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Volume 16, Number 15,
Issue of August 1, 1996
pp. 4543-4550
Copyright ©1996 Society for Neuroscience
Phenotypic Alteration of a Human BK (hSlo) Channel by
hSlo Subunit Coexpression: Changes in Blocker
Sensitivity, Activation/Relaxation and Inactivation Kinetics, and
Protein Kinase A Modulation
Steven I. Dworetzky,
Christopher G. Boissard,
Janet T. Lum-Ragan,
M. Craig McKay,
Debra J. Post-Munson,
Joanne T. Trojnacki,
Chia-Ping Chang, and
Valentin K. Gribkoff
Central Nervous System Drug Discovery, Bristol-Myers Squibb
Pharmaceutical Research Institute, Wallingford, Connecticut 06492
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
A human homolog of the large-conductance calcium-activated
potassium (BK) channel  subunit (hSlo) was cloned, and
its effects on a human BK channel (hSlo) phenotype are
reported. Coexpression of hSlo and hSlo, in
both oocytes and human embryonic kidney 293 cells, resulted in
increased Ca2+ sensitivity, marked slowing of BK
channel activation and relaxation, and a significant reduction in slow
inactivation. In addition, coexpression changed the pharmacology of the
BK channel phenotype: hSlo-mediated currents in oocytes were
more sensitive to the peptide toxin iberiotoxin than were
hSlo + hSlo currents, and the potency of
blockade by the alkaloid BK blocker tetrandrine was much greater on
hSlo + hSlo-mediated currents compared with
hSlo currents alone. No significant differences in the
response to charybdotoxin or the BK channel opener NS1619 were
observed. Modulation of BK channel activity by phosphorylation was also
affected by the presence of the hSlo subunit. Application
of cAMP-dependent protein kinase increased
POPEN of hSlo channels, but
decreased POPEN of most hSlo + hSlo channels. Taken together, these altered
characteristics may explain some of the wide diversity of BK channel
phenotypes observed in native tissues.
Key words:
BK channels;
coexpression;
hSlo;
modulation;
channel blockers;
BK phenotypes
INTRODUCTION
Potassium channels are the most diverse class of
ion channels. The voltage-dependent potassium
(K+) channels achieve phenotypic variation
in part through the evolution of gene families coding for similar
channels (Pongs, 1992 ; Salkoff et al., 1992; Chandy and Gutman, 1995),
unlike another important class of K+ channels,
the large-conductance calcium (Ca2+)-activated
K+ (BK) channels (Robitaille and Charlton, 1992;
Bielefeldt and Jackson, 1993). BK channels are regulated by both
voltage and internal calcium concentration; these factors, together
with their large single-channel conductance, make them very effective
regulators of cell excitability, particularly adapted to monitoring and
indirectly regulating calcium entry. BK channels have multiple
phenotypes (Latorre et al., 1989; Reinhart et al., 1989; Wang and
Lemos, 1992), but, to date, only one gene has been identified (Atkinson
et al., 1991; Adelman et al., 1992; Butler et al., 1993; Dworetzky et
al., 1994; Pallanck and Ganetzky, 1994). At least two phenotypes exist
in rat brain: type I BK channels (fast-gating, BK toxin-sensitive
channels activated by cAMP-dependent protein phosphorylation) and type
II BK channels (relatively toxin-insensitive, slowly-gating, blocked by
the alkaloid tetrandrine, with activity reduced by cAMP-dependent
protein phosphorylation) (Reinhart et al., 1989, 1991; Wang and Lemos,
1992). Alternative exon splice variation confers different properties
on BK channels, particularly in relation to calcium sensitivity, but
this does not seem to be sufficient to account for these major
phenotypic classes (Lagrutta et al., 1994; Tseng-Crank et al., 1994).
Regulatory () subunits have been shown to alter rates of
inactivation of voltage-dependent K+ channels
(Rettig et al., 1994), and a recently cloned bovine BK channel subunit (Knaus et al., 1994) was found to increase the sensitivity of
cloned BK channels to Ca2+ (McCobb et al., 1995;
McManus et al., 1995) and to the BK channel opener DHS-1 (McManus et
al., 1995). In this study, we report the cloning of a human BK subunit (hSlo) and describe how coexpression alters the
biophysical and pharmacological characteristics of a cloned human BK
channel.
MATERIALS AND METHODS
Cloning of the human subunit. PCR primers were
designed adjacent to the 5 and 3 end of the bovine subunit
sequence (Knaus et al., 1994) and used to amplify partial clones by PCR
(45 sec, 94°C; 60 sec, 45°C; 75 sec, 72°C; 35 cycles) from
reverse-transcribed human tracheal poly A+ RNA.
The resulting DNA fragment was isolated, purified, random-prime-labeled
with deoxycytidine [ -32P]triphosphate, and used to
screen a human uterus cDNA library (Clontech, Palo Alto, CA). The
filters were hybridized in 30% formamide, 2X PIPES, and 1% SDS at
42°C, and washed in 0.1× SSC, 0.1% SDS at 42°C. Resulting
positive plaques were plate-purified, and the inserts were isolated by
EcoRI restriction enzyme digestion. Positive clonal inserts
were ligated into pBluescriptKS+ (Stratagene, La
Jolla, CA), and both strands were sequenced by dideoxy termination
reactions. To remove the 5 and 3 untranslated regions of the
hSlo clone, synthetic oligonucleotide primers were
designed to amplify only the hSlo coding region with a T3
RNA polymerase and Kozak consensus sequence (Kozak, 1987) added to the
5 end [forward primer;
5-CGCAATTAACCCTCACTAAAGGGCGCCACCATGGTGAAGAAGCTGGTGATGGCC-3 and
reverse primer; 5-GCATGGATGGATGTCACTACTTCTGG-3]. The
hSlo PCR product was subcloned into the pCRII TA cloning
vector (Invitrogen, San Diego, CA), sequenced to verify possible PCR
amplification errors, and used for preparation of cRNA. The original
hSlo insert was also subcloned into pcDNA3 (Invitrogen)
for mammalian expression in human embryonic kidney (HEK) 293 cells. The
hSlo clone was tested for expression by in
vitro transcription and translation (not shown). The Genbank
accession number for the hSlo clone is U38907.
Expression and recording in Xenopus oocytes. The
hSlo and hSlo cDNAs were linearized with the
restriction enzyme NotI and BamHI, respectively,
and in vitro transcribed using the mMessage mMachine T3 RNA
polymerase kit according to the manufacturer's instructions (Ambion,
Austin, TX). The cRNAs were solubilized in RNase-free water and stored
at  70°C at a concentration of 1.5 µg/µl. The hSlo
construct used in the present experiments was originally cloned from a
human brain substantia nigra cDNA library (Dworetzky et al., 1994) and
did not contain any of the previously identified alternative splice
exons (Dworetzky et al., 1994; Tseng-Crank et al., 1994). Frog oocytes
were prepared and injected using standard techniques (Colman, 1984). In
hSlo + hSlo coexpression experiments, each
oocyte was injected with ~50 nl of the appropriate cRNA, resulting in
total injections of 100 nl/oocyte. Injection of equivalent amounts of
hSlo cRNA (50 nl) supplemented with 50 nl of RNase-free
water did not alter the characteristics of hSlo currents.
After injection, oocytes were maintained at 17°C in ND96 medium
consisting of (in mM): 90 NaCl, 1.0 KCl, 1.0 CaCl2, 1.0 MgCl2, 5.0 HEPES, pH 7.5. Horse serum and penicillin/streptomycin, both 5% of
final volume, were added as supplements to the incubation medium.
Electrophysiological recording commenced 2-6 d after cRNA injection.
Before the start of an experiment, oocytes were placed in a recording
chamber and incubated in modified Barth's solution (MBS) consisting of
(in mM): 88 NaCl, 2.4 NaHCO3, 1.0 KCl, 10 HEPES, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, pH 7.5. Oocytes were impaled with
electrodes (1-2 M ), and standard, two-electrode voltage-clamp
techniques were used to record whole-cell membrane currents (Stuhmer,
1992; GeneClamp 500, Axon Instruments, or Turbo TEC01C, Adams and
List). Voltage-clamp protocols typically consisted of a series of
voltage steps 100-750 msec duration in 20 mV steps from a holding
potential of 60 mV to a maximal potential of 100-140 mV; records
were digitized at 5 kHz and stored on a computer with pClamp 6.0 or
AxoData software (Axon Instruments, Foster City, CA).
Expression and recording in HEK 293 cells. HEK 293 cells
were plated on poly-D-lysine-coated coverslips at
10-20% confluency and transiently transfected 3 d later with
either hSlo (1 µg DNA/35 mm dish) or hSlo + hSlo (0.75 µg of each DNA/35 mm dish) by the
lipofectamine method according to the manufacturer's instructions
(Life Technologies, Gaithersburg, MD). Whole-cell and excised patch
voltage-clamp recordings were made with standard techniques (Hamill et
al., 1981) 24-72 hr after transfection. An Axopatch 200 amplifier and
pClamp 6.0 software (Axon Instruments) were used for all recordings.
The bath solution for both whole-cell and outside-out patch recordings
contained (in mM): 145 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES,
pH 7.4. The internal solution for whole-cell recordings was (in
mM): 140 KCl, 20 MOPS, 0.2 K2EGTA, 0.174 CaCl2 (1 µM estimated
[Ca2+]free), pH 7.2. The
pipette-filling solution and the bath solution used for inside-out
excised patch recordings contained (in mM): 140 KCl, 20 MOPS, 1 K2EGTA, pH 7.2. CaCl2 was added to adjust free
[Ca2+]. Pipettes (2.5-5.0 M in bath
solution) were pulled from borosilicate glass, coated with SYLGARD, and
fire-polished. Whole-cell currents were evoked by step depolarization
of the membrane potential from 60 to 100 mV in 20 mV increments.
Leakage currents, which were negligible, were uncorrected, and series
resistance was compensated ~80%. Data were filtered at 2 or 5 kHz
and digitized at 10 or 40 kHz. Ensemble averages of excised patch
records were made by repetitively stepping (50-200 sweeps) the
membrane potential of outside-out patches from 60 to 40 mV every 2 sec. Capacitance and leak compensation were made with amplifier
controls. Records were filtered at 1-2 kHz and digitized at 10 kHz.
The control free [Ca2+] was determined to be
0.79 µM; this and all free
[Ca2+] were determined by a Fura-2 fluorometric
assay in accord with the manufacturer's instructions (Molecular
Probes, Eugene, OR). Average ensemble and whole-cell currents were best
fit by a least-squares minimized, two-component exponential function of
the form y = A1 × exp[(t t0)/ 1] + A2 × exp[(t t0)/2] + C, where A is the current amplitude, is the
time constant, and C is the asymptotic current value. In
most cases, two-tailed t tests were used to determine
statistical significance of the differences in time-to-peak,
1, and 2 values.
Blocker and opener pharmacology. All drug solutions were
introduced into the bath by gravity flow. Iberiotoxin (IbTX) and
charybdotoxin (ChTX) (Peptides International, Louisville, KY) were
prepared as aqueous stocks and diluted in MBS before use; tetrandrine
(Aldrich, Milwaukee, WI) was prepared as a stock solution in
dimethylformamide or dimethylsulfoxide (DMSO) and likewise diluted in
the appropriate solution just before use. For IbTX and ChTX
experiments, maximal suppression was defined as the peak suppression
observed in response to 100-250 nM IbTX or
250-500 nM ChTX. These concentrations were
sufficient to block all of the expressed current. Blockers were applied
for at least 10 min. Logistic fits of the concentration-response
relationships and EC50 estimates were obtained
with KaleidoGraph software (Abelbeck). The BK channel opener NS1619
(Olesen et al., 1994) was made in-house, dissolved in DMSO (20 mM stock), and diluted to final concentration
just before use.
Protein kinase A modulation. The effects of exposure to the
catalytic subunit of the cAMP-dependent protein kinase A (PKA)
(Promega, Madison, WI) on inside-out patches containing either
hSlo or hSlo + hSlo bathed with
symmetrical 140 mM KCl solutions were measured by
continuously monitoring steady-state changes in
NPopen
(NPopen = I/i;
n = number of active channels;
Popen is the open probability, I
is the mean current, and i is the single-channel current
amplitude; bin width 5 sec) before and during application of 60 nM PKA in the bath solution. The bath solution
also contained 500 µM
Mg2+-ATP throughout the experiment. The free
[Ca2+] of the bathing solution was 4.52 µM. Holding voltage was adjusted so that some
channel closures to baseline were observed in the control solution.
Recordings were filtered at 1 kHz and digitized at 10 kHz.
RESULTS
subunit cloning
hSlo was cloned by reverse transcription of human
tracheal tissue and amplification of the cDNA via PCR protocols; the
design of the oligonucleotide primers was based on the reported bovine
sequence (Knaus et al., 1994). The amplified human DNA fragment was
then used as a probe to screen a human uterus cDNA library. This
library was used because of the low abundance of subunit message
present in brain (our unpublished results). Numerous positive plaques
were identified, and of the four clones carried through plaque
isolation, all proved to be subunit homologs with various amounts
of 5 and 3 untranslated sequence. Alignment of the human and bovine
protein sequences shows 85% identity (Fig. 1). The rat
brain and smooth muscle subunit sequences, which are highly
homologous to the human and bovine sequences, were identical to each
other (our unpublished results), and there is unlikely to be any
difference between the human smooth muscle and human brain subunit.
Fig. 1.
Predicted primary sequence of a cloned human BK
channel subunit (hSlo) and its alignment with a
bovine subunit. The identical amino acids are boxed
together, resulting in 85% identity between these sequences with the
putative M2 transmembrane domain being completely conserved.
The putative transmembrane domains M1 and M2 are
marked within the consensus sequence by the black
boxes.
[View Larger Version of this Image (47K GIF file)]
Activation/relaxation and inactivation kinetics
In general, hSlo produced rapidly activating currents
in response to depolarization, and partial slow inactivation was
observed with long voltage pulses. Coexpression of hSlo and
the hSlo subunit produced more slowly activating currents
in which slow inactivation was greatly reduced (see below).
Coexpression of hSlo with the voltage-dependent
K+ channels KV1.3,
KV1.4, and KV1.5 produced
no detectable difference in activation, inactivation, or peak amplitude
values of currents mediated by these channels (data not shown). Oocytes
injected with hSlo alone did not produce any detectable
current, and there was no noticeable alteration in the characteristics
of the native oocyte current.
Although steady-state channel kinetics could not be studied because of
the large number of channels in most excised patches, we could directly
measure the activation kinetics of hSlo and hSlo + hSlo. Excised and whole-cell patch-clamp recordings
from transiently transfected HEK 293 cells and two-electrode
voltage-clamp recordings in oocytes demonstrated that activation of
hSlo channels was fast, whereas activation of
hSlo + hSlo channels was significantly slower
(Fig. 2, Table 1; all
values are mean ± SEM throughout). Current relaxation was
similarly influenced by hSlo coexpression (Table 1). The
difference in activation kinetics was maintained when examined at
multiple voltage-step values (60 mV to 60, 80, and 100 mV, whole-cell
patch recording in HEK 293 cells). At 60, 80, and 100 mV, respectively
(n = 5), hSlo current activation time
constants were 1 = 1.57 ± 0.20 msec,
1.25 ± 0.18 msec, and 0.89 ± 0.10 msec, and
2 = 20.96 ± 5.59 msec, 16.13 ± 7.63 msec, and 10.22 ± 4.87 msec, whereas corresponding values
for hSlo + hSlo (n = 5) were
1 = 10.22 ± 2.74 msec, 9.98 ± 2.12 msec, and 6.64 ± 1.39 msec, and 2 = 77.49 ± 24.20 msec, 84.91 ± 28.40 msec, and 56.10 ± 28.18 msec (repeated measures ANOVA; 1:
F = 10.61, p < 0.0001;
2: F = 3.69, p = 0.016). In oocytes, a similar difference between the experimental
groups was observed when time to peak current was measured at voltage
steps from 60 mV (hold) to 60-140 mV in 20 mV increments (data for
steps to 140 mV shown in Table 1; F = 72.4, p < 0.0001). Slow current inactivation was
significantly reduced and slowed in oocytes and HEK 293 cells
expressing hSlo + hS3lo (Figs. 2,
3), and hSlo currents in oocytes recovered
from inactivation significantly more slowly (Fig. 3).
Fig. 2.
Activation records for hSlo and
hSlo + hSlo BK channels in oocytes and HEK 293 cells. Activation of hSlo + hSlo BK currents
was significantly slower than currents mediated by hSlo
expression in Xenopus oocytes (A) and HEK 293 cells (B; whole-cell patch recordings, C;
ensemble average currents from excised membrane patches, D;
excised patch recordings, single traces). Currents were
normalized for presentation. Peak current values in A, 6.4 µA for hSlo and 4.6 µA for hSlo + hSlo; currents shown are in response to a depolarizing
step from 60 to 80 mV. In B, 17.1 nA for hSlo
and 11.7 nA for hSlo + hSlo; currents shown
resulted from voltage steps from 60 to 100 mV. In C, 114 pA for hSlo and 355 pA for hSlo + hSlo. Horizontal axis scale: 20 msec for A and
B, 40 msec for C. The currents in C
and D resulted from voltage steps from 60 to 40 mV. The
pulse duration for the multichannel recordings in D is 200 msec; note that the patches contain different (and large) numbers of
channels; initial amplitude (i.) is 110 pA. No significant
difference was observed in the amplitudes of hSlo or
hSlo + hSlo currents in either expression
system after comparable incubation periods; likewise, no difference was
observed in the single-channel slope conductance of channels coded by
these constructs when currents from patches with a small number of
channels were recorded (hSlo = 286 ± 12 pS,
n = 5; hSlo + hSlo = 289 ± 6 pS, n = 4). Note that deactivation of
hSlo + hSlo currents was also slower, as
indicated in A and visible in C and D
(see Table 1).
[View Larger Version of this Image (12K GIF file)]
Fig. 3.
Slow current inactivation reduction in oocytes
expressing hSlo + hSlo relative to those
expressing hSlo. A, In a paired-pulse paradigm,
to examine the rate of recovery from inactivation, a 1 sec voltage step
(60 mV hold to 100 mV) was used as the ``conditioning'' stimulus to
produce a significant level of inactivation (time 0, end of the
conditioning pulse), followed at an increasing interval by a single
identical ``test'' voltage step. Initial inactivation was measured by
comparing the early peak current of the conditioning voltage step with
the residual current at the end of the first step. Peak current
amplitudes of the subsequent step in each paired episode were used to
measure recovery. The conditioning step produced significantly greater
levels of inactivation of hSlo currents compared with
expressing hSlo + hSlo currents (t
test; p < 0.001), and recovery was significantly
slower (repeated measures ANOVA, F = 15.9, p = 0.005). Full recovery was not achieved by 7.5 sec
after the conditioning pulse. With longer single voltage steps (10 sec), a significant increase in the time course of inactivation was
observed (hSlo 1 = 309.7 ± 36.2 msec, n = 5; hSlo + hSlo
1 = 702.0 ± 88.2 msec, n = 5; p = 0.003, two-tailed t test;
2 values not reported because of contamination
by slowly activating native current). B, Examples of current
inactivation and its recovery resulting from the paired-pulse paradigm
(8 sweeps) in oocytes expressing hSlo or hSlo + hSlo. The level of inactivation was independent of
current amplitude (expression level) within the normal limits
encountered in this study. There was no inactivation of native
Ca2+-activated Cl
current, and currents represent IbTX-sensitive current components,
after subtraction of residual currents in supramaximal IbTX.
[View Larger Version of this Image (16K GIF file)]
As reported previously, coexpression of mSlo and
bSlo (bovine) resulted in a significant increase in the
Ca2+ sensitivity of the expressed BK channels
(McCobb et al., 1995; McManus et al., 1995). Similar results were
obtained with hSlo and hSlo coexpression. This
was observed as a leftward shift in the conductance/voltage
(g/V) relationship (Fig. 4A) and a
reduction in the half-maximal activation voltage for any tested
Ca2+ concentration (Fig. 4B).
Fig. 4.
Coexpression of hSlo + hSlo increased the sensitivity of BK channels to
intracellular Ca2+. A, Conductance
(g/gmax)/voltage (g/V) plots
generated for hSlo (i.) and hSlo + hSlo (ii.) in 28.2 µM
Ca2+ (a) and 0.79 µM Ca2+ (b);
recordings obtained from inside-out excised patches from transiently
transfected HEK 293 cells in response to voltage ramps (100 to 100 mV, 4 sec duration; data from a minimum of 25 ramps per patch). Note
that at both concentrations of intracellular
Ca2+, the g/V relationship for
hSlo + hSlo is shifted to the left, indicating
increased Ca2+ sensitivity. Curves were generated
with a standard Boltzmann relationship in which
g/gmax = (1 +exp[(V1/2 Vm)/K])1.
B, The half-maximal activation values
(V1/2) for three concentrations of
intracellular Ca2+ are plotted for
hSlo and hSlo + hSlo;
V1/2 values were consistently lower (as
plotted) for hSlo coexpressed with the hSlo
subunit.
[View Larger Version of this Image (21K GIF file)]
Pharmacology of hSlo- and hSlo + hSlo-mediated currents
To determine whether differences existed in the pharmacology of
hSlo- and hSlo + hSlo-mediated
currents, we applied IbTX (Galvez et al., 1990; Giangiacomo et al.,
1992) and ChTX (Miller et al., 1985; Sugg et al., 1990), which are
high-affinity peptidyl blockers of the rat brain type I BK channel
(Reinhart et al., 1989), and tetrandrine, an alkaloid blocker of the
rat brain type II BK channel (Wang and Lemos, 1992), to oocytes and/or
HEK 293 cells expressing these constructs. A marked difference in the
sensitivity of the currents to both IbTX and tetrandrine was observed.
The currents in oocytes expressing hSlo were significantly
more sensitive to IbTX than those in oocytes expressing hSlo + hSlo (Fig. 5A;
EC50 = 11.4 nM for
hSlo, EC50 = 163.7 for hSlo + hSlo). Less effect of coexpression was observed in the
responses to ChTX; a small increase in sensitivity was noted in the
oocytes expressing hSlo + hSlo (Fig.
5B), but these effects were not significant. The blockade by
both peptides occurred more slowly in oocytes expressing
hSlo + hSlo, although this was not quantified.
Application of the alkaloid tetrandrine to HEK 293 cells expressing
either hSlo or hSlo + hSlo revealed
a concentration-dependent depression of BK current; however, the
potency of blockade was much greater on hSlo + hSlo-mediated currents (Fig.
6A). In addition, tetrandrine was shown to
exacerbate the slower activation kinetics of the hSlo + hSlo currents; this was particularly evident in the
oocytes, in which tetrandrine produced a marked slowing of the current
activation, with a significant increase in the latency to current peak
(Fig. 6B). No consistent differences were observed in the
concentration-response (1.0-100 µM)
relationship of the benzimidazolone BK channel opener NS1619 (Olesen et
al., 1994) between the two types of BK channels (Fig.
7). This was consistent with an earlier report that
openers of this class, unlike DHS-1, did not distinguish between types
of native BK channels (McKay et al., 1994).
Fig. 5.
IBTX and ChTX pharmacology. A,
Application of the BK channel-blocking peptide IbTX to oocytes
expressing hSlo or hSlo + hSlo
revealed that coexpression with the hSlo subunit resulted
in a nearly 10-fold decrease in the sensitivity to IbTX blockade;
n = 5-10 oocytes/IbTX concentration. Maximal effect
was defined as the effect produced by incubation in 100-250
nM IbTX; maximal levels of suppression did not
differ significantly between hSlo and hSlo + hSlo. B, Application of the peptidyl blocker
ChTX did not reveal a significantly different profile of blockade for
hSlo and hSlo + hSlo. Maximal
effect was defined as the response to 250-500 nM
ChTX, and maximal levels of effect did not differ between
hSlo and hSlo + hSlo.
[View Larger Version of this Image (19K GIF file)]
Fig. 6.
Tetrandrine pharmacology in oocytes and HEK 293 cells. A, Suppression of hSlo-mediated whole-cell
membrane currents in HEK 293 cells by the alkaloid type II BK blocker
tetrandrine was reduced relative to the degree of block observed in
cells transfected with hSlo + hSlo;
n = 5-9 cells/tetrandrine concentration. The fitted
curves suggest multiple affinity interactions of tetrandrine with both
constructs; absolute maximal levels of tetrandrine block could not be
determined because of limited solubility. The traces are
examples of the relative effects of 2.5 µM
tetrandrine on whole-cell HEK 293 BK membrane currents. Note that
tetrandrine slowed activation of both types of current, but this effect
was most pronounced with a greater level of block produced in cells
expressing hSlo + hSlo. B, A
pronounced increase in the latency to current peak was observed only in
oocytes expressing hSlo and hSlo + hSlo when exposed to tetrandrine. Traces
depict the effect of 50 µM tetrandrine
(b.) on BK currents in oocytes expressing hSlo
(top traces; i.), or hSlo + hSlo
(bottom traces; ii.), relative to control values
(a.). The dotted trace in the bottom set
is the control hSlo trace for comparison. Tetrandrine
had no effect on the current activation in the hSlo oocyte,
but it significantly delayed the peak of the current in the oocyte
expressing hSlo + hSlo
(EC50 = 8.5 µM). The
currents are the response to voltage steps from a holding potential of
60 to 100 mV, and only the initial portions of the normalized
currents are depicted.
[View Larger Version of this Image (24K GIF file)]
Fig. 7.
Effects of the application of the BK channel
opener NS1619. At a range of NS1619 concentrations, oocytes injected
with either hSlo or hSlo + hSlo
cRNA showed similar levels of activation of whole-cell BK currents.
Measured currents represent the response to a voltage step from 60 to
140 mV. A minimum of five oocytes were used for each concentration of
NS1619.
[View Larger Version of this Image (17K GIF file)]
Modulation of hSlo- and hSlo + hSlo-mediated currents by PKA
BK channels are modulated by cAMP-dependent protein
phosphorylation and dephosphorylation, with most type I channels
increasing Popen in response to
phosphorylation and most type II channels showing decreased activity
when phosphorylated (Reinhart et al., 1989, 1991). The catalytic
subunit of PKA was applied to the cytosolic side of hSlo and
hSlo + hSlo channels in excised membrane
patches from transfected HEK 293 cells. The hSlo channels (6 of 6) showed a marked and reversible increase in
Popen after exposure to PKA, whereas the
majority of hSlo + hSlo channels (5 of 8)
showed a reduction in Popen (Fig.
8).
Fig. 8.
Modulation of hSlo and hSlo + hSlo channels by exposure to PKA. A,
Application of the catalytic subunit of PKA produced an increase in
NPopen when applied to the cytosolic
side of an inside-out membrane patch excised from an HEK 293 cell
transiently expressing hSlo. B, PKA reduced
NPopen in a patch excised from an HEK 293 cell transiently coexpressing hSlo + hSlo. The
mean increase in NPopen (averaged over the
entire 240 sec control period vs the 240 sec PKA application) for
hSlo patches was 97.1 ± 35.9% (SEM all) above control
values (n = 6; increases seen in all 6 patches); the
mean decrease in NPopen recorded from
hSlo + hSlo patches was 19.0 ± 15.6%
(n = 8; 5 decreased, 1 no change, 2 increased, all data
included in average; the difference was significant, p < 0.03, two-tailed t test). Partial recovery after removal
of PKA was observed in some patches during a 240 sec wash period (data
not shown).
[View Larger Version of this Image (22K GIF file)]
DISCUSSION
The slower activation of hSlo + hSlo
currents is unlikely to result from the increased
Ca2+ sensitivity of these channels in the
presence of the hSlo subunit. A recent report (DiChiara
and Reinhart, 1995) showed that increasing Ca2+
concentrations produced more rapid activation of dSlo and
hSlo BK channels; assuming that increased
Ca2+ sensitivity of hSlo in the
presence of the hSlo subunit acted like an apparent
increase in Ca2+ concentration relative to
hSlo channels when all other variables were equivalent, a
decrease in activation time would be expected, rather than the observed
increase. Increasing the step voltage likewise was shown to reduce
activation time for hSlo (DiChiara and Reinhart, 1995).
Although this was observed for both constructs, the significant
difference in activation time and time to peak current was independent
of the step voltage. The increased time to deactivation of
hSlo + hSlo, however, was consistent with an
increase in Ca2+ sensitivity in the presence of
the subunit (DiChiara and Reinhart, 1995), and we therefore cannot
eliminate this as the cause of the slower deactivation/relaxation.
In some important respects, coexpression produced BK channels and
currents consistent with Type II BK channel characteristics, whereas
hSlo channels had some characteristics consistent with a
Type I classification. The hSlo channels had greater
sensitivity to IbTX, faster activation kinetics, and channel activity
was upregulated by PKA, which are type I characteristics. The
hSlo + hSlo channels, on the other hand, were
less sensitive to IbTX, had much slower activation kinetics, and were
downregulated by PKA application, which are type II characteristics
(Reinhart et al., 1989, 1991). The relatively greater effect of
tetrandrine on hSlo + hSlo currents was also
phenotypically similar to type II BK channels. In a previous study,
coexpression of a bovine subunit (bSlo) and a mouse
Slo homolog (mSlo) resulted in an increase in
Ca2+ sensitivity (McManus et al., 1995), which we
likewise observed with coexpression of hSlo and
hSlo. Although they reported that coexpression resulted
in the disappearance of brief closed events, they did not report
detailed changes in channel kinetics or differences in sensitivity to
toxin blockade. This may be the result of the constructs expressed;
although mammalian Slo proteins are highly homologous (e.g.,
mSlo vs hSlo, 98% sequence identity; Dworetzky
et al., 1994), it seems that Slo subunit sequences do not
have the same degree of identity (Fig. 1), and these differences may be
sufficient to affect expression and/or function. In at least two
characteristics, however, we found variance with the original
characterization of type I and type II channels. Type I channels were
found to be somewhat more sensitive to
[Ca2+]in (Reinhart et
al., 1989), which would be inconsistent with hSlo being the
type I BK channel, based on our data and that of the previous study. In
addition, the similar levels of block by ChTX suggests that this
classification is not applicable to all of the BK channel phenotypic
alterations produced by Slo subunit coexpression.
However, we must also keep in mind that the original classification
resulted from the characterization of rat brain BK channels, and
differences with the human (hSlo) phenotype could underlie
the observed variance.
Although we observed protein expression of hSlo and
hSlo separately in HEK 293 cells (data not shown), we
have not demonstrated coexpression of both proteins within the same
cell independent of electrophysiological recordings. Therefore, we
could not eliminate recordings in which coexpression did not seem to be
successful. This had the result of making the effects of coexpression
seem more variable (note increased SEM values for most measures in
cells expressing hSlo + hSlo) and may have
been a particular problem in the PKA experiments, in which the
consequence of coexpression was a change in direction of modulation
rather than in degree of effect. Nevertheless, these results indicate
that in some important characteristics hSlo resembled the
rat brain type I BK channel, whereas coexpression of the
hSlo subunit altered some characteristics of the
expressed hSlo channel phenotype to a type II profile.
It was difficult to directly compare the quantitative measurements of
our cloned expressed BK channels with those reported for native type I
and type II BK channels. In particular, relatively few studies have
focused on native populations of type I and type II BK channels, and
experiments in these cases were generally performed in different
systems and under different experimental conditions. Nevertheless, in
the PKA experiments, we observed close to a doubling of
Popen when hSlo channels were
exposed to PKA, which is very similar to the values reported for the
type I channel recorded from lipid bilayers (Reinhart et al., 1991). In
the hSlo + hSlo experiments, when PKA was
applied to these channels, a small but significant decrease in
Popen was observed that was not as large as
the downregulation for the type II channels recorded in lipid bilayers.
However, given that two of the patch recordings showed an increase
after exposure to PKA, the results suggest the presence of a mixed
population of channels, at least with respect to their response to
phosphorylation, which may reflect the presence of both hSlo
channels with and without coexpression of hSlo despite
cotransfection. If the channels in which PKA did not produce an
upregulation of Popen are considered alone,
the PKA-induced decrease in Popen in
patches from hSlo + hSlo-transfected cells
would then closely resemble the comparable reduction for native type II
channels (Reinhart et al., 1991). In addition, estimated
EC50 values for hSlo-expressing cells
for BK channel blockers are within an order of magnitude of values
similarly obtained for native (toxin-sensitive and presumably type I)
BK channels (Reinhart et al., 1989; Giangiacomo et al., 1992). However,
the hSlo + hSlo toxin curves in Figures 5 and
6 seem to be shallow compared with hSlo alone and do not
reflect the degree of insensitivity expected of a pure population of
type II BK channels. The shallow curves may suggest the presence of two
binding sites, again suggestive of a mixed population of channels, at
least in the oocytes. Regardless of the exact degree or correspondence
between cloned hSlo and hSlo + hSlo
BK channels and any previously described BK channel classification
scheme, it is clear from the data presented in this study that the
association of the BK-specific hSlo subunit confers
significant phenotypic alteration to hSlo channels. Thus,
the hSlo subunit is a likely major source of observed
phenotypic variation in native BK channel populations, but is not the
only source of BK channel diversity.
In conclusion, the changes in phenotype produced by this single known
Slo subunit are significant and are manifest in several
modalities. When coupled with changes produced by alternative splice
variation, the contribution of the hSlo subunit forms
part of the molecular basis for native BK channel diversity. A better
understanding of the interactions of these channels and their
regulatory subunit(s) should provide insight into the structural
determinants of BK channel modulation and its functional consequences
on cellular physiology.
Note added in proof: Subsequent to the
submission of this manuscript, Meera et al. (1996) report that
coexpression of hSlo and hSlo produced an
increase in current activation time constants and an increase in
Ca2+ sensitivity.
FOOTNOTES
Received March 7, 1996; revised May 3, 1996; accepted May 7, 1996.
Correspondence should be addressed to Dr. Valentin K. Gribkoff, Central
Nervous System Drug Discovery, Department 404, Bristol-Myers Squibb
Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT
06492.
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E. S. L. Faber and P. Sah
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R. Brenner, T. J. Jegla, A. Wickenden, Y. Liu, and R. W. Aldrich
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S.-N. Wu, H.-F. Li, and Y.-C. Lo
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E M C Jones, M Gray-Keller, and R Fettiplace
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B. S. Rothberg and K. L. Magleby
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X.-M. Xia, J. P. Ding, and C. J. Lingle
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A R Evans, M R Vasko, and G D Nicol
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K. Ramanathan, T. H. Michael, G. Jiang, H. Hiel, and P. A. Fuchs
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D.H. Cox, J. Cui, and R.W. Aldrich
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M. Hanner, W. A. Schmalhofer, P. Munujos, H.-G. Knaus, G. J. Kaczorowski, and M. L. Garcia
The beta subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin
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