Membrane Biology Group (M.J.S., R.R.D., A.G.C., L.T.)
Department of Biomedical Sciences University of Edinburgh Medical
School Edinburgh, Scotland, UK, EH8 9AG
Medical Research
Council Brain Metabolism Unit (F.A.A.) Edinburgh, Scotland, UK, EH8
7NA
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ABSTRACT |
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INTRODUCTION |
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In mouse AtT20 corticotropes large conductance calcium- and voltage- activated potassium (BK) channels play a pivotal role in the regulation of ACTH secretion (4, 5). In this system the stimulatory neuropeptide, CRH, potently inhibits BK channels through protein kinase A (PKA)-mediated phosphorylation of the BK channel complex (5, 6). Importantly, glucocorticoids prevent PKA inhibition of BK channels, and this action of the steroid is pivotal to early glucocorticoid inhibition of ACTH secretion in this system (4, 5, 6).
In contrast to the multiple gene families of other voltage-dependent
cation channels, the pore-forming -subunits of BK channels are
encoded by a single gene, Slo (7, 8), that undergoes
extensive, hormonally regulated, alternative RNA splicing (9, 10).
These splice variants give rise to channels with different functional
properties, including calcium sensitivity, single-channel conductance,
and regulation by intracellular signaling pathways (11, 12, 13, 14). In
addition, association of
-subunits with accessory subunits may
result in differential channel properties or modify their cellular
distribution (15, 16, 17, 18).
Mouse corticotrope BK channels display considerably higher calcium
sensitivity (6) than previously identified -subunits cloned from
mouse brain. Furthermore, in common with native BK channels from
several cell types of the anterior pituitary (5, 19, 20), mouse
corticotrope BK channels are inhibited by PKA-mediated phosphorylation,
whereas the majority of brain BK channels are activated by PKA (21, 22).
Elucidation of the molecular components of BK channels in specific cell
types is beginning to reveal the molecular basis for their functional
role in fundamental physiological processes (23, 24). To further
address the functional role, and molecular regulation, of BK channels
in mouse corticotrope cell function, we have 1) characterized and
cloned the BK channel -subunit splice variants expressed in mouse
AtT20 D16:16 corticotropes, and 2) directly compared the functional
characteristics (calcium sensitivity and regulation by PKA) of these
identified subunits expressed in HEK 293 cells with native AtT20 D16:16
BK channels.
This report represents the first corticotrope ion channel to be
functionally characterized at the molecular level. We demonstrate that
mouse corticotrope BK channels are composed of -subunits expressing
the mouse homolog of the previously described cysteine-rich STREX-1
exon (10, 25). Furthermore, the STREX-1
-subunit variants expressed
in HEK 293 cells are inhibited by PKA-mediated phosphorylation.
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RESULTS AND DISCUSSION |
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Functional Characterization of Mouse Corticotrope -Subunits
Expressed in HEK 293 Cells
Expression of the STREX-1 and ZERO variants of mouse AtT20
corticotrope BK channel -subunits in HEK 293 cells resulted in large
outward macropatch voltage- and calcium-activated potassium currents
(Fig. 2
). In mock transfected HEK 293,
under identical recording conditions, outward macropatch calcium- or
voltage-activated potassium currents were not observed (Fig. 2A
).
Furthermore, RT-PCR (not shown) and Western blot (Fig. 1D
) analysis did
not reveal endogenous BK channel expression in this cell line.
Transfection of cloned mouse AtT20 corticotrope BK channel
-subunits
lacking the proposed initiator methionine (8, 26) in HEK 293 cells did
not result in functional BK channel expression (M. J. Shipston, R.
Duncan, and L. Tian, unpublished data).
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The slope conductance of homomeric unitary STREX-1 channels
[127.2 ± 4.2 picosiemens (pS), n = 8] in
physiological potassium gradients was not significantly different from
the slope conductance of native mouse AtT20 corticotrope channels
recorded under identical conditions (125.2 ± 3.6 pS, n = 8,
Fig. 2D). In contrast, the slope conductance of the homomeric ZERO
channel variant was significantly greater (145.3 ± 3.1 pS, n
= 7) than either STREX-1 or native mouse AtT20 corticotrope channels
(not shown).
ß-Subunits Are Not Integral Components of BK Channels in Mouse
AtT20 Corticotropes
Taken together, these data suggest that native mouse AtT20
corticotrope BK channels are largely composed of the -subunit
STREX-1 variant. However, in some systems, including vascular smooth
muscle cells (24), BK channel
-subunits functionally associate with
regulatory ß-subunits that confer enhanced calcium sensitivity
compared with the
-subunit expressed alone (16, 30). To establish
whether the enhanced calcium sensitivity of native mouse AtT20
corticotrope is a result of expression of the STREX-1 exon rather than
association of ZERO variants with ß1- or
ß2-subunits, we assayed the sensitivity of native mouse
AtT20 BK channels for the triterpenoid glycoside, DHS-1 (16, 30).
Previous studies have shown that submicromolar concentrations of DHS-1
activates BK channels only when the ß1 (or
ß2)-subunit is functionally coupled to
-subunits (16, 25, 30). Coexpression of the ZERO
-subunit variant with
ß1-subunit in HEK 293 cells resulted in a leftward shift
of the half-maximal activation potential by approximately 60 mV to
35.3 ± 7.4 mV, (n = 3) at 0.1 µM
[Ca2+]i compared with the ZERO variant alone
(see Fig. 2B
). Application of 100 nM DHS-1 to the
intracellular face of patches containing ZERO +
ß1-subunit resulted in robust activation (259.0 ±
86.7%, n = 4) of channel activity in all patches tested (Fig. 3
, A and B) that reversed to control upon
washout. In contrast, under identical recording conditions, application
of 100500 nM DHS-1 to the intracellular face of patches
from native mouse AtT20 BK channels or HEK 293 cells expressing the
STREX-1
-subunit variant or ZERO subunits alone (Fig. 3B
) had no
significant effect on BK channel activity. Previous studies (25) have
reported that ß1-subunits increase the sensitivity of the
STREX-1 variant to [Ca2+]i and DHS-1.
Furthermore, RT-PCR analysis using degenerate primers based on the
published mouse, human, and bovine ß1-subunit sequences
(16, 31) support our functional data that native mouse AtT20
corticotropes do not express endogenous ß1-subunits (Fig. 3C
). As a positive control for the RT-PCR screen, RNA from HEK 293
cells cotransfected with the ZERO
-subunit and
ß1-subunit showed robust expression (Fig. 3C
). Thus, as
DHS-1 has no effect on endogenous BK channel activity,
ß1-subunit transcripts are not expressed, and native
AtT20 BK channels do not inactivate (5, 6, 32), our data suggests that
the previously described ß1- or ß2-subunits
are not integral components of native AtT20 corticotrope BK channels
(16, 30).
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Application of 1 mM cAMP to the intracellular face of
patches, in the presence of Mg-ATP, resulted in a robust inhibition of
channel activity at both the macropatch and unitary current level
(Figs. 4 and 5
) in all patches tested. In macropatch
recordings maximal inhibition (mean percentage change in pretreatment
steady-state outward current, Io, was -47.5 ± 3.6%,
n = 4, P < 0.01 determined at + 40 mV) was
observed 10 min after application of cAMP to the intracellular face of
the patch at all potentials examined (Fig. 4
, AC). The inhibitory
effect of cAMP was completely blocked by the specific PKA inhibitor
peptide, PKI(5-24) (mean percentage change in
Io was 5.5 ± 13.2%, n = 3). Furthermore, in the
absence of ATP, cAMP alone had no significant inhibitory effect.
Indeed, removal of ATP resulted in a small, but significant, increase
in steady-state outward current by 24.6 ± 11.9%, n = 4,
P < 0.05 after 10 min (not shown). In an inside-out
patch containing a single STREX-1 BK channel, application of cAMP to
the intracellular face of the patch resulted in a robust inhibition of
single-channel mean open probability (Po) with a time
course similar to that observed for macropatch records (Fig. 5
, AC).
Furthermore, in three other patches in which unitary currents could be
resolved, cAMP significantly inhibited NPo
(P < 0.01), an effect that was blocked by
PKI(5-24) (n = 2 patches; not shown).
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Mouse Corticotrope BK Channels Are Predominantly Composed of the
STREX-1 -Subunit Variant
The data presented in this report provide the first molecular and
functional characterization of an ion channel expressed in anterior
pituitary corticotropes. Several lines of evidence suggest that mouse
AtT20 corticotrope BK channels are composed of -subunits containing
the mouse homolog of the previously described 59-amino acid,
cysteine-rich, STREX-1 exon at splice site 2 (10, 25).
First, cDNA cloning and RT-PCR splice site analysis revealed
robust expression of STREX-1 containing -subunits in mouse AtT20
corticotropes. Importantly, an identical STREX-1 exon is expressed in
normal adult mouse anterior pituitary gland and a highly homologous
exon (2 amino acid differences out of 59) is expressed in rat anterior
pituitary cells. Second, functional expression of the STREX-1
-subunit in HEK 293 cells revealed channels with almost identical
calcium sensitivity, unitary conductance, and inhibition by PKA as for
the majority (> 90%) of native AtT20 D16:16 BK channels under
identical recording conditions. In contrast, expression of the ZERO
-subunit variant resulted in channels with higher single-channel
slope conductance and significantly lower calcium sensitivity compared
with the STREX-1
-subunit or native AtT20 BK channels. Finally,
RT-PCR analysis and functional assays using the triterpenoid glycoside,
DHS-1, revealed that the high calcium sensitivity of native mouse AtT20
corticotrope BK channels is not a result of association of
-subunits
with previously described ß-subunits (16, 30).
Importantly, the STREX-1 -subunit variant, as for endogenous BK
channels in mouse AtT20 corticotropes (6), is inhibited by
PKA-dependent protein phosphorylation. Indeed, several putative PKA
consensus phosphorylation sites can be assigned from primary sequence
data that are distributed across the C-terminal intracellular domain.
However, we cannot exclude that the STREX-1
-subunit associates with
unidentified regulatory subunits, in native AtT20 D16:16 corticotropes
or HEK 293 cells, that confer sensitivity to PKA inhibition.
Increasing evidence suggests that the STREX-1 or STREX-2 (STREX) exon
(10, 25) is widely expressed in neuroendocrine cells including
pancreatic ß-cells and adrenal chromaffin cells (25, 27) as well as
anterior pituitary corticotropes (this study). Thus, the STREX exon may
be a common feature of neuroendocrine cells in which BK channel
-subunits retain a high sensitivity to calcium in the absence of
functional interaction with ß-subunits, as described for vascular
smooth muscle cells (24). Intriguingly, the rat STREX exons expressed
in adrenal chromaffin cells is hormonally regulated by the stress axis
and has been proposed to be expressed in excitable cells associated
with enhanced repetitive action potential firing (10, 23). Thus, the
STREX variants in other components of the stress axis, including
anterior pituitary cells, may also be under dynamic long-term
regulation as well as involved in the short-term regulation of
corticotrope function by CRH and other signaling pathways (5, 6, 32).
Elucidation of the molecular components of mouse corticotrope BK
channels reported in this study should allow us to define further the
functional role, and molecular regulation, of these important multiple
coincidence detectors in anterior pituitary corticotropes.
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MATERIALS AND METHODS |
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Isolation of cDNAs Encoding Mouse Corticotrope BK Channels
An RT-PCR strategy was used to amplify cDNAs encoding the entire
ORF of mouse anterior pituitary AtT20 D16:16 corticotrope BK channel
-subunits. Briefly, total cellular RNA from approximately
107 mouse AtT20 D16:16 corticotropes was isolated using
standard techniques (34) and mRNA purified using poly (A) quik columns
(Stratagene, La Jolla, CA) according to the
manufacturers instructions. One microgram of this RNA was used as
template in a first strand cDNA synthesis directed from an anchored
deoxyoligo d(T) primer
(5'-TTCTAGAATTCAGCGGCCGC(T)30N1N2),
using Superscript II reverse transcriptase (Life Technologies, Inc., Paisley, UK). The resultant cDNA was diluted and used in a
PCR to amplify the entire ORF using forward (spanning initiator
methionine: 5'-GAT GGA TGC GCT CAT CAT MCC G) and reverse (spanning
stop codon: 5'-CTG GGA TAG GCA TTA TCC GGC TCA) deoxynucleotides, with
Expand polymerase (Roche Diagnostics Ltd, Lewes,
UK). PCR product(s) were ligated to a T/A vector (pCR2.1,
Invitrogen, San Diego, CA) and completely sequenced on
both strands (OSWEL DNA Services, Southampton, UK). The STREX-1 variant
sequence has been deposited in Genbank, accession number: AF156674.
Splice Variant Analysis
First-strand cDNA from mRNA isolated from AtT20 D16:16 cells and
adult mouse anterior pituitary glands was generated as described above.
Analysis of splice sites was performed using primers encompassing
splice sites 13: forward (5'-CAG AGT CAA GAT AGA GTC AGC); reverse
(5'-AAG TGG CAT CAC CAG GTT CCG) and for splice site 4: forward (5'-GAT
ACT TCG CTT CAG GAC AAG G); reverse (5'-AAT GTC TGC GGA GTG CTG TAG C).
In some experiments a nested PCR approach, with external primers that
spanned the pore region and splice sites 14 in the first round
amplification, followed by a second round amplification with the above
internal primer sets, was used to confirm splice variants (not shown).
PCR products were characterized by gel electrophoresis and restriction
digestion, and products of interest were ligated into the T/A vector
pCR2.1 (Invitrogen) and sequenced on both strands.
For RT-PCR analysis of ß1-subunit expression, degenerate primers were designed against the published bovine, human, and mouse ß1-subunits (16, 31). Forward primer: 5'-ATG GKR AAG AAG CTG GTG ATG GCC; reverse primer: 5'-TCT GRG CCG CCA GGA TGG; and PCR products analyzed as above.
Construction of Expression Plasmids and Expression in HEK 293
Cells
Subcloning of the entire ORF of AtT20 BK channel subunits into
pcDNA3.1 resulted in low expression of channels in HEK 293 cells
compared with channel constructs containing additional 5'- and
3'-untranslated region (UTR) (14). To improve channel expression
HindIII-NheI restriction fragment(s) from AtT20
BK channel clones, spanning splice sites 13, were ligated into the
HindIII-NheI site of the mbr5 mslo (8)
variant (Genbank accession number: L16912). The
HindIII-NheI sites are conserved in all
mslo variants, and constructs were generated from the
mbr5-BSmxt plasmid construct, a generous gift from Dr Lawrence Salkoff
(Washington University, St. Louis, MO) (8). The mbr5 clone is identical
in sequence to characterized mouse corticotrope clones except that
splice sites 14 do not contain inserts and mbr5 contains additional
5'- and 3'-UTR. Thus mbr5 is identical to the ZERO variant (see
Results) isolated from AtT20 D16:16 corticotropes. The
KpnI-XbaI fragment of mbr5 encompassing the
entire ORF and additional 5'- and 3'-UTR and containing either the
AtT20 null splice site 2 (ZERO, see Results) or containing
the AtT20 STREX-1 splice site 2 insert (STREX-1) were subcloned into
the mammalian expression vector pcDNA3.1+ or pcDNA3.1+ zeo respectively
(Invitrogen BV, Leek, The Netherlands) for expression in
HEK 293 cells.
The bovine tracheal smooth muscle cell BK channel ß1-subunit (ß1) cDNA was kindly provided by Dr. Reid J. Leonard (Merck Research Laboratories, Rahway, NJ) (16). The EcoRI-NotI fragment encoding the entire ORF was subcloned into the mammalian expression vector pcDNA3.1+ zeo (Invitrogen BV).
For transient transfections, HEK 293 cells were seeded onto glass
coverslips 24 h before transfection at 40% confluency and
transfected with 1 µg of the respective expression plasmid using
lipofectamine (Life Technologies, Inc.) essentially as
described by the manufacturer. For cotransfections,
ß1-subunit DNA was transfected at a 5-fold excess over
the -subunit. Cells were used 2472 h after the start of
transfection at between 4080% confluency. Stable cell lines were
created as above by seeding in 24-well cluster dishes
(Costar, Cambridge, MA), and stable transformants were
selected for zeocin resistance using 0.2 mg/ml zeocin
(Invitrogen).
Electrophysiology
BK channels in AtT20 D16:16 corticotropes or cloned channels
expressed in HEK 293 cells were analyzed under voltage-clamp in the
excised inside-out configuration of the patch clamp technique at room
temperature (2024 C) using physiological potassium gradients
essentially as previously described (6, 14). The pipette solution
(extracellular) contained (in millimolar concentration): 140
NaCl, 5 KCl, 0.1 CaCl2, 5 MgCl2, 20 glucose, 10
HEPES, pH 7.4. The bath solution (intracellular) contained (in
millimolar concentration): 140 KCl, 5 NaCl, 2 MgCl2, 1 or 5
(1,2-bis-O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA), 30 glucose, 10 HEPES, pH 7.35, with free calcium
[Ca2+]i buffered to the concentration
indicated in the respective figure legend and calculated as previously
described (35). For [Ca2+]i greater than, or
equal to, 1 µM, dibromo-BAPTA was used as the calcium
buffer.
Data acquisition and voltage protocols were controlled by an Axopatch
200 A or B amplifier and pCLAMP6 software (Axon Instruments Inc.,
Foster City, CA). All recordings were sampled at 10 kHz and filtered at
2 kHz. For patches in which unitary currents could be resolved,
channels were voltage clamped at the potential indicated in the
respective figure legend. Mean steady-state single-channel open
probability (Po) was determined from at least 30 sec of continuous
recording under each experimental condition. For macropatch recordings,
outward BK currents were evoked by 500-msec step depolarizations
(-50 to + 80 mV), and the steady-state current amplitude, averaged
from five consecutive depolarizations 450 msec into the pulse, was
determined at each potential. For macropatch recordings with seal
resistances > 10 G leak subtraction was not routinely applied.
Pipettes were manufactured from Garner no. 7052 glass, coated with
sylgard, and had typical resistances of between 210 M
in bath
solution after fire polishing. Patches containing a single BK channel
(verified at + 80 mV and 10 µM
[Ca2+]i) were extremely rare (<0.5% of
total patches) even with pipette resistance values > 10 M
.
Additional analysis and curve fitting was performed using Igor Pro3.1 (Wavemetrics Inc., Lake Oswego, OR). Conductance values (G) were calculated from peak current (I) measured at 450 msec into the voltage pulse using the relationship G = I/(V-Ek) where V is the activation potential and Ek is the calculated potassium reversal potential. Conductance-voltage curves were fit with a single Boltzmann function, G(V) = Gmax/(1+ exp(V50 - V)/k), where Gmax corresponds to the maximal conductance, V50 is the voltage for half-maximal activation, V is the activation potential, and k is the slope factor reflecting the voltage dependence of conductance.
Western Blotting
Partially purified membrane homogenates from AtT20 D16:16 cells
were prepared by homogenizing approximately 107 cells on
ice in the following (in millimolar concentration): 150 KCl, 5 EGTA, 2
MgCl2, pH 10.6, containing 12 U/ml aprotinin, 5 µg/ml
leupeptin, 6 mM 4-(2-aminoethyl)benzenesulfonyl
fluoride, and 4 mM Pepstatin A followed by two
freeze thaw cycles. After centrifugation for 5 min 1,000 x
g at 4 C, the resultant supernatant was pelleted at
40,000 x g to give the crude membrane fraction.
Protein samples (1040 µg) were separated on a 10% SDS gel and
electroblotted to Immobilon polyvinylidene fluoride membranes.
Membranes were blocked for 2 h at room temperature with PBS
containing 0.1 mM EDTA, 0.1% Triton X-100, pH 7.4
(PBS-TE), and 5% (wt/vol) low-fat milk (Marvel). Blots were incubated
overnight at 4 C with a 1:2000 dilution of the affinity-purified
antibody slo(913-926) [directed toward residues
913926 of the pore-forming
-subunit of mouse brain BK channels
(28)] in PBS-TE containing 1% wt/vol Marvel. Blots were washed five
times with PBS-TE and incubated for 45 min at room temperature with
horseradish peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ; 1:5000 final dilution) in
PBS-TE containing 5% (wt/vol) Marvel. After five washes in PBS-TE,
blots were incubated with enhanced chemiluminescence (ECL) reagents
(Amersham Pharmacia Biotech) according to the
manufacturers protocol and blots were exposed to ECL film in the
linear response range (Amersham Pharmacia Biotech).
Reagents
ATP magnesium or potassium salt, was from Sigma-Aldrich Co. (St. Louis, MO) and stored as buffered 1 M stock
solutions at -20 C before use. ATP and cAMP were buffered in bath
solution to pH 7.35 and applied to the intracellular patch by gravity
perfusion with 10 volumes of bath solution at a flow rate of 12
ml/min. PKI5-24, BAPTA, dibromo-BAPTA, and Fura-2 were
obtained from Calbiochem (Nottingham, UK). DHS-1 was a
generous gift of Dr. Owen McManus (Merck Research Laboratories, Rahway,
NJ). The C-terminal slo(913-926) antibody was a
generous gift of Dr. Hans-Guenther Knaus (University of Innsbruck,
Innsbruck, Austria). All other reagents, unless otherwise stated, were
of the highest analytical grade available from Sigma-Aldrich Co. Ltd., Poole, UK, or Merck Ltd., Poole, UK.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by The Wellcome Trust (Ref: 046787/Z) and The Physiological Society.
1 Present address: Organon Laboratories Ltd., Newhouse,
Lanarkshire, ML1 5SH, Scotland, UK.
Received for publication April 22, 1999. Revision received June 9, 1999. Accepted for publication June 28, 1999.
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REFERENCES |
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