarization of intact cells and do not appear to contribute to macroscopic currents (Lhuillier and Dryer 1999). Their functional significance, if any, remains unknown. However, macroscopic KCa contributes to spike repolarization and afterhyperpolarization in CG neurons (Dryer et al. 1991), and therefore differences in the properties of large-conductance KCa channels could contribute to the physiological differences between choroid and ciliary neurons.

Our previous studies on the developmental regulation of KCa channels and macroscopic KCa have focused on cells that, based on their size, are likely to consist primarily of ciliary neurons. Given the results in ciliary neurons, we hypothesized that KCa regulation in developing choroid neurons would follow a similar pattern, i.e., that expression of an identical population of KCa channels would require trophic factors secreted from target cells in the smooth muscle choroid layer. Here we show that both of these hypotheses are incorrect. Instead, developmental expression of macroscopic KCa in choroid neurons appears to be cell-autonomous, and certainly proceeds normally in the absence of target-derived factors. By contrast, functional expression of KCa in ciliary neurons is not cell-autonomous and requires exposure to trophic factors that are present in the iris, but that cannot be detected in the choroid smooth muscle layer. Moreover, the kinetic properties of large-conductance KCa channels, once they are present in the plasma membrane, are different in the two cell types. Specifically, choroid cells express large-conductance KCa channels with substantially slower kinetics than those of ciliary neurons. This conclusion is based on several independent kinetic methods, including analyses of macroscopic current fluctuation, activation, and deactivation, as well as more direct measurements in inside-out patches. These differences in macroscopic behavior can provide an explanation for at least some of the intrinsic differences in spike discharge between choroid and ciliary neurons. It is also possible that differences in gating properties and regulation of functional expression are consequences of a common molecular mechanism.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation and culture

Ciliary ganglion neurons were dissociated at embryonic day 9 (E9) or E13 as described in detail previously (Cameron et al. 1998, 1999; Dourado and Dryer 1992; Subramony et al. 1996). Cells isolated at E13 were used for electrophysiology within 2 h of isolation. Cells isolated at E9 were examined at various times up to 4 days after isolation, as indicated. Dissociated cells were grown on poly-D-lysine-coated glass coverslips in a culture medium consisting of Eagle's minimal essential medium supplemented with 10% heat-inactivated horse serum, 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 40 ng/ml recombinant rat ciliary neurotrophic factor (R and D Systems, Minneapolis, MN). In some experiments, E9 CG neurons were cultured for 12 h in media that contained iris extracts, choroid extracts, or both, as indicated. To prepare extracts, the iris and the choroid layer were dissected out of chick embryos at E9, placed in ice-cold Earle's balanced salts (1 iris or choroid per ml), and homogenized in a glass homogenizer. Extracts were centrifuged at 15,000 g for 1 h at 4° to remove membranous fractions. Protein concentrations in all extracts were adjusted to 230-260 µg/ml by addition of Earle's balanced salts. Iris extracts were added to cell culture media at a concentration of 3% (vol/vol). Choroid extracts were added to media at various concentrations as indicated.

Immunocytochemistry

Somatostatin staining was performed essentially as described by Darland and Nishi (1998). Briefly, CG neurons were dissociated at E13, fixed for 30 min in Zamboni's solution (4% paraformaldehyde, 15% picric acid, and 0.1 M sodium phosphate buffer, pH 7.4), rinsed in PBS, and blocked overnight in 10% horse serum, 0.5% Triton X-100, and 0.2% sodium azide in PBS. Cells were then incubated overnight at room temperature (22°C) with monoclonal rat anti-somatostatin (Accurate Chemical and Scientific, Westbury, NY, cell line YC7; 1:100 dilution in blocking solution without detergent). Cells were rinsed and incubated with PBS containing 10% H2O2 and 30% ethanol to inactivate endogenous peroxidase, and staining was revealed by the ABC method (Vectastain kit, Vector Laboratories Burlingame, CA) using diaminobenzidine as a chromogen according to the manufacturer's directions. No signal was obtained from control CG cultures treated with preabsorbed primary antibody or with no primary antibody.

Electrophysiology and data analysis

Whole cell recordings were made using standard methods as described in detail previously (Cameron et al. 1998, 1999; Dourado and Dryer 1992; Lhuillier and Dryer 2000; Subramony and Dryer 1997; Subramony et al. 1996). In most experiments, currents were evoked by depolarizing steps to 0 mV from a holding potential of -40 mV in normal and Ca2+-free external saline. Normal external saline consisted of 145 mM NaCl, 5.4 mM KCl, 5.4 mM CaCl2, 0.8 mM MgCl2, 5 mM D-glucose, 13 mM HEPES-NaOH, and 250 nM tetrodotoxin, pH 7.4. Nominally Ca2+-free external saline was the same except that it contained 5.8 mM MgCl2 and no added CaCl2. Net Ca2+-dependent outward currents (KCa) were then obtained by digital subtraction (control - Ca2+-free) and normalized for cell size by measuring soma surface area for each cell as described previously (Cameron et al. 1998, 1999; Dourado and Dryer 1992; Subramony et al. 1996). For steady-state fluctuation analysis, currents in each cell were evoked by a series of 10 depolarizing steps of 2.2 s duration to various command potentials in normal saline, followed by 10 identical steps after application of Ca2+-free saline. Data were digitized at 2 kHz, and the DC components of the evoked currents were removed. The steady-state portions of the evoked currents were cosine-tapered (w{n} = cos {pi n/N}), and power spectra were computed from 4,096 digital samples using routines implemented in Pclamp software (Clampfit v. 8.0, Axon Instruments, Foster City, CA). Spectra obtained for currents evoked by each of the 10 depolarizing steps were averaged, and the resulting mean spectra were subtracted (control spectrum - Ca2+-free spectrum). This served to remove contributions from instrumentation noise and other voltage-evoked ionic currents (except for Ca2+ currents), and to thereby isolate the mean power spectrum of macroscopic KCa. The resulting KCa power spectra were smoothed by adjacent point averaging and then fitted with Lorentzian curves of the form S(f) = S(0)/[1 + (2pi fctau )2] as described in the text using a Levenburg-Marquardt least-squares algorithm (Microcal Origin, v 6.0, Northampton, MA). Single Lorentzian curves provided excellent fits to the KCa power spectra at all command potentials, a finding consistent with our previous studies with cell-attached patches in which we found that only a single population of large-conductance KCa channels became active on depolarization of intact cells, as well as single-exponential macroscopic tail currents over a wide range of test potentials (Lhuillier and Dryer 1999). This spectral subtraction procedure allows calculation of variance contributed by those macroscopic currents that depend on external Ca2+, which with these voltage-clamp protocols consist of voltage-activated Ca2+ currents and KCa. To determine whether Ca2+ currents make a significant contribution to subtracted power spectra, a series of similar protocols were carried out in the presence of 10 mM tetraethylammonium, which causes a complete blockade of KCa (Dryer et al. 1991) and which therefore allows for isolation of spectral components contributed by Ca2+ currents. Increases in variance contributed by Ca2+ currents were undetectable in macroscopic measurements, a result consistent with the very low unitary conductance of these channels (Marrion and Tavalin 1998). Consequently, the subtracted power spectra reflect gating of BK type KCa channels. Deactivation kinetics were analyzed from Ca2+-dependent tail currents recorded at various membrane potentials after a step pulse to 0 mV from a holding potential of -40 mV (Lhuillier and Dryer 1999). The decay phases of tail currents were fitted with single-exponential curves using Clampfit software.

Inside-out patch recordings were made by standard methods as described in detail previously (Cameron et al. 1998; Lhuillier and Dryer 1999). Briefly, patches were excised into Ca2+-free salines containing 10 mM EGTA, and KCa channels were activated by bath application of salines containing 5 µM free Ca2+. The holding potential was +25 mV throughout. Single-channel data were filtered at 2 kHz with a 4-pole Bessel filter and stored on magnetic videotape in FM mode for off-line digitization (2 kHz) and analysis using Pclamp software. Idealized traces were generated using a half-threshold crossing procedure that ignored transitions of <0.5 ms duration, and idealized data were used to construct open-time histograms and to calculate Po. All statistical analyses were carried out using Statistica software (v5.1, Statsoft, Tulsa, OK). Throughout, error bars represent SE. Data were analyzed by one-way ANOVA followed by Scheffé's multiple range test (where comparisons were made between multiple groups), Student's unpaired t-test (for comparisons between 2 groups), and linear regression analysis. Cell size distribution data were analyzed by the Kolmogirov-Smirnov one-sample test for deviations from a unimodal Gaussian distribution. Throughout, P < 0.05 was regarded as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of isolated choroid and ciliary neurons

In intact ganglia, the two populations of CG neurons can be distinguished on the basis of size, ultrastructure, and somatostatin immunoreactivity. Choroid neurons express somatostatin, whereas ciliary neurons do not, and the majority of choroid neurons are smaller than ciliary neurons, although there is overlap in their size distributions (Darland and Nishi 1998; De Stefano et al. 1993; Epstein et al. 1988). In particular, De Stefano et al. (1993) used ultrastructural criteria to show that somatostatin is a highly specific marker for choroid neurons. In our initial studies, we measured neuronal diameters in acutely isolated preparations of embryonic day 13 (E13) CG neurons (Fig. 1A). The distribution of neuronal diameters was then fitted with a Gaussian distribution or as the sum of two Gaussians. Attempts to fit the observed distribution with a single Gaussian were unsatisfactory, and the raw data were significantly (P < 0.05) different from a unimodal normal distribution (Kolmogorov-Smirnov 1-sample test). However, better fits were obtained with the sum of two approximately equally weighted Gaussians with means of 14.5 and 19.8 µm (Fig. 1A). To determine whether these two groups correspond to choroid and ciliary neurons, preparations of dissociated E13 CG neurons were stained for somatostatin immunoreactivity (Fig. 1B), and the size distributions of labeled and unlabeled neurons were determined. The resulting distributions correspond closely to the two Gaussians observed in unstained cells (Fig. 1C). In these preparations, 114 of 116 neurons with diameters <= 14 µm expressed somatostatin, a marker for choroid neurons. In contrast, none of the 108 cells with diameters >= 17 µm expressed somatostatin, indicating that these represent an enriched population of ciliary neurons. To minimize the possibility of sampling error in physiological experiments, neurons with diameters <= 12 µm were considered to be choroid neurons, neurons with diameters >= 20 µm were considered to be ciliary neurons, and cells with intermediate diameters were not studied.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 1. Criteria for identification of choroid and ciliary neurons. A: distribution of neuronal diameters of ciliary ganglion (CG) neurons isolated acutely at embryonic day 13 (E13). Adequate fit of the distribution requires the sum of 2 Gaussian curves (shown superimposed) suggesting 2 size classes of CG neurons. These data are significantly different from a unimodal normal distribution (Kolmogorov-Smirnov 1-sample test). B: immunocytochemical staining for somatostatin in acutely dissociated E13 CG neurons. Note robust staining of the small CG neuron (choroid cell) but not the large CG neuron (ciliary neuron). Calibration bar indicates 20 µm. C: size distribution of somatostatin-positive choroid cells (left) and somatostatin-negative ciliary cells (right) in a preparation of CG neurons isolated acutely at E13. In subsequent experiments, neurons with diameters <= 12 µm were considered to be choroid neurons, whereas neurons with diameters >= 20 µm were considered to be ciliary neurons.

Differences in macroscopic KCa kinetics in choroid and ciliary neurons

We have previously shown that macroscopic KCa in CG neurons reaches maximum current density by E13 (Dourado and Dryer 1992). Here we have observed that the current density of macroscopic KCa is not significantly different in choroid and ciliary neurons isolated acutely at this developmental stage (Fig. 2). However, the kinetic properties of the underlying channels are different in the two cell types. This was established in part by three different types of macroscopic measurements. Whole cell recordings have the advantage of sampling only those channels that contribute to macroscopic currents [i.e., KCa channels that are properly co-localized with voltage-dependent Ca2+ channels in the plasma membrane (see Gola and Crest 1993; Lhuillier and Dryer 1999; Marrion and Tavalin 1998)]. This recording configuration also avoids any changes in KCa channel properties that could occur as a result of patch excision.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Expression of macroscopic KCa in choroid and ciliary neurons isolated acutely at E13. Traces are whole cell currents evoked by depolarizing steps (shown above traces) in normal and Ca2+-free salines as indicated. Typical traces are shown for a ciliary neuron (left) and a choroid neuron (right). Mean KCa current densities and SE from a large number of choroid and ciliary neurons are shown below, and are not significantly different in the 2 cell types. Numbers above bars denote number of cells tested.

In the first approach, the kinetic properties of whole cell KCa were determined by steady-state fluctuation analysis. If one assumes that channels gate independently, then it is possible to extract information about the underlying kinetics from spectral density analysis of macroscopic current fluctuations (Anderson and Stevens 1973). Although fine details of the kinetics are masked by bandwidth limitations inherent in whole cell recording, this type of analysis has been successfully used to resolve kinetic differences associated with developmental changes in the subunit stoichiometry of ligand-gated ionic channels (e.g., Fischbach and Schuetze 1980; Vicini and Schuetze 1985). In our experiments, currents in each CG cell were evoked by a series of 10 depolarizing step pulses of 2.2-s duration to various holding potentials from a holding potential of -40 mV in the presence of Ca2+. These protocols were then repeated in the absence of external Ca2+ (Fig. 3, A and B) as described previously (Cameron et al. 1998, 1999; Dourado and Dryer 1992; Dourado et al. 1994; Subramony et al. 1996). The mean power spectra of macroscopic current fluctuations were calculated for currents evoked in the presence and absence of Ca2+, and the resulting spectra were subtracted digitally to determine the Ca2+-dependent component of the variance associated with evoked steady-state outward currents (Fig. 3, C and D). The subtracted power spectra were fitted with Lorentzian curves of the form S(f) = S(0)/[1 + (2pi fctau )2], where S(f) is power as a function of frequency, S(0) is the extrapolated power at frequency = 0 Hz, fc is the frequency at which power is S(0)/2, and tau  is a time constant defined as 1/2pi fc and related but not precisely equal to the mean open-time. In both populations of neurons, single Lorentzian curves provided excellent fits to the subtracted power spectra, suggesting that a kinetically homogeneous population of channels underlies macroscopic KCa (Fig. 3, C and D). These panels show spectra calculated for currents evoked by a depolarizing step to 0 mV, and they indicate that KCa channels have different kinetic properties in the two populations of cells. The mean time constant of fitted Lorentzian curves calculated from pulses to 0 mV in a large number of choroid cells was more than twice that of ciliary cells (Fig. 3E). These differences are statistically significant and are not a consequence of different probabilities of channel opening (Po). It is possible to determine Po during a depolarizing step by means of binomial statistics (Anderson and Stevens 1973). Thus i = (1 - Po) sigma I2I, where i is the unitary current flowing through a single open KCa channel, sigma I2 is the variance of the macroscopic fluctuations associated with KCa gating, and µI is the mean Ca2+-dependent outward current. Independent measurements with cell-attached patches indicate that i = 6.5 pA at 0 mV with close to physiological ionic gradients (Lhuillier and Dryer 1999). Current variance associated with KCa gating (sigma I2) is simply the integral of the fitted Lorentzian curve, which is given by S(0)pi fc/2. The mean current (µI) is the difference in net outward current evoked in the presence and absence of Ca2+. From these parameters, the mean Po during a step pulse to 0 mV was calculated to be 0.92 ± 0.06 in ciliary cells, and 0.95 ± 0.07 in choroid cells. These results are not significantly different and indicate that step pulses to 0 mV evoke comparable KCa activation in the two cell types. In a separate set of experiments, similar analyses were carried out on both cell types using a range of depolarizing steps (to -25 mV, 0 mV, and +25 mV) from a holding potential of -40 mV. The resulting time constants derived from power spectral analysis were significantly (P < 0.05) longer in choroid cells than in ciliary cells at all three membrane potentials (Fig. 3F). However, these differences were more apparent at more depolarized potentials, i.e., the voltage dependence of KCa gating appears greater in choroid neurons than in ciliary neurons.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3. Steady-state fluctuation analysis indicates that KCa channels in E13 ciliary and choroid neurons have different kinetic properties. Representative high-gain AC-coupled traces of macroscopic currents at 0 mV are shown for a ciliary neuron (A) and a choroid neuron (B) in the presence and absence of external Ca2+ as indicated. Each trace shows 2 s of data acquired at a steady-state level of current. Note increase in "noise" for currents evoked in the presence of Ca2+. Mean steady-state Ca2+-dependent current was 1.48 nA for the ciliary neuron and 1.12 nA for the choroid neuron. Subtracted power spectra (control spectrum - Ca2+-free spectrum) are shown for the same cells in C and D. Power spectra are shown with superimposed fitted single Lorentzian curves with the indicated time constants. E: mean time constants and SE obtained from KCa power spectra obtained at 0 mV command potential in a large group of choroid and ciliary neurons. Mean time constant for macroscopic KCa in choroid neurons is significantly greater than that of ciliary neurons. F: mean time constants and SE derived from KCa power spectra in a different set of choroid neurons () and ciliary neurons () computed from currents evoked by steps to -25, 0, and +25 mV. Note significantly greater mean time constant in choroid neurons at all 3 command potentials, and greater difference at more positive membrane potentials.

This pattern can also be seen in analyses of KCa deactivation kinetics in the two cell types. For these experiments, KCa was activated by a 25-ms step to 0 mV from a holding potential of -40 mV, at which time the membrane was stepped through a series of test potentials between -70 and -20 mV. This protocol was repeated in normal and Ca2+-free salines, and the resulting Ca2+-dependent currents were obtained by digital subtraction (Fig. 4A). The decay phases of the KCa tail currents were fitted with single-exponential curves, which provided good fits to the data in both cell types as described previously for ciliary neurons (Dryer et al. 1991; Lhuillier and Dryer 1999). As with fluctuation analysis, the resulting deactivation time constants were significantly longer in choroid neurons than in ciliary neurons, but only at -30 and -20 mV. Tail-current decay time constants were not significantly different in the two cell types at more negative membrane potentials, an observation consistent with the trends noted above in fluctuation analysis.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Differences in kinetics of macroscopic KCa activation and deactivation in E13 ciliary and choroid neurons. In this figure, current traces are plotted on different ordinate scales to facilitate comparison of macroscopic kinetics. A: macroscopic KCa deactivates more rapidly in ciliary neurons. Traces to left show net Ca2+-dependent currents evoked by a 25-ms step to 0 mV followed by step pulses through a series of test potentials (-20 to -70 mV) in ciliary and choroid neurons, as indicated. Decay phases of tail currents are shown with superimposed fitted single-exponential curves. Graph to right shows plot of mean tail-current decay time-constant vs. membrane potential in ciliary neurons (open circle ) and choroid neurons (). Error bars are SE. Note significantly (P < 0.05) shorter mean tail-current decay time-constants ciliary neurons at -30 and -20 mV. B: macroscopic KCa activates more rapidly in ciliary neurons. Traces to left show typical net Ca2+-dependent currents evoked by step pulse to 0 mV in ciliary and choroid neurons as indicated. Rising phases of currents from several cells were fitted with single-exponentials (not shown), and resulting mean time-constants and SE are shown in bar graph to the right. Note significantly (P < 0.05) faster KCa activation in ciliary neurons.

Significant differences in macroscopic KCa behavior in the two cell types also appeared in analyses of activation kinetics derived from test pulses to 0 mV from a holding potential of -40 mV (Fig. 4B). The time course of activation was fitted with a single-exponential. Activation of KCa was several-fold slower in choroid neurons than in ciliary neurons, and the differences in the resulting time constants are statistically significant (P < 0.01). Macroscopic KCa is the largest outward current that can be evoked from normal resting potential in both cell types, and these data therefore provide a mechanism for the different spike durations in the two cell populations.

Differences in single-channel KCa kinetics in choroid and ciliary neurons

These analyses were carried out using two different single-channel recording configurations. In one set of experiments, the kinetic properties of KCa channels were determined in inside-out patches excised from choroid and ciliary neurons. This method has the advantage of examining KCa channel gating more directly under controlled membrane potential and [Ca2+]i, but has the disadvantage of sampling all KCa channels in the patch membrane, whether or not they would normally contribute to macroscopic currents. In these experiments, patches were excised into a Ca2+-free bath saline (containing 10 mM EGTA) and held at +25 mV. KCa channels were quiescent in Ca2+-free bath saline but became active on bath application of salines containing 5 µM free Ca2+ (Fig. 5). These conditions were chosen because they yielded a mean Po > 0.90, comparable to the gating conditions of the macroscopic measurements described above (Fig. 6, A and B). Kinetic analyses were performed on patches that had one, or at most two, large-conductance KCa channels, and that did not contain other detectable channel types. In particular, patches that contained intermediate-conductance KCa channels were excluded, as these channels do not appear to contribute to macroscopic currents and are not activated by depolarization of intact CG neurons (Lhuillier and Dryer 1999). The unitary conductance of single large-conductance KCa channels, derived from all-points histograms, was indistinguishable in ciliary and choroid neurons. In both cell types, this was 120 pS with [K+]o = 37.5 mM and [K+]i = 150 mM (data not shown). Open-time distributions were constructed from idealized data ignoring transitions of <0.5 ms duration. Single exponential curves provided excellent fits to the resulting distributions in both cell types. The open-time histograms shown in Fig. 6, A and B, were constructed from patches that contained only one KCa channel based on maximum current amplitudes observed at high Po. It should be noted that the time constants of the fitted exponential curves were significantly longer in choroid cells than in ciliary cells (Fig. 6, A and B). Mean time constants pooled from several excised patches were in good agreement with mean time constants obtained from fluctuation analysis (Fig. 6C).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 5. Examples of large-conductance KCa unitary currents in inside-out patches excised from identified CG neurons isolated at E13. Representative 2-s traces from a ciliary neuron (A) and a choroid neuron (B) before and after bath application of 5 µM Ca2+ as indicated. Closed and open states are indicated to the left of the traces. Patches were held at +25 mV. Note that these patches were quiescent in Ca2+-free bath salines, and robust activation of a single KCa channel after Ca2+ application. Note also the greater frequency of channel closure in the presence of Ca2+ in the patch excised from the ciliary neuron.



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 6. Different mean open-times of large-conductance KCa channels in inside-out patches excised from E13 ciliary and choroid neurons. A: properties of KCa unitary currents in a ciliary neuron. Trace to the left shows 200-ms inset of data from a patch recorded in the presence of 5 µM free Ca2+ at +25 mV. Middle graph shows probability of KCa opening (Po) obtained from 10 s of continuous data in the presence of 5 µM free Ca2+. Graph to the right shows open-time histogram constructed from this patch, with superimposed fitted single-exponential curve with a time constant of 6.0 ms. B: properties of KCa unitary currents in a choroid neuron. Note substantially longer time constant (18.7 ms) of single-exponential fit to the open-time histogram. Data in A and B are from patches that contained only 1 KCa channel. C: mean time constants of open-time histograms and SE obtained from several ciliary and choroid neurons as indicated. These means are significantly different and indicate substantially longer mean KCa open-time in choroid neurons.

Different modes of KCa regulation in developing choroid and ciliary neurons

We have previously shown that large CG neurons isolated at E9 and allowed to develop in culture for 4 days do not express macroscopic KCa at significant levels, in distinct contrast to CG neurons isolated acutely at E13 (Cameron et al. 1998, 1999; Dourado and Dryer 1992; Subramony and Dryer 1997; Subramony et al. 1996). Application of iris extracts (Subramony et al. 1996) or target-derived differentiation factors (Cameron et al. 1998, 1999) restores normal expression of KCa in CG neurons developing in vitro. The majority of those recordings were made from cells that, based on their size, were likely to be ciliary neurons. To determine whether the two cell types achieve their mature (E13) current density by different developmental mechanisms, we initially determined whether the two cell types acquire robust macroscopic KCa at the same developmental stages. Approximately half of the CG neurons at E9 are comprised of choroid cells (Landmesser and Pilar 1974). Therefore robust expression of KCa in E9 choroid neurons would predict a distinctly bimodal distribution of current densities and an inverse correlation between current density and cell size in the entire population of CG neurons. This was not observed in a large number of CG neurons isolated acutely at E9 (E9 + 0 in vitro cells), with care taken to sample neurons spanning the full range of neuronal diameters apparent in these preparations (Fig. 7A). Instead, the distribution of current densities was unimodal, and there was no correlation between current density and cell size either within a group or when the entire population of CG neurons was considered. These data indicate that very few cells of either type express KCa at significant densities by E9, in contrast to E13, when both cells express robust KCa at a comparable current density (Fig. 2). Does the developmental expression of KCa in choroid neurons require inductive interactions, as observed previously for ciliary neurons? To test this hypothesis, CG cells were isolated at E9, a stage prior to significant synapse formation with target tissues in either cell type. Neurons were then allowed to develop in vitro for 4 days in standard culture medium (E9 + 4 in vitro cells), at which time KCa expression was determined by means of whole cell recordings. As with our previous studies, we found that KCa was either undetectable or expressed at very low density in E9 + 4 in vitro ciliary neurons. In contrast, macroscopic KCa expression was robust in E9 + 4 in vitro choroid neurons and the current density was comparable to that observed in CG neurons isolated acutely at E13 (Fig. 7B). These differences are statistically significant. As expected from these data, the distribution of KCa densities in the entire sample of E9 + 4 in vitro CG neurons was clearly bimodal with a statistically significant inverse correlation between cell size and KCa current density when the entire population of CG cells was considered (Fig. 7C). There was no correlation between cell size and current density within a group. In other words, qualitatively different distributions of current density were observed in E9 + 4 in vitro CG neurons compared with cells isolated acutely at either E9 or E13. These results support the hypothesis that KCa is regulated cell-autonomously in small choroid neurons, and clearly indicate different KCa regulatory mechanisms in developing choroid and ciliary neurons.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7. Different modes of developmental regulation of macroscopic KCa in ciliary and choroid neurons. CG neurons were isolated at E9 and used for electrophysiology immediately after isolation (E9 + 0 in vitro cells) or maintained in vitro for an additional 4 days (E9 + 4 in vitro cells) in the absence of target-derived factors. A: current density distribution for whole cell KCa appears unimodal in acutely isolated E9 + 0 in vitro CG neurons (left), and the raw data are not significantly different from a normal distribution (Kolmogorov-Smirnov 1-sample test). There is no correlation between cell size and KCa current density at this stage of development (right), indicating that neither neuronal population expresses substantial macroscopic KCa at E9. B: mean whole cell KCa current density is significantly greater in E9 + 4 in vitro choroid neurons than in ciliary neurons. C: the entire data set shown in B is plotted as a histogram (left) showing bimodal KCa current density distribution that is significantly different from a normal distribution (Kolmogorov-Smirnov 1-sample test). There is a significant negative correlation between cell size and KCa current density (right), further indicating a significant difference between choroid and ciliary neurons. Solid line is best fit by linear regression of these data.

Differences in KCa stimulating activity of choroid coat and iris/ciliary body extracts

Given the differences in the functional regulation of KCa expression in choroid and ciliary neurons, it was of interest to determine whether choroid neuron target tissues in the eye express KCa stimulatory factors. We have previously shown that ciliary neurons that develop in vitro can be induced to express macroscopic KCa by iris extracts or by target-derived factors such as TGFbeta 1/4 (Cameron et al. 1998, 1999; Subramony et al. 1996). In the present experiments, we prepared extracts of the choroid smooth muscle layer and the iris/ciliary body in parallel, using procedures described previously (Cameron et al. 1998, 1999; Subramony et al. 1996). CG neurons were isolated at E9 and cultured for 12 h in the presence or absence of choroid extract and/or iris extract. Recordings were then made from CG neurons, at least one population of which express functional KCa in response to iris extracts (Cameron et al. 1998, 1999; Subramony et al. 1996). As described previously, iris extracts evoked robust stimulation of KCa expression in CG neurons, probably owing to the ciliary neurons present in these cultures (Fig. 8). In contrast, application of choroid extracts at several concentrations did not evoke KCa expression in CG neurons.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 8. Effects of choroid smooth muscle extracts and iris extracts on KCa expression in CG neurons. CG neurons were dissociated at E9 and cultured for 12 h in the absence or in the presence of target tissue extracts at the concentrations indicated. KCa density was then determined by whole cell recordings. Treatment with iris extracts, but not choroid layer extracts, evokes a robust stimulation of KCa.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Choroid and ciliary neurons of the chick CG are both derived from cranial neural crest and undergo key developmental events at the same embryonic stages (reviewed in Dryer 1994). However, the two cell populations of cells differ in several mature phenotypic traits (De Stefano et al. 1993; Dryer 1994; Epstein et al. 1988; Marwitt et al. 1971; Ullian et al. 1997). Some of these, such as differences in neuropeptide expression, are regulated by cell-cell interactions during development (Coulombe and Nishi 1991; Coulombe et al. 1993; Darland and Nishi 1998; Darland et al. 1995). In this study, we have shown that the biophysical properties of large-conductance (BK-type) KCa channels, and the mechanisms controlling their developmental expression, are different in choroid and ciliary neurons of the chick CG. The unitary conductance of KCa channels is the same in both cell types. However, ciliary neurons express KCa channels with a relatively short open-time, which is associated with more rapid macroscopic activation and deactivation kinetics in ciliary neurons. Moreover, functional expression of KCa channels in ciliary neurons is dependent on cell-cell interactions during development, whereas choroid neurons regulate these channels by an apparently cell-autonomous mechanism. Consistent with this, KCa stimulatory factors are present in the iris, one of the target tissues of ciliary neurons, but are not expressed in the choroid smooth muscle layer, the target tissue of the choroid neurons.

This pattern may not be unique to the chick CG. Multiple cell populations with distinct electrophysiological properties are present in many autonomic ganglia and central nuclei. To cite just one example, B-cells and C-cells within bullfrog paravertebral sympathetic ganglia receive afferents from different spinal segments and innervate different target tissues (Dodd and Horn 1983; Horn et al. 1988; Smith 1994). These cells also have different conduction velocities, action potential waveforms, and intrinsic patterns of repetitive spike discharge (Smith 1994). Differences in the developmental expression of ionic channels may often occur in neighboring neurons that project to physiologically distinct target cells. In such cases, differential regulation of ion channel expression by inductive cell-cell interactions may provide a mechanism to ensure that the intrinsic properties of pre- and postsynaptic elements are appropriately matched. In this regard, we have observed that expression of macroscopic KCa is regulated by soluble trophic factors in essentially all chick lumbar paravertebral sympathetic neurons, but that only a subpopulation of these respond to nerve growth factor (Raucher and Dryer 1995).

The intrinsic properties of ciliary and choroid neurons are well matched to those of their respective target tissues. Specifically, ciliary neurons need to be able to discharge spikes at a sustained high frequency (>100 Hz) because tetanic contractions of their striated muscle targets in iris require stimulation at this range of frequencies (Pilar and Vaughan 1969). The relationship between stimulus frequency and contraction has not been studied in choroid smooth muscle, but ocular smooth muscles in other species exhibit maximum contractions at stimulation frequencies as low as 10 Hz (Suzuki 1983). It is not known whether KCa kinetics can account for these differences in CG neuron repetitive firing. However, the slower macroscopic kinetics of KCa in choroid neurons would predict a longer duration spike duration, as is observed (Dryer 1994). Other aspects of the behavior of choroid and ciliary neurons are quite similar under voltage clamp, and this is the first consistent difference that we have observed over many years of investigation. In this regard, it bears noting that neurons whose dominant outward currents exhibit more rapid activation and deactivation kinetics tend to have shorter duration action potentials and are able to follow high frequencies of synaptic stimulation (reviewed in Gan and Kaczmarek 1998; Rudy et al. 1999).

The molecular basis for the different developmental and kinetic properties of KCa channels in the two CG cell types is not known, but it is very plausible that these observations share a common mechanistic basis. One possibility is that the two cell types express different slo splice variants. Cell-specific expression of different slo variants has been observed in cochlear hair cells of the chick (Navaratnam et al. 1997; Rosenblatt et al. 1997) and turtle (Jones et al. 1999), as well as in the mammalian CNS (Tseng-Crank et al. 1994) and in Drosophila (Becker et al. 1995). Differences in KCa kinetics in hair cells are known to have large effects on oscillatory membrane behavior (reviewed in Fettiplace and Fuchs 1999). We have previously detected two different slo partial cDNAs in the chick CG. These cDNAs correspond to transcripts that differ by the presence or absence of a single 28 amino acid exon present in one isoform but not the other (Subramony et al. 1996). This alternative exon is located close to the C-terminus, and variations in this portion of the molecule do not produce particularly large differences in the kinetics of SLO channel alpha subunits expressed in heterologous systems (Ramanathan et al. 1999, 2000). However, it is certainly possible that variability in this portion of the channel molecule could result in differences in plasma membrane targeting. Preliminary data from single-cell RT-PCR indicate that both cell types express the smaller of these two slo splice variants, but to date these experiments have been inconclusive as to possible differential expression of the larger variant (data not shown).

It is also possible that the two populations of CG neurons differentially express auxiliary (beta) subunits that result in different KCa kinetics (Ramanathan et al. 1999, 2000) and/or developmental regulatory mechanisms. This hypothesis is especially attractive because avian beta subunits produce rather more substantial effects on KCa kinetics (Ramanathan et al. 1999, 2000), and could also play a role in membrane targeting. In this regard, we have recently obtained evidence that a significant component of the trophic regulation of KCa expression in ciliary neurons entails trafficking of preexisting intracellular KCa channels to the plasma membrane. Moreover, target-derived factors such as TGFbeta 1, which cause robust stimulation of functional KCa expression in ciliary neurons, do not affect expression of slo transcripts in those cells (Lhuillier and Dryer 2000). Therefore it is possible that auxiliary subunits selectively expressed in one CG cell type regulate plasma membrane insertion and the kinetics of KCa channels. There is precedent in the literature for these types of mechanisms. For example, two different proteins isolated from Drosophila have been shown to interact with SLO channels and inhibit their insertion into the plasma membrane (Schopperle et al. 1998; Xia et al. 1998). One of these, known as dSLIP1, can also interact with mammalian SLO channels (Xia et al. 1998). Vertebrate homologues of dSLIP1 have not been identified, but it is certainly possible that multiple SLO interacting proteins exist, some of which could act selectively on specific SLO isoforms. These types of interactions are not unique to KCa channels. For example, auxiliary subunits of various voltage-activated K+ channels appear to regulate both the gating kinetics and the efficiency of plasma membrane insertion in heterologous systems (e.g., Fink et al. 1996; Salinas et al. 1997; Shi et al. 1996).

In summary, we have shown that two closely related populations of parasympathetic neurons that innervate different target tissues express large conductance KCa channels with different gating properties. These neurons regulate the functional expression of KCa channels by different mechanisms, which may allow for appropriate matching between the intrinsic firing properties of the neurons and the physiological properties of their target tissues.


    ACKNOWLEDGMENTS

We thank R. Nishi for assistance with somatostatin immunocytochemistry.

This work was supported by National Institutes of Health Grants NS-32748 to S. Dryer and MH-12758 to J. Cameron.


    FOOTNOTES

Address for reprint requests: S. E. Dryer (E-mail: SDryer{at}UH.EDU).

Received 13 April 2000; accepted in final form 10 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society