Bovine Versus Rat Adrenal Chromaffin Cells: Big Differences in BK Potassium Channel Properties

Peter V. Lovell, Dustin G. James, and David P. McCobb

Department of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lovell, Peter V., Dustin G. James, and David P. McCobb. Bovine Versus Rat Adrenal Chromaffin Cells: Big Differences in BK Potassium Channel Properties. J. Neurophysiol. 83: 3277-3286, 2000. Both bovine and rat adrenal chromaffin cells have served as pioneering model systems in cellular neurophysiology, including in the study of large conductance calcium- and voltage-dependent K+ (BK) channels. We now report that while BK channels dominate the outward current profile of both species, specific gating properties vary widely across cell populations, and the distributions of these properties differ dramatically between species. Although BK channels were first described in bovine chromaffin cells, rapidly inactivating ones were discovered in rat chromaffin cells. We report that bovine cells can also exhibit inactivating BK channels with varying properties similar to those in rat cells. However, a much smaller proportion of bovine cells exhibit inactivating BK current, the proportion of the total current that inactivates is usually smaller, and the rate of inactivation is often much slower. Other gating features differ as well; the voltage dependence of channel activation is much more positive for bovine cells, and their rates of activation and deactivation are faster and slower, respectively. Modeling studies suggest that channel heterogeneity is consistent with varying tetrameric combinations of inactivation-competent versus -incompetent subunits. The results suggest that chromaffin BK channel functional nuances represent an important level for evolutionary tailoring of autonomic stress responses.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Decades-old studies of adrenal catecholamine systems highlighted dramatic species differences in adrenal capacity and in relative proportions of epinephrine (EPI) and norepinephrine (NE) containing chromaffin cells (Coupland 1953, 1965; West 1955). Although the chromaffin cell has become a premier model for mechanistic studies of neurosecretory effector mechanisms, comparative studies of chromaffin function have languished. We argue that, in the current climate of definitive characterization of structure-function relationships of ion channels, the BK (for big K+) calcium- and voltage-gated potassium channels in chromaffin cells provide a unique opportunity to relate channel structure-function, and equally importantly, its regulation, to broader issues of neural system function, and its evolutionary significance. No neural cell is less-invasively isolated from adult animals with its entire complement of channels ready for physiological and molecular studies. Moreover, chromaffin cells are perhaps unique in the degree to which their voltage-dependent K+ currents are dominated by a narrowly defined, and itself uniquely distinct, class of channels. With all BK channels apparently encoded by the Slo gene, functional bases for BK channel heterogeneity are being rapidly attributed to aspects of structural variation, including alternate splicing and varying association with accessory subunits, bringing within reach an integrative stretch from gene to system-level function.

BK channels were first described in bovine chromaffin cells (Marty 1981; Marty and Neher 1985). A functional variant exhibiting rapid intrinsic inactivation with constant calcium and depolarization has since been thoroughly characterized in rat adrenal chromaffin cells (Lingle et al. 1996; Solaro et al. 1995). Evidence for similar inactivating BKi channels has also been presented for pancreatic beta  islet cells (Li et al. 1999; Smith et al. 1990), hippocampal pyramidal neurons (Hicks and Marrion 1998; McLarnon 1995), and bovine chromaffin cells (Solaro and Lingle 1992); however, characterization of bovine BK channels is incomplete.

Even nuances of BK gating are likely to effect firing responses of chromaffin cells. Comparisons between subsets of rat chromaffin cells preferentially expressing either BKi or BKs currents suggested corresponding differences in repetitive firing properties; the former sustained better repetitive firing during a depolarizing stimulus (Solaro et al. 1995). However, the improved repetitive firing may be attributed to slower deactivation kinetics, characteristic of the BKi channels, rather than to inactivation per se. A slower deactivation in BK channels should give rise to a larger afterhyperpolarization (AHP), facilitating repetitive firing by hastening the recovery of Na+ and Ca2+ channels from inactivation accrued during an action potential. Similarly, the rate of BK channel activation should also affect the AHP, since it will affect the number of channels that manage to open during a brief action potential.

Heterogeneity in activation and deactivation gating may be derived in part from alternative splicing at one site in transcripts of Slo, the only known gene-encoding channels of this type (Jones et al. 1999a,b; Ramanathan et al. 1999; Saito et al. 1997; Xie and McCobb 1998). Oocyte expression of the two splice variants that are abundant in chromaffin cells reveals that inclusion of an optional exon ("STREX") results in a negative shift in the voltage dependence of activation and slows deactivation, compared with its omission in the "ZERO" form (Xie and McCobb 1998). Intriguingly, this splicing decision appears to be subject to regulation by the hypothalamic-pituitary-adrenocortical stress axis (HPA), raising the possibility of hormonal regulation of firing properties (Xie and McCobb 1998). The recent discovery that a beta  subunit can confer inactivation on BK channels (Wallner et al. 1999; Xia et al. 1999) suggests that accessory subunit(s) underlie some heterogeneity in BK gating.

In the present study, we describe profound differences in the distribution of BK gating properties between rat and bovine chromaffin cells. A modeling approach similar to that proposed by Ding et al. (1998) is also described, addressing issues of channel stoichiometry and testing a model of chromaffin cell heterogeneity in bovine and rat adrenal glands. We discuss the implications of species-specific patterns in relation to BK structure-function relationships, catecholamine secretion, and the possibility of an endocrine organizational influence on chromaffin BK properties.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chromaffin cell isolation and culture

Medullary tissue from 17 bovine (Cudlin's Meat Market, Newfield, NY) and 12 Sprague Dawley rat (Charles River Laboratories, Wilmington, MA) adrenal glands was dissected from surrounding cortical tissues, minced in sterile Hank's saline (GIBCO, Grand Island, NY), and incubated in collagenase B (Boehringer Mannheim, Indianapolis, IN; 1.5 mg/ml in Hank's) for 60 min at 37°C with gentle agitation. Minced tissue was then washed in Ca2+- and Mg2+-free Hank's saline (GIBCO), and transferred into trypsin (GIBCO; 0.125% in Ca2+- and Mg2+-free Hank's) for 30 min at 37°C. Following several washes in prewarmed cell culture medium (GIBCO; RPMI 1640 with 10% horse serum, 5% fetal calf serum, 2 U/ml penicillin-G, 2 µg/ml streptomycin sulfate, 100 U/ml nystatin), tissue was triturated with fire-polished pasteur pipettes to dissociate chromaffin cells. One hundred microliters of the cell suspension was then aliquoted into 15-mm glue rings in 35-mm plastic dishes (Falcon 3001, Fisher Scientific, Pittsburgh, PA) coated with collagen (Vitrogen, Collagen Corporation, Carlsbad, CA; 0.6 mg/ml in ddH2O) or poly-D-lysine (Sigma, St. Louis, MO; 0.01% in ddH2O). Cell cultures were maintained at 37°C in a 5% CO2 atmosphere and used for several days.

Electrophysiological methods

Single-channel and macroscopic currents were recorded using standard patch-clamp techniques as described previously (Hamill et al. 1981; Sakmann and Neher 1985). In short, patch electrodes (3-6 MOmega ) were pulled from borosilicate capillary glass (World Precision Instruments, Sarasota, FL; 1.5 mm ID, 1.12 mm OD) and coated with silicone elastomer (Sylgard 184; Dow Corning, Midland, OH) to decrease capacitance. Voltage-clamp electrophysiology data were collected with a List EPC-7 patch-clamp amplifier using standard clamp protocols designed with Pulse software (Heka Elektronik, Lambrecht, Germany) for the Macintosh Power PC 8100/80. Data were acquired and digitized at 20 kHz sampling rate with an ITC-16 A/D converter (Instrutech, Great Neck, NY), and stored on a Macintosh Power PC. Off-line analysis of clamp data were performed using custom software in Igor Pro (Wavemetrics, Lake Oswego, OR).

All experiments were conducted at room temperature, 20-22°C. Inside-out and outside-out patches were pulled following applied suction and seal resistances of 3-6 GOmega . Inside-out patches were excised in the presence of zero [Ca2+] as described below. Solution exchange and drug applications were accomplished with a seven-barrel gravity-fed perfusion pipette.

Solutions

Symmetrical K+ solutions were used throughout to eliminate a potassium driving force and allow any DC offset to be cancelled at 0 mV. For inside-out patch recordings, the usual pipette and bath saline solutions contained (in mM) 160 KCl and 10 HEPES, pH adjusted with KOH to 7.4 to make 6 µM free [Ca2+]. Free [Ca2+] was confirmed using an ORION 97-20 Ionplus Ca2+ selective (Orion Research, Beverly, MA) calibrated against a standard [Ca2+] series (0.1 M Ca2+ stock, Orion Research) and adjusted as necessary. Zero Ca2+ solution contained an additional 5 mM EGTA (Sigma). Type XI trypsin (0.5 mg/ml; Sigma) was perfused onto inside-out patches for trypsin experiments. For outside-out recordings, perfused and pipette salines were the same as those used in the pipette for inside-out patches. When pharmacology experiments were performed, charybdotoxin (CTX; 10 nM; Alomone Labs, Jeruselum, Israel), or tetraethylammonium (TEA+; 1 mM; ICN Biomedicals, Aurora, OH) were added to the bath-perfused salines and pH adjusted to 7.4. Osmolarity of salines were measured by dew point osmometer and adjusted to 300 osmM.

Data collection and statistical analysis

Single- and multi-channel currents were linear leak subtracted and ensemble currents fit using a Levenberg-Marquardt search algorithm to obtain nonlinear least-square measurements of kinetic parameters. The phenotype of BK currents recorded from patches containing 5-30 channels was found to accurately represent currents expressed in the whole chromaffin cell. This was confirmed by comparing currents from as many as three patches pulled from a single cell. The fraction of inactivated current (BKi/BKtotal) was measured by calculating a ratio of the BK current at 350 ms as a function of the estimated peak BK current. Estimates of BK tail current decay were made using Levenberg-Marquardt search algorithm for both single and double exponential fits to at least 70% of the waveform. In most cases single exponential fits were found to be acceptable. Likewise, the time constants of inactivation and activation were measured using a similar technique. The calcium-voltage dependence of activation (G-V) and inactivation (Hinf) were evaluated by measuring peak amplitudes at increasing voltage steps of 20 mV, converting these to conductances by dividing out the driving force, and fitting G-V and Hinf curves to a single-term Boltzmann of the form
<IT>G</IT><SUB><IT>V</IT><SUB><IT>m</IT></SUB></SUB><IT>=</IT><IT>G</IT><SUB><IT>max</IT></SUB><IT>/1+exp</IT>[(<IT>V</IT><SUB><IT>m</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>0.5</IT></SUB>)<IT>/s</IT>]
with parameters for maximum conductance (Gmax), voltage of half-activation (V0.5), and slope (s; the steepness of the voltage dependence of activation, in mV/e-fold change in voltage). Statistical comparisons of the fraction of inactivated current, time constant of inactivation, time constant of deactivation, and voltage of half-activation were evaluated using a Mann-Whitney U test for mean differences (alpha  = 0.05). Unless otherwise noted in the text, quantitative analyses were performed on data collected from patches held at -100 mV; earlier data from patches held at -80 mV showed very similar inactivating component for both species, on average. Correlations between BK channel properties were made using a Spearman Rank correlation test (alpha  = 0.05).

Fitting current waveforms

BK current waveforms were simulated with a modified Hodgkin-Huxley (1952) activation/inactivation model. Individual BK channels were modeled as tetramers comprised of varying combinations of independent subunit-entities that were, dichotomously, either inactivation-competent or inactivation-incompetent (after Ding et al. 1998 and MacKinnon et al. 1993). Five-channel stoichiometries were assumed to be functionally distinct; the two-channel configurations with adjacent and opposite subunit arrangements were assumed to have identical properties. The channel form with no inactivation-competent subunits did not inactivate. For those with one or more, the inactivation rate increased linearly with increasing number of inactivation-competent subunits. Total currents then represent the binomial expansion from overall subunit breakdown (percentages, types, and total number) as proportionate summations of the five-channel stoichiometries. Parameters used in the simulation were identical for models of bovine and rat BK currents. The single-channel peak current (Imax = 22.4 pA) was estimated from a 280-pS channel conductance activated at +80 mV in a symmetrical K+ solution. Time constants of inactivation (tau i) were determined for each of the five-channel stoichiometries by a linear interpolation between empirical exponential fits of the average fastest and slowest single-channel currents showing apparently different rates of inactivation. According to the model presented by Ding et al. (1998), time constants of activation (tau m) were fixed at 2.5 ms for each of the five channel types. Hence a channel with four inactivation domains (4 bki subunits, to distinguish it from BKi channels) was estimated to have a tau m of 2.5 ms and tau i of 25 ms with a cooperativity factor of 1.0. Rate constants for channels with a decreasing proportion of bki subunits were fixed at the following values in ms: (3 bki) tau i = 33.3; (2 bki) tau i = 50; (1 bki) tau i = 100; (0 bki) tau i = infinity .

A subset of BK currents recorded from bovine and rat inside-out patches were fit to the model described above using a Levenburg-Marquardt search algorithm, and number of channels (N) and percentage of bki subunits was determined. Error analysis of fits was performed using calculated residuals and a standard error term. Small patches (<3 channels) were excluded from the analysis.

The model was extended to address the issue of whether the modeled distributions of inactivation-competent subunits represent single, unimodally varying populations, or alternatively, bimodal distributions of cell types. A standard angular transformation [angle = arc sine (squareroot of %bki)] of the raw fit data were used to spread the variance around the 0 and 100% constraining boundaries. The mean and standard deviation of the transformed data were used to generate a gaussian distribution. Values from the gaussian distribution were then reverse transformed for comparison with a histogram of the raw data.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bovine chromaffin cells express inactivating and noninactivating BK channels

While the kinetics of inactivation vary widely, bovine adrenal chromaffin cells can express rapidly inactivating BK currents similar to those that have been characterized in rat chromaffin cells (Solaro et al. 1995). We find that approximately 10% (4 of 48 at Vhold = -100 mV) of bovine cells exhibit rapid and virtually complete inactivation of BK current, while many more exhibit a significant component of inactivating BK current in addition to a noninactivating component. The percentages of patches exhibiting exclusively BKi or BKs type currents, as compared with a mix, are shown in Table 1.


                              
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Table 1. Number of excised inside-out patches containing only BKi, BKs, or both BKi and BKs in bovine and rat chromaffin cells

BK channels are defined by the synergistic effects of submembrane calcium and membrane depolarization on channel activation, as well as a large single-channel conductance. BK currents were found to dominate the voltage-dependent outward current profile in bovine chromaffin cells, as has been shown true for rat cells (Solaro et al. 1995). In some inside-out patches exposed to 6 µM Ca2+, depolarizing steps from very negative holding potentials (Vhold -100 mV) activated approximately 280 pS K+ channels, which in ensemble records exhibited virtually complete inactivation with a time constant as low as 20 ms (Fig. 1C). The same patch exhibited very few channel openings in a single trace shown in zero [Ca2+] (Fig. 1C). As with rat chromaffin patches, the properties of calcium-dependent K+ current activation and inactivation in bovine cells were dependent on membrane voltage (Fig. 1, A and B). Further confirming the identification, inactivating K+ currents recorded in outside-out patches were reduced approximately 93% by 1 mM TEA+ (n = 10) and 80% by 10 nM CTX (n = 5; Fig. 1, E and D), values characteristic for rat and bovine BK channels (Ding et al. 1998; Neely and Lingle 1992a). Moreover, as has been observed for BKi channels in rat chromaffin cells (Ding et al. 1998; Solaro and Lingle 1992), exposure of inside-out patches to the protease trypsin resulted in a gradual removal of inactivation, typically accompanied by a 30-60% increase in peak BK current amplitude (Fig. 1F).



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Fig. 1. Many bovine chromaffin cells express rapidly inactivating, voltage- and calcium-dependent BK current. A: predominantly BKi currents elicited by steps to +80 mV following 350-ms conditioning prepulses to potentials indicated to the left. In B, maximal current amplitudes from the patch in A were normalized to the peak current activated at +80 mV and plotted as a function of the conditioning potential. Boltzmann functions were used to fit the voltage dependence of activation (tau m) and inactivation (tau h). In C through F, ensemble averaged currents are shown from patches with between 10 and 30 channels with currents elicited by steps to +80 mV from holding potentials indicated. C: rapid and nearly complete inactivation of currents recorded during exposure of an excised inside-out patch to 6 µM Ca2+ were virtually absent in the single trace shown in zero Ca2+. Reintroduction of the Ca2+ solution quickly restored the currents (not shown). In D and E, partially inactivating current recorded from outside-out patches were reversibly reduced by 80% in 10 nM charybdotoxin (CTX) and 93% by 1 mM TEA+, respectively (wash outs not shown). F: 5-min application of trypsin (0.5 mg/ml) to the cytoplasmic face of an inside-out patch exhibiting mixed BK currents abolished inactivation and increased the amplitude of BK current by approximately 30%.

The inactivation properties of BK currents varied over a wide range between individual cells in both bovine and rat chromaffin cell populations. Patches with just one or two channels revealed unequivocally that individual channels exhibit wide variability in the rate at which they enter the inactivated state (Fig. 2). Channels in some patches inactivated relatively quickly during the 350-ms holding step (Fig. 2A), while others showed delayed entry into the inactivated state (Fig. 2B) or even a complete lack of inactivation (noninactivating channel in Fig. 2C). Ensemble averages of the traces shown in A-C further reveal qualitative differences in both the proportion and rate of inactivation, the fastest of which decayed in 53 ms (patch in A), the slowest in 101 ms (patch in B; Fig. 2D).



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Fig. 2. Inactivation kinetics vary between single BK channels in bovine chromaffin patches. A: rapid inactivation exhibited in a patch containing probably just 2 channels. All patches in Fig. 2 were inside-out, with both faces exposed to the same K+ solution containing 6 µM Ca2+. Steps to +80 mV were separated by 3 s at -100 mV. A representative subset of the numerous traces obtained from each patch is shown. B: slower inactivation exhibited in a single-channel patch. C: a patch containing at least 1 inactivating and 1 noninactivating BK channel. D: ensemble averages representing 99, 35, and 99 sweeps from patches in A through C. Averaged traces from patches in A and B were fit with a single exponential function yielding time constants of inactivation of 53 and 101 ms, respectively.

Distributions of BK inactivation properties differ dramatically between species

While the range of properties observed in patches was similar between bovine and rat chromaffin cells, the distribution of BK channel properties across the respective cell populations differed strikingly in measures of activation, deactivation, and inactivation gating. Differences in the representation of inactivation were most immediately apparent (Table 1 and Fig. 3). Similar to previous categorical descriptions (Solaro et al. 1995), we found that rat chromaffin patches expressing exclusively BKi current constituted 67.1% of the 70 patches sampled (including only patches for which the holding potential was -100 mV from 9 animals). BKi and BKs currents were defined as exhibiting at least 90% of complete inactivation or noninactivation, respectively, 350 ms after the start of a depolarizing test step to +80 mV (note that 350 ms is well beyond the time required for complete inactivation in the vast majority of BKi cells). An additional 31.4% of rat patches expressed a substantial inactivating component, while only a small minority (1.4% BKs) exhibited virtually no inactivation over 350 ms. This categorization is in stark contrast with that for 48 bovine chromaffin patches treated with the same voltage protocol and identical solutions. Only a small minority of patches (8.3%) could be defined as exclusively BKi, a substantial fraction exhibited virtually no inactivation (20.8% BKs), and the majority exhibited partial inactivation (70.8%). Thus rat adrenals have eight to nine times as many cells expressing purely BKi currents as do bovine adrenals. A similar breakdown was observed for additional patches held at -80 mV before the test step, as detailed in Table 1. We further calculated the average proportion of the peak BK current that had inactivated by 350 ms (BKi/BKtotal) in each patch, a measure of the fraction, but not the kinetics of inactivation. We found considerable variation in proportion of BKi/BKtotal across the populations, the bovine adrenal distribution exhibiting a positive skew in favor of a noninactivating component, while the rat showed nearly the opposite skew (Fig. 3B). Average values for rat and bovine patches (89.5 ± 2.0 vs. 42.5 ± 4.6%, mean ± SE, respectively) were significantly different according to a nonparametric Mann-Whitney U test for mean differences (P < 0.0001).



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Fig. 3. Rate and extent of BK current inactivation both differ strikingly between rat and bovine chromaffin cells. A: representative examples of BK currents recorded from rat and bovine chromaffin cells (left and right panels, respectively), illustrating the variety and range of BK current profiles. All patches were inside-out, exposed to identical symmetrical K+ solutions with 6 µM Ca2+, and contained 10-20 channels. Traces are ensemble averages of between 35 and 99 steps. The time constant of inactivation as measured by a single exponential function is shown to the top right of each trace. B: frequency distributions of values of BKi/BKtotal were very different for the 2 species, with a much higher proportion of the 93 rat patches showing complete or nearly complete inactivation, while the 104 bovine patches were more typically dominated by sustained BK current (P < 0.0001). C: the average rate of inactivation was determined by fitting a single exponential function to the inactivating component of ensemble currents in a subset of patches that excluded those exhibiting either very slow or noninactivating currents. Measured rates differed significantly between species (P = 0.0001) for 71 bovine and 90 rat patches (Vh = -100 and -80.)

In accordance with measures of the proportion of inactivating current at 350 ms, bovine cells exhibit extreme variation in the rate at which they inactivate (tau i). To measure this value, we fit curves to the current decay from the peak with a single exponential equation of the form: I(t) = I0 * exp(-t/tau i). Although the inactivation process in these patches is likely to be comprised of multiple exponential components, derived from single channels expressing very different rates of inactivation, single exponential functions fit the traces closely, and were used for comparison, according to precedent (Ding et al. 1998). Patches exhibiting exceedingly slow or no inactivation were excluded from the analysis of average inactivation rates. Bovine patches from 17 animals (n = 71) showed significantly slower average kinetics of inactivation (91.2 ± 8.2 ms) than comparable rat patches (n = 90; 56.0 ± 3.4 ms; P = 0.0001, Fig. 3C). Species differences in the kinetics of inactivation support a comparative binomial modeling analysis to explain variation in BK channel inactivation rates, as discussed below.

Rates of BK current activation and deactivation differ between rat and bovine chromaffin cells

The rate of BK channel activation and deactivation may be critical in chromaffin cell repetitive firing. Because Solaro et al. (1995) previously noted that the deactivation rate for the currents in BKi-dominated cells was typically slower than that for BKs-dominated cells, and that this dichotomy apparently paralleled repetitive firing characteristics, we wished to examine whether or not deactivation and activation kinetics were similar for bovine cells that tend to be dominated by BKs currents. We found that BK currents in bovine patches exhibited a more rapid deactivation than those in rat patches. Deactivation kinetics were measured from tail currents that followed a repolarizing step to -80 mV following a 15-ms step to +90 mV, sufficient to ensure complete activation of both BKi and BKs channels (see Fig. 4A). A single exponential fit of the form: I(t) = I0 exp(-t/tau d) adequately described the process and was used for direct comparisons. The time constant of deactivation (tau d) of bovine cells averaged 2.7 ± 0.4 ms (n = 32; Fig. 4B). This was a significantly faster than the rat average of 4.7 ± 0.3 ms (n = 62; P < 0.0001). Although activation rate measurements are potentially complicated by temporal overlap with the inactivation process, exponential fits to the activation phase of currents revealed faster activation kinetics for rat (2.0 ± 0.3 ms; n = 46) than bovine cells (5.4 ± 1.0 ms; n = 29). As observed for the time constant of inactivation, differences in activation and deactivation (Fig. 4C) between species were large despite rather wide variation across cells within a species.



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Fig. 4. Bovine chromaffin BK currents exhibit significantly faster kinetics of deactivation than rat chromaffin BK currents. Inward tail currents were elicited by steps to -80 mV from a brief 15-ms step to +90 mV (voltage protocol shown below currents in A; solutions as described in Figs. 2 and 3; all patches inside-out). In A, representative BK tail currents are shown for a bovine (tau d = 1.8 ms) and a rat patch (tau d = 5.1 ms). Tail currents were fit to a single exponential of the form [1 - exp (-t/tau d)] to determine the rate of channel closure. B: mean ± SE values for tau d (P < 0.0001). C: frequency distributions illustrate the very different distributions of deactivation rates for bovine (n = 32) and rat (n = 62) patches.

Bovine and rat adrenal chromaffin cells exhibit differences in their voltage dependence of activation

Alternative splice variants of the Slo gene exhibit marked differences in the voltage dependence of activation at a given calcium concentration (Saito et al. 1997; Xie and McCobb 1998), and these differences accompany differences in deactivation kinetics. To determine whether species differences in deactivation are likewise accompanied by differences in the voltage dependence of gating, we ran a series of steps to increasingly positive test potentials, measured peak conductances, and fit a single boltzmann to G-V plots (Fig. 5). The V0.5 for 20 bovine patches averaged 38.3 ± 5.6 mV, as compared with -0.5 ± 3.7 mV for 21 rat patches (Fig. 5B; P < 0.0001). The steepness of the voltage dependence also differed between rat and bovine cell populations (25.1 ± 1.4 and 18.7 ± 1.5 mV/e-fold change in conductance, respectively; P = 0.0014).



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Fig. 5. Bovine and rat chromaffin BK currents differ in the voltage dependence of activation gating. A: bovine BK currents were activated in 6 µM Ca2+ according to the voltage protocol shown above current traces. A series of currents activated at successive voltage steps are shown for BKs-dominated (top), mixed (middle), and BKi-dominated (bottom) patches. Bovine and rat BK current series like those in A were recorded in identical solutions for each patch, peak current values in each trace plotted as a function of holding potential, and peak values fitted with a single Boltzmann equation. Three parameters were obtained: voltage of half-activation (V0.5), slope factor, and maximum conductance (Gmax). In C, composite G-V curves were generated by independently averaging V0.5 and slope coefficients from 20 bovine and 22 rat patches. Peak conductances were normalized by the respective peak conductance obtained with a step to +100 mV. Bovine BK currents were half-activated at significantly more positive voltages than those recorded in rat patches (P < 0.0001). Horizontal error bars at the point of half-amplitude represent the standard error of the mean for V0.5. The steepness of the voltage dependence of activation also exhibited significant variation, as explained further in RESULTS.

The voltage dependence of activation and the kinetics of deactivation were found to covary significantly across the two-species range according to a Spearman Rank correlation (P < 0.0001; Fig. 6A). The pattern of faster deactivation with more negative activation was expected, since shifting the voltage dependence in the negative direction should stabilize the open configuration in preference to the closed. Despite the interdependence of the properties; however, the high degree of variability may in part reflect the possibility that kinetics and voltage dependence can be differentially affected, for example, by association with beta  subunits (Dworetzky et al. 1996; Ramanathan et al. 1999). Similarly, the fraction of the total BK current that inactivated in 350 ms was also correlated with activation voltage dependence (Fig. 6B) and deactivation kinetics (Fig. 6C; P < 0.0001). Thus patches with negative V0.5 values and long tau d values tended to have large inactivating components.



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Fig. 6. Features of BK channel gating, and voltage dependence covary, suggesting an intradependence of key BK channel components. The gating kinetics and voltage dependence of BK currents from bovine and rat patches were measured and correlated with one another in a pairwise fashion. Lines or curves were fit to represent apparent trends. A: the rate of deactivation (tau d) correlates negatively with the voltage of half activation (V0.5) in 19 rat and 20 bovine patches. In B, the proportion of BKi/BKtotal is predicted by the voltage dependence of half activation. C: the fraction of inactivated current approaches the ceiling of 1 as deactivation time increases.

Modeling variation in rates of inactivation across species

Exposure of the intracellular face of native BK channels to trypsin results in a progressive slowing of inactivation (Ding et al. 1998). This led the Lingle group to propose a model wherein variability within the rat chromaffin population was derived from binomial variation in the ratio of inactivation-competent and -incompetent subunits contributing to the tetrameric structure of the BK channels. Although it now seems likely that inactivation is conferred on Slo gene-derived BK channels by an accessory subunit related to the recently cloned beta 2 (Wallner et al. 1999; referred to as beta 3 by Xia et al. 1999), for the purposes of modeling, it is formally equivalent to consider inactivation competence as being conferred by alpha (presumably products of Slo, the only known gene encoding BK-type channels) or beta subunits, provided that each alpha associates with a single beta. Because the bovine population exhibits many more cells with slower and incomplete inactivation than rat, the bovine system provides an important opportunity to further test the model.

Using a similar approach to Ding et al. (1998), we fit ensemble currents from widely varying patches to a mathematical function in which the total current as a function of time is represented as the sum of five current components [each with Hodgkin and Huxley (1952) activation and inactivation rate parameters]. With two subunit types forming tetramers, five distinct channel configurations are possible, assuming that the two configurations having two like subunits either adjacent or opposite are functionally indistinct. We assume one inactivation-competent subunit confers measurable inactivation on a channel, and that the inactivation rate increases linearly with increasing number of competent subunits in one channel. Of the four inactivation rate parameters (for channels with 1-4 inactivation-competent subunits), the extremes roughly equate to the fastest and slowest average rates observed in ensemble currents from rat and bovine patches, and the intermediates are linearly interpolated between them. Despite measurable variability in channel activation, we used a fixed value for each of the channel types described by the model to constrain fit approximations to the kinetics of inactivation. Thus the binomial distribution constrains the relative proportions of the five channel types for a given overall ratio of the two subunit types. Patches with few enough channels that events could be unequivocally attributed to single channels were too infrequent to test for discrete kinetic values, particularly considering the difficulty in counting channels in patches with channels that inactivate rapidly. By letting only the overall ratio and the total number of channels vary during the curve fitting iterations, while the five linearly related parameter sets were invariant, ensemble currents from most multi-channel patches were well described. Figure 7A shows examples of fits to three patches with widely varying proportions of inactivating and noninactivating BK current. Fit deviations to patches did not vary in a consistent pattern across the range of subunit proportions, and decreased with increasing number of channels in the patch. As expected, frequency distributions of estimated values of the percentage of inactivation-competent subunits in 57 rat and 38 bovine patches differed markedly (Fig. 7B; histograms), with bovine cells predicted to express lower proportions of the inactivation competent subunit (18.1 ± 23.1 %bki expressed as the means ± SD) than rat (58.7 ± 24.7 %bki).



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Fig. 7. Differences in bovine and rat inactivation kinetics are predicted by a 2-subunit model of heteromultimeric channel assembly. Rat and bovine BK currents from excised inside-out patches were activated by a step to +80 mV from a holding potential of -100 mV in 6 µM [Ca2+]. Traces were fit with an equation modeling the binomial expansion of proportions of inactivation-competent and -incompetent subunits into 5 channel stoichiometries. Fixed parameters were interpolated for each channel stoichiometry as described in METHODS. The number of channels and percentage of inactivation-competent subunits were free parameters. A: 3 2nd order Savitsky-Golay smoothed ensemble traces (thick line) differing in kinetics of inactivation were fit with the model function (thin line). The predicted percentage of inactivation competent subunits (%bki) and channel number (N) for each patch are shown with each ensemble trace. In B, the distributions of the percentage of inactivation-competent subunits are shown for 38 bovine (top) and 57 rat (bottom) patches. The average predicted percentages of inactivation competent subunits were (18.1 ± 23.1%; mean ± SD) for bovine and (58.7 ± 24.7%). To determine whether or not the underlying population structure reflected a unimodal distribution, the mean and variance of the proportional data (%bki) was used to construct normal gaussian distributions (dotted lines).

With currents from each cell represented by a single value describing the proportion of inactivating subunits, we then used the model to address a separate, higher-level issue; whether the chromaffin population within a species represents a single, unimodally varying population, or alternatively, might be dichotomous with respect to inactivation. To address this, the mean and standard deviation from the transformed values for the binomial proportions of inactivation-competent subunits were used to generate unimodal gaussian distributions (Fig. 7B; dashed lines). Assuming a single bovine cell population with a normal distribution around the mean, we predicted that the number of cells expressing almost exclusively BKs channels should be on the order of 6.39 of 38 (16.8% of patches; Fig. 7B; top panel). The occurrence of cells with purely noninactivating channels (18 of 38 or 47%) therefore greatly exceeds that expected. For the rat chromaffin cell population included in this data set, the number of pure BKs cells was only slightly higher than predicted. However, from other recordings not included because the solutions were different, pure BKs proportion was nearer to 10%. This percentage is similar to the 9.2% reported by Solaro et al. (1995). Moreover, pure BKs patches were probably underestimated in our later experiments. Even a small inactivating component made it easier to establish that a patch was inside-out, and provided full electrical access. We also suspect BKs expressing cells may have a lower density of BK channels, making it more difficult to get an acceptable patch. Together these arguments lead us to favor the hypothesis that both rat and bovine populations are dichotomous with respect to inactivation, with one subset expressing strictly noninactivating channel subunits and the other expressing both types. Considerable variation in the relative abundance of inactivating subunits occurs in the inactivating subset of both species, with a very different mean for each. Species also appear to differ in the relative size of the two cell subsets, with bovine having many more cells belonging to the exclusively noninactivating category.


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Species differences in BK function

BK currents of both rat and bovine chromaffin cells dwarf other voltage-dependent K+ currents. Like rat, bovine cells express BK channels showing wide variation in gating properties, with variability in kinetics of activation, deactivation, and inactivation gating and in voltage- and calcium-dependent gating. However, despite overlapping ranges, big differences in the representation of functional properties characterize the two species' chromaffin populations, raising intriguing questions about the molecular bases of channel heterogeneity, its regulation, and ramifications for neurosecretory function. Most strikingly, patches with only noninactivating currents are fourfold more abundant in bovine than rat patches, whereas those with virtually complete inactivation are much more abundant in rat. Patches with mixed and intermediate properties, typically including both slowly and virtually noninactivating components, constitute a majority in bovine, but not rat cells. Rat channels activate at more negative voltages (more than 30 mV with [Ca2+]i at 6 µM), activate faster, and deactivate more slowly than bovine chromaffin BK channels, on average.

Implications for the molecular bases of functional variety

In both species, individual channels can be observed that are noninactivating, rapidly inactivating, and having intermediate rates of inactivation. BK-beta 2, a recently cloned accessory subunit with an N-terminal inactivation "ball," produces inactivation in oocyte-expressed Slo channels that is very similar to that observed in rat and bovine chromaffin cells (Wallner et al. 1999; Xia et al. 1999). It therefore seems likely that beta 2 or related proteins will encode their BKi channels. If we assume that Slo tetramers can associate in a 1:1 stoichiometry with a beta  homologue (Knaus et al. 1994a,b), variability in inactivation rates may be explained as a function of the number of alpha  subunits paired with an inactivation-competent beta  subunit. This modeling approach satisfactorily simulates widely varying currents from the two species. Thus the data are consistent with tetrameric channels comprised of just inactivation-competent and -incompetent subunits, as postulated by Ding et al. (1998). Our findings motivate a search for quantitative differences between species in the molecular expression of inactivation-conferring beta subunit(s), perhaps with an inverse pattern of expression of a beta  form that does not confer inactivation.

Rat and bovine chromaffin cell populations have long been considered dichotomous with respect to catecholamine phenotype, comprised of EPI and NE secreting subsets (Coupland 1953, 1965; West 1955). Although the rat chromaffin population was originally described as dichotomous with respect to the expression of inactivating versus noninactivating currents (BKi vs. BKs) (Solaro et al. 1995), the rarity of purely BKs expressing cells and the existence of a significant number of cells with a small noninactivating component in a mostly inactivating current observed with more extensive sampling raised doubts that a categorical distinction could be made (Lingle et al. 1996; Solaro et al. 1995). Using the tetramer model to estimate the proportion of inactivation-competent and -incompetent channels that might underlie individual current traces, we observed a much larger proportion of pure BKs patches for the bovine population than would be expected from a unimodally varying population with a mean similar to that observed. We suggest that there is a subset of cells in bovine, and perhaps rat as well, that expresses no inactivating subunits, in addition to a subset that expresses inactivating subunits to varying degrees. Whether this pattern aligns with NE versus EPI phenotype is under investigation.

beta 2, like beta 1, can shift the G-V curve for Slo activation in the negative direction (Dworetzky et al. 1996; Jones et al. 1999a,b; McCobb et al. 1995; McManus et al. 1995; Meera et al. 1996; Oberst et al. 1997; Ramanathan et al. 1999; Saito et al. 1997; Wallner et al. 1999; Xia et al. 1999), and thus species differences in beta  expression may contribute to G-V differences as well as to inactivation differences. However, the reported species differences in activation and deactivation are also similar to those between patches from Xenopus oocytes injected with "STREX" and "ZERO" splice variants of Slo (Jones et al. 1999a,b; Ramanathan et al. 1999; Saito et al. 1997; Xie and McCobb 1998). The STREX exon was so named because its inclusion is promoted by the HPA stress-hormone cascade (Lai et al. 1999; Xie and McCobb 1998). Intriguingly, serum levels of cortisol are at least one order of magnitude lower in bovine than its equivalent steroid, corticosterone, in rat. Significantly, we find that, relative to ZERO, the STREX exon is much less abundant in bovine than rat (Chatterjee et al. 1999). Thus part of the species differences in BK gating probably results directly from species differences in splicing of the Slo gene, and this, we hypothesize, may be a consequence of species differences in stress axis set-point. How much of the species difference in G-V can be attributed to Slo splicing as compared with beta  association remains to be determined.

Both Slo splicing and beta  subunit association can also affect the kinetics of activation and/or deactivation of Slo channels under given voltage and calcium conditions. Effects of beta 2 on activation kinetics have not yet been determined; however, beta 1 markedly slows activation even while shifting the G-V in the negative direction (Dworetzky et al. 1996). By contrast, STREX inclusion hastens activation in conjunction with a negative shift in G-V. The faster activation in rat cells compared with bovine cells that we observe would suggest that Slo splicing exerts more control over activation kinetics than beta  subunits do, and/or that the beta  subunits expressed in chromaffin cells of rat, at least, do not slow activation appreciably. STREX and inactivation-conferring beta  subunits both slow deactivation, and thus both probably contribute to the slower deactivation typical of rat cells. Correlations between V0.5 values, time constants of deactivation, and fraction of current inactivating (or rates of inactivation) were apparent when considered across the two species, but were loose. Although measurement error contributes to the variation, a lack of precise coupling between expression of molecular determinants such as beta  and STREX is suggested.

Do BK functional differences affect catecholamine responses?

The enhanced repetitive firing of a subset of rat chromaffin cells has been tentatively attributed to slower BK channel deactivation (Lingle et al. 1996; Solaro et al. 1995). Thus slow closure during the repolarization phase of the action potential is thought to augment the amplitude and duration of the brief AHP. This in turn may facilitate subsequent firing by freeing Na+ and Ca2+ channels from inactivation. Our comparative data suggest that rat BK currents will begin to turn on earlier, and will do so faster, which should speed repolarization, reducing the number of Na+ and Ca2+ channels entering the inactivated state, and augmenting the AHP to facilitate recovery for those that do inactivate. The combined effects should confer measurably greater repetitive firing properties on rat than bovine cells, other things being equal. Of course, repetitive firing properties are determined by an interactive set of channel types. Differences in properties of other channels (Gandía et al. 1995; Hollins and Ikeda 1996), coupling interactions between BK channels and calcium channels (Prakriya and Lingle 1999), BK properties not measured here, and the relative abundance of channel types may contribute to species differences, or even compensate for differences in BK gating. Recording in whole cell mode, we find that repetitive firing properties exhibit a lability reminiscent of Ca2+ channel rundown, suggesting that calcium buffering and Ca2+ channel modulation are critical. The perforated-patch recording mode is probably least disruptive and is being used to determine whether the BK species differences do produce robust differences in excitability.

What purpose might be served by differences in inactivation properties? The preceding arguments would suggest that BK channel inactivation would negatively affect repetitive firing and catecholamine output. On the other hand, by decreasing action potential duration BK channels should decrease Ca2+ entry during an action potential. Channel inactivation might then augment secretion. Thus a key issue concerns the occurrence of BK channel inactivation, in vivo. The sensitivity of the inactivation process to depolarization and intracellular [Ca2+]i (Ding et al. 1998; Solaro and Lingle 1992; Solaro et al. 1995) make this difficult to predict. However, since inactivation is too slow to take effect during a single action potential, accumulation of inactivation during a train of action potentials may be necessary for effects on action potential waveform or repetitive firing. Interestingly, muscarinic acetylcholine receptor activation may be more efficacious than depolarization in bringing about BK inactivation (Herrington et al. 1995; Neely and Lingle 1992a,b). Such inactivation might serve as a negative-feedback suppressor of adrenal over-stimulation, the need for which would be greater in cells with greater repetitive firing properties deriving from the combination of facilitated activation and slower deactivation. In this context, variation in activation and inactivation might be roughly, but not precisely, commensurate. This idea is consistent with the pattern of BK channel properties observed when comparing two very different species. We postulate that several features of BK channel gating serve to adapt chromaffin cell excitable properties for autonomic responses that are quite different for a large, grazing ruminant than for a more active and excitable rodent. Integrative approaches will be needed to understand the precise consequences of differences in BK properties between species, and the regulatory factors driving those differences.


    ACKNOWLEDGMENTS

We thank Drs. Ron Harris-Warrick, Bruce Johnson, and Jason MacLean for many helpful discussions, and G.-J. Lai, G. Dernick, E. Carpenter-Hyland, A. Thabet, O. Ulukpo, K. Margot, and B. Thangam for technical assistance. We also thank Cudlin's Meat Market for generously providing bovine adrenals.

This work was supported by a grant from the American Heart Association. P. V. Lovell was supported by a National Institute of Mental Health training grant.


    FOOTNOTES

Address for reprint requests: D. P. McCobb, Dept. of Neurobiology and Behavior, W153 Mudd Hall, Cornell University, Ithaca, NY 14853.

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 24 August 1999; accepted in final form 28 February 2000.


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