Department of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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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.
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INTRODUCTION |
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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
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
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.
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METHODS |
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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 M
) 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 G. 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
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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
(
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
(
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
m of 2.5 ms and
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)
i = 33.3; (2 bki)
i = 50; (1 bki)
i = 100;
(0 bki)
i =
.
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.
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RESULTS |
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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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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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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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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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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 (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/
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/
d)
adequately described the process and was used for direct comparisons.
The time constant of deactivation (
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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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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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 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
d values tended to have large
inactivating components.
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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
2 (Wallner et al.
1999
; referred to as
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).
|
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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DISCUSSION |
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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-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
2 or related proteins will encode their
BKi channels. If we assume that Slo tetramers can associate in a 1:1 stoichiometry with a
homologue (Knaus et al. 1994a
,b
), variability in inactivation
rates may be explained as a function of the number of
subunits
paired with an inactivation-competent
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
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.
2, like
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
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
association remains to be determined.
Both Slo splicing and subunit association can also
affect the kinetics of activation and/or deactivation of Slo
channels under given voltage and calcium conditions. Effects of
2 on
activation kinetics have not yet been determined; however,
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
subunits do,
and/or that the
subunits expressed in chromaffin cells of rat, at
least, do not slow activation appreciably. STREX and
inactivation-conferring
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
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.
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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.
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FOOTNOTES |
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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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REFERENCES |
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