From the Membrane Biology Group, Division of Biomedical Sciences,
University of Edinburgh Medical School, Edinburgh EH8 9XD,
Scotland, United Kingdom and Laboratory of Signal
Transduction, NIEHS, National Institutes of Health, Research
Triangle Park, North Carolina 27709
Received for publication, November 15, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Large conductance, calcium- and voltage-activated
potassium (BK) channels control excitability in many tissues and are
regulated by several protein kinases and phosphatases that remain
associated with the channels in cell-free patches of membrane. Here, we
report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase (PKA) to
associate with the channel and regulate its activity. A synthetic
polypeptide encompassing the central d position leucine residues
in LZ1 blocks the regulation of recombinant mouse BK channels by
endogenous PKA in HEK293 cells. In contrast, neither an
alanine-substituted LZ1 peptide nor a peptide corresponding to another,
more C-terminal putative leucine zipper, LZ2, had any effect on
regulation of the channels by endogenous PKA. Mutagenesis of the
central two LZ1 d position leucines to alanine in the BK channel also
eliminated regulation by endogenous PKA in HEK293 cells without
altering the channel sensitivity to activation by voltage or by
exogenous purified PKA. Inclusion of the STREX splice insert in the BK
channel protein, which switches channel regulation by PKA from
stimulation to inhibition, did not alter the requirement for an intact
LZ1. Although PKA does not bind directly to the channel protein
in vitro, mutation of LZ1 abolished co-immunoprecipitation
of PKA and the respective BK channel splice variant from HEK293 cells. Furthermore, a 127-amino acid fusion protein encompassing the functional LZ1 domain co-immunoprecipitates a PKA-signaling complex from rat brain. Thus LZ1 is required for the association and regulation of mammalian BK channels by PKA, and other putative leucine zippers in
the BK channel protein may provide anchoring for other regulatory enzyme complexes.
Reversible protein phosphorylation represents a fundamental
cellular regulatory mechanism to control the activity and function of
plasma membrane ion channels (1). The co-ordination, specificity, and
compartmentalization of ion channel regulation by reversible protein
phosphorylation is facilitated by assembly with signaling complexes
comprising cognate protein kinases and protein phosphatases. Assembly
of ion channels with signaling complexes typically results from
multiple protein-protein interactions mediated by distinct interaction
domains (2) allowing signaling molecules to interact directly with an
ion channel or indirectly as part of a higher order complex.
Large conductance calcium- and voltage-activated potassium (BK)
channels have been widely exploited as models of ion channel regulation
by reversible protein phosphorylation; however, the molecular basis for
kinase and phosphatase assembly with the BK channel is largely unknown
(1, 3). BK channels play a central role in the regulation of cellular
excitability because they are activated directly by both voltage and
intracellular free calcium (4-6) and potently modulated by reversible
protein phosphorylation (1). For example, they provide a dynamic link
between electrical and chemical signaling events in cells, are major
determinants of vascular smooth muscle tone (5, 7), and regulate action potential duration and frequency as well as neurotransmitter and hormone release in neurons and endocrine cells (6, 8). A single gene
(KCNMA1) encodes for the pore-forming Recently a structural motif, the leucine zipper (LZ), originally
described in classes of DNA-binding proteins (20), has been reported to
play an important role in coordinating both the assembly of ion
channels as well as their interaction with protein kinase and protein
phosphatase signaling complexes (21-24). In several of these channels
the catalytic subunit of PKA (PKAc) is targeted to the channel through
a protein kinase A-anchoring protein (AKAP). The AKAP acts as an
adapter protein by binding to both the LZ domain of the channel and the
regulatory subunits of PKA. In this report we identify a
putative LZ domain for BK channel assembly with a PKA signaling
complex essential for the functional regulation of mammalian BK
channels by PKA-dependent phosphorylation.
Construction of BK Channel Splice Variant and Mutant cDNA
Constructs--
The cloning and sub-cloning of the mouse BK channel
splice variants ZERO and STREX into the mammalian expression vector
pcDNA3 or pcDNA3.1+ (Invitrogen) have been described previously
(17, 25). A C-terminal hemagglutinin (HA) tag was introduced into each
channel construct by replacing the normal stop codon with a sequence
encoding the HA tag from a mouse BK channel construct kindly provided
by Dr. Yi Zhou and Prof. Irwin B Levitan. The C terminus is thus
identical to the mouse mbr5 (GenBankTM accession number
GI:347144) sequence present in the original clones apart from the HA
tag (REVEDECYPYDVPDYA*), where the italicized
amino acids indicate HA-tag amino acids replacing the normal stop
codon. Amino acid numbering in the subsequent text and figures is in
accordance with the amino acid sequence of the mouse mbr5 clone
(accession number: GI:347144; the start methionine being
M1ELEH) for consistency.
Alanine substitutions in the third and fourth LZ1 d position
leucine residues (Fig. 1: amino acids Leu-530 and Leu-537) was
performed by site-directed mutagenesis using a single mutagenic primer
set with the QuikChange system according to the manufacturer (Stratagene, La Jolla, CA) to generate the HA-tagged LZ1
mutant channels ZEROL530A/L537A and
STREXL530A/L537A.
Thioredoxin fusion proteins were generated by PCR and subcloning
fragments into the pBAD/Thio-TOPO vector (Invitrogen). Fusion proteins
were constructed with an N-terminal His-patch (HP)-thioredoxin fusion and C-terminal V5 and hexahistidine epitopes to facilitate purification and immunoprecipitation. All soluble fusion proteins were
induced and purified from BL21 RIL Escherichia coli using standard methods. All immunoprecipitations using thioredoxin fusion proteins were performed with the intact HP-thioredoxin fusion, because
cleavage of the thioredoxin fusion resulted in proteins that were
largely insoluble.
The LZ1489-616 thioredoxin fusion protein was designed to
span the LZ1 domain from the end of the predicted regulator of
potassium conductance (RCK) domain Competing Peptides--
Leucine zipper competing peptides were
synthesized by Genemed Synthesis (South San Francisco, CA) and used at
a final bath concentration of 25-80 µM. Concentrations
of peptide <5 µM were largely ineffective (data not
shown). The LZ1 peptide
(DQSCLAQGLSTMLANLFS) corresponds to amino acids 523-539, spanning the 2nd, 3rd, and 4th
d positions in the mouse 5-heptad repeat LZ1 motif (Fig.
1a) except that an N-terminal aspartate (D) residue was included to improve water solubility. The corresponding alanine substituted peptide (Ala-LZ1) was identical except that the 2nd, 3rd,
and 4th LZ1 heptad repeat d residues were replaced by
alanine
(DASCLAQGASTMLANAFS). The
LZ2 peptide (DLRAVNINLCDMCVILS)
corresponds to conserved residues 818-834 within a putative C-terminal
4-heptad repeat LZ motif in the mouse BK channel (Fig.
1a).
HEK293 Cell Culture and Transfection--
HEK293 cells were
subcultured essentially as previously described (17, 25) except with
one modification whereby cells were placed in serum-free (ITS,
Invitrogen) medium 24 h before experiments. Briefly, cells were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum in a humidified atmosphere of 95% air, 5%
CO2 at 37 °C. Cells were routinely passaged every 3-7
days using 0.25% trypsin in Hanks' buffered salt solution containing
0.1% EDTA. For immunoblotting studies cells were grown to 70-80%
confluence in 75-cm2 flasks. For electrophysiological
assays cells were plated on glass coverslips in 6-well cluster dishes.
Twenty-four hours before the experiment cells were washed, and medium
was replaced with Dulbecco's modified Eagle's medium containing ITS
serum replacement (Invitrogen). For transient transfections of BK
channels HEK293 cells were seeded onto glass coverslips in 6-well
cluster dishes at a density to allow cells reaching 40-60% confluence
after 24 h. Cells were then transfected with 1 µg of the
respective cDNA using LipofectAMINE (Invitrogen) in Dulbecco's
modified Eagle's medium essentially as described by the manufacturer.
After 5 h medium was supplemented with 10% fetal calf serum,
which was replaced after 24 h, and electrophysiological recordings
made 24-72 h post-transfection. Stable cell lines were also created by
selection and maintenance for Zeocin or Geneticin resistance using 0.2 mg ml Pull-down and Immunoprecipitations--
Immunoprecipitation (IP)
of HA-tagged channels or PKA was performed using transient or stably
expressing HEK293 cell lines essentially as previously described (17).
Briefly, cells were solubilized in radioimmunoassay buffer containing
50 mM Tris-HCl, pH 7.5, 0.5 mM
MgCl2, 0.2 M NaCl, 10 mM
EDTA, 20 mM sodium pyrophosphate, 100 mM sodium
fluoride, 1 mg/ml bovine serum albumin, 1% (v/v) Triton X-100, and
protease inhibitors (Roche Molecular Biochemicals). Insoluble material
was removed by centrifugation (10,000 × g, 15 min,
4 °C), and the lysate was pre-cleared by incubation with 20 µl of
50% (v/v) protein G-Sepharose with agitation for 1 h at 4 °C.
PKA- or HA-tagged channels were immunoprecipitated from the cleared
lysate with the respective antibody (prebound to 40 µl of 50% (v/v)
protein G-Sepharose) for 4 h at 4 °C. IP antibodies used were
mouse anti-HA monoclonal antibody (clone: 12CA5, Roche Diagnostics) for
HA-tagged channels, a sheep anti-PKAc polyclonal antibody (ab365 (29),
a generous gift from Dr Roger A Clegg), or a rabbit anti-PKAc
polyclonal antibody (Santa Cruz Biotechnology Inc.). The
immunoprecipitate was washed 5 times with 1 ml of radioimmunoassay buffer before SDS-PAGE analysis.
For Western blot detection a rabbit anti-HA polyclonal (Y-11, Santa
Cruz Biotechnology, Inc.) and the above sheep anti-PKAc polyclonal were
used as described in figure legends (Figs. 4 and 5). IP of
thioredoxin fusion proteins of the BK channel intracellular C terminus
employed a mouse monoclonal anti-V5 antibody (Invitrogen). Detection
was by enhanced chemiluminescence.
Patch Clamp Electrophysiology--
All experiments were
performed in the inside-out configuration of the patch clamp technique
at room temperature (20-24 °C) using physiological potassium
gradients essentially as described previously (17). The pipette
solution (extracellular) contained 140 mM NaCl, 5 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 20 mM glucose, 10 mM HEPES, pH 7.4. The bath solution (intracellular)
contained 140 mM KCl, 5 mM NaCl, 2 mM MgCl2, 1 or 5 mM BAPTA, 30 mM glucose, 10 mM HEPES, 1 mM ATP,
pH 7.3, with free calcium [Ca2+]i buffered to 0.2 µM unless indicated otherwise. For assays in which the
catalytic subunit of PKA (PKAc, Promega, Madison, WI) was applied
directly to patches, patches were exposed to the above intracellular
bathing solution containing 0.1 mM dithiothreitol during
control and PKAc application to exclude effects dues to dithiothreitol
(30) present in purified PKAc preparations. 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. After patch
excision channel activity was allowed to stabilize for at least 10 min
(typically 10-15 min after excision), and stability plot experiments
demonstrated that BK channel activity was stable for >1 h under the
recording conditions used (data not shown) in the absence of channel
modulators. Application of cAMP or other reagents to the intracellular
face of patches was by gravity-driven perfusion (10 volumes of the
recording bath solution (bath volume, 0.5 ml) by gravity-driven
perfusion at a flow rate of 1-2 ml/min) or direct application to the
bath. Channel activity was determined during 30-s depolarizations to +40 mV.
Single-channel open probability (Po) was derived
either from single-channel analysis using pSTAT for patches with <4
channels or, in the case of patches with >4 channels, by an integration-over-baseline algorithm using Igor Pro 4.1 (WaveMetrics, Lake Oswego, OR). In the latter case N Po (number of functional channels × open probability of channel) values were determined as follows. All-point histograms were plotted to obtain the
"offset," i.e. leak current, as well as the
single-channel current amplitude from the peak intervals. After
subtraction of the offset from the traces these were integrated over
0.5-60-s segments. The integral divided by integration time and
single-channel current amplitude gives NPo.
To determine mean percent (%) change in channel activity after a
treatment in patches with low to moderate levels of channel expression,
mean Po or N × Po was
averaged from several minutes of recording at +40 mV immediately before
and 10 min after the respective drug treatment. Mean change in activity was expressed as a percentage (%) of the pretreatment control ±S.E.
In the respective figure legends and text (see "Results") a
positive percentage (%) change in activity reflects activation, whereas a negative percentage (%) change reflects channel inhibition.
Putative LZ Motifs in Mammalian BK Channels--
The amino acid
sequence of LZ motifs is typically characterized by a seven-residue
(heptad) repeat (commonly denoted abcdefg, see Fig.
1, a and b) with
positions a and d in each heptad repeat occupied
by hydrophobic residues (20, 31). Leucine provides the most
thermodynamically stable residue at position d (32).
However, significant deviations in amino acid sequence from this
"classical" leucine zipper motif may exist, for example, deletions
or insertions of individual residues within the heptad repeat or the
presence of polar residues at position a or d
(22, 31, 32, 34).
Allowing for such variability, manual inspection of the C-terminal
amino acid sequence of mammalian BK channels revealed several putative
LZ motifs. A C-terminal LZ motif (LZ1, Fig. 1, a and b, between residues 513 and 548 of the murine BK channel
variant mbr5 (accession number GI:347144 (9)) contains five heptad repeats downstream of residues that contribute to the proposed "fixed
interface" of the BK channel regulator of K channel conductance (RCK,
or tetramerization) domain (26-28). Although the second d residue of the five-heptad LZ1 repeat is glutamine (Q) and the fifth
a residue is non-hydrophobic, the stability of
"prototypical" LZ domains with a Gln (Q) residue at a single
d position is, paradoxically, not significantly compromised
compared with isoleucine or valine substitutions (32). Comparison of
the amino acid sequence of mammalian BK channels with the structure of
the RCK domain in calcium-activated potassium (MthK) channels from Methanobacterium thermoautotrophicum suggests that LZ1 forms
the
A second putative four-heptad repeat LZ domain (LZ2: residues 816-843,
Fig. 1a) is positioned between splice site 2 and the conserved PKA consensus site at serine residue Ser-899 (see Fig. 1a). At least two further three-heptad repeats may also be
present in the C terminus.
To investigate whether LZ1 or LZ2 plays a functional role in targeting
PKA to BK channels we examined the regulation of two distinct murine BK
channel splice variants, STREX and ZERO, that are differentially
regulated by PKA-dependent phosphorylation when expressed
in HEK293 cells (17). Two functional strategies were exploited, first,
by designing competitive peptide inhibitors of LZ1 and LZ2 interactions
in an attempt to disrupt PKA regulation of STREX and ZERO channels,
respectively, and second, by investigating PKA-dependent
regulation of STREX and ZERO channels in which candidate LZ1 motifs
identified in the peptide inhibitor screen were disrupted by mutating
the third and fourth d position leucine residues (Leu-530
and Leu-537) to alanine.
LZ1 Is Required for PKA Regulation of the ZERO Splice
Variant--
Because the LZ1 domain is conserved in mammalian BK
channels and distinct BK channel splice variants may be differentially regulated by PKA (17), we addressed whether the LZ1 motif was required
for regulation of distinct splice variants by endogenous PKA. To assay
for regulation of BK channel splice variants by endogenous PKA we
applied cAMP to the intracellular face of isolated inside-out patches
from HEK293 cells to activate PKA closely associated with the channel
as previously reported (17). The mouse ZERO variant of BK is activated
by PKA closely associated with the channel and dependent upon a
C-terminal serine residue (Ser-899) conserved in all mammalian BK
channel splice variants (17, 25). C-terminal HA-tagged ZERO channels
were stimulated upon application of cAMP to the intracellular face of
inside-out patches from HEK293 cells only in the presence of Mg-ATP
(48.6 ± 8.0%, n = 8, Fig. 2) in a similar fashion to untagged
channels (17). cAMP activation of ZERO channel activity was not
observed in the presence of 25-80 µM LZ1 competing
peptide (mean change in activity was
To address whether mutation of the LZ1 motif prevents transduction of
the effect of PKA-mediated phosphorylation of the mutant channel rather
than preventing interaction of PKA with the channel, we applied the
purified catalytic subunit of PKA (PKAc) to the intracellular face of
patches containing ZEROL530A/L537A channels. Application of
PKAc resulted in a 49.3 ± 5.2%, (n = 3)
activation of ZEROL530A/L537A channels, similar to that
upon activation of endogenous PKAc with cAMP. Taken together these data
suggest that the LZ1 motif is a common site required for targeting of
PKA to murine ZERO variant BK channels.
LZ1 Is Required for PKA-dependent Inhibition of the
STREX Splice Variant--
To examine whether the conserved LZ1 domain
is also required for regulation of other BK channel splice variants by
endogenous PKA, we examined the regulation of the mouse STREX variant
expressed in HEK293 cells. STREX channels are identical to ZERO apart
from inclusion of a 59-amino acid insert (17, 25). The mouse STREX splice variant is potently inhibited by PKA closely associated with the
channel complex and dependent upon a conserved serine residue within
the STREX insert (17). STREX channels expressing a C-terminal HA tag
were similarly inhibited (mean % change in activity was
To address whether the action of cAMP is dependent upon targeting of
endogenous PKAc with the channel via a "typical" protein kinase
A-anchoring protein, as for several other types of ion channel
(21-23), we examined cAMP regulation in the presence of the
AKAP-competing peptide, ht31 (33). In parallel experiments, cAMP-dependent inhibition of STREX channel activity in the
presence of ht31 peptide was not significantly different from that with cAMP alone (mean % inhibition was PKAc Docking with Mouse BK Channel Variants Mediated via a LZ1
Complex--
The catalytic subunit of PKA (PKAc) has previously been
shown to co-immunoprecipitate with both the ZERO and STREX BK channel splice variants (17). Because PKA regulation of both ZERO and STREX
splice variants was dependent upon a functional LZ1 motif we addressed
whether alanine substitution of the third and fourth d
position LZ1 leucine residues prevented co-immunoprecipitation of PKAc
with either splice variant subunit. PKAc co-immunoprecipitated with
STREX or ZERO channels upon pull-down of the HA epitope-tagged channels
expressed in HEK293 cells (Fig.
4a) as previously reported for
the untagged splice variants (17). However, PKAc did not co-IP with
either alanine-substituted LZ1 BK channel splice variant (Fig.
4a). Furthermore, association of PKAc with either STREX or
ZERO channels did not require interactions mediated via PKA regulatory
subunits because an antibody that IPs PKAc that is dissociated from its
regulatory subunits (29) resulted in robust co-IP of the respective BK
channel variant (Fig. 4b). Similar co-IP was observed with
anti-PKAc antibodies that IP the holoenzyme (not shown). Furthermore,
co-IP was not dependent upon channels being phosphorylated at the
putative PKA consensus sites because PKAc co-immunoprecipitated with
the STREX channel in which both the STREX (STREX Ser-4) and
conserved (Ser-899) PKA consensus serine residues were mutated to
alanine (STREXS4A:S899A construct, Fig. 4b).
This suggests that LZ1 is required for targeting of PKAc to the channel
and that targeting is independent of substrate recognition.
Because the LZ1 motif is immediately downstream of the core RCK domain
(also referred to as the tetramerization domain) (26-28) mutation of
this site may disrupt the C-terminal conformation of the channel, thus
preventing association of a PKAc signaling complex with other domains
in the channel. Indeed, in Drosophila PKAc binds directly
with the C terminus of the channel at a region close to the
Drosophila equivalent (Ser-942) of the conserved mammalian
PKA consensus site at mammalian serine residues Ser-899 (3, 19). To
directly test whether the LZ1 domain in mouse BK channels is required
as a targeting motif for a PKAc signaling complex we generated soluble
thioredoxin C-terminal fusion proteins encompassing the LZ1 domain
(LZ1489-616 construct, Fig. 5a) as well as fusion proteins
encompassing LZ2, the STREX alternative site of splicing, and the
conserved PKA consensus motif at Ser-899 (ZERO579-984 and
STREX579-984 constructs respectively, Fig. 5a).
All assays were performed with the thioredoxin fusions because cleaved
proteins were largely insoluble.
In vitro pull-down assays using the LZ1489-616,
ZERO579-984 or STREX579-984 fusion proteins
as bait failed to pull down recombinant purified PKA catalytic subunit
(Fig. 5, b and c). However, using solubilized
whole rat brain lysate (Fig. 5, b and c) or
HEK293 cell lysate (not shown) resulted in specific pull down of PKAc
with the LZ1489-616 fusion protein (Fig. 5b)
but not with either the ZERO579-984 or
STREX579-984 fusions or thioredoxin alone (Fig.
5c). To confirm that a functional LZ1 domain is required for
PKAc complex assembly with the LZ1489-616 fusion protein
we mutated the third and fourth d position LZ1 leucine
residues (Leu-530 and Leu-537) within the LZ1489-616 fusion protein to alanine. This construct was expressed at equivalent levels as the wild type fusion protein; however,
mLZ1489-616 failed to pull down PKAc from brain lysates.
Taken together these data suggest that a functional LZ1 domain is
required for assembly of a PKAc signaling complex, dependent upon an as
yet unidentified adapter protein(s), with mammalian BK channels.
Increasing evidence suggests that LZ domains play an important
role in both the assembly of ion channel signaling complexes as well as
ion channel assembly per se (21-24). Allowing for known variations in the amino acid sequence of classical leucine zipper motifs, manual inspection of the intracellular C-terminal domain of
mammalian BK channels revealed at least four possible LZ domains with
LZ1 and LZ2 (Fig. 1a), displaying the highest criteria for functional LZ domains.
In this paper we have identified, using both competing peptide and
mutagenesis studies, a leucine zipper motif present in BK channels that
is required for the targeting and functional regulation of mammalian BK
channels by PKA. Using a combination of functional and biochemical
assays we demonstrate that LZ1, but not LZ2, is required for BK channel
assembly with a PKA signaling complex. First, short competing peptides
designed to disrupt specific LZ domain interactions blocked
PKA-dependent regulation of both STREX and ZERO BK channel
variants. In these assays we stimulated PKA activity intimately
associated with the channel by applying cAMP to the intracellular face
of isolated inside-out patches. As previously reported (17) the effects
of cAMP in this system are dependent upon the presence of Mg-ATP, and
the actions of cAMP are completely abolished by the PKA inhibitor
peptide PKI5-24. Although STREX channels are inhibited
whereas ZERO channels are activated by PKA closely associated with the
channel (17), the short LZ1-competing peptides effectively blocked
PKA-dependent regulation of either splice variant. In
contrast, alanine-substituted LZ1-competing peptides or peptides
directed against LZ2 were not able to block PKA-dependent
regulation of the respective BK channel splice variant. Secondly,
disruption of the LZ1 domain in STREX or ZERO by mutating the third and
fourth d position leucine amino acids to alanine (Leu-530
and Leu-537) prevented channel regulation by endogenous PKA. A
functional LZ1 is not required for the transduction of the effect of
PKA-dependent phosphorylation because application of
purified PKAc to patches containing the LZ1 mutant channels was still
able to regulate channel activity. This supports the hypothesis that
PKA-dependent phosphorylation rather than PKA binding to
the channel complex per se is responsible for PKA regulation
of mammalian BK channels. Third, although PKAc co-immunoprecipitates
with both wild type STREX and ZERO channels, pull-down of PKAc was not
evident with either splice variant when the respective LZ1 motif was
mutated. Finally, although PKAc does not directly interact with a
fusion protein incorporating LZ1, a PKAc signaling complex can be
isolated from native brain tissue using a fusion protein that contains
an intact LZ1 domain but not an intact LZ2 domain. Although we cannot
exclude that LZ1 is required for correct conformation of a binding site
within the 127-amino acid LZ1489-616 fusion protein rather
than acting as the direct PKAc signaling complex interaction motif, our
data strongly support the hypothesis that LZ1 functions as a conserved
motif for directing assembly of a PKAc signaling complex with mammalian
BK channels.
The adapter protein linking PKAc with the BK channel has not been
identified; however, three independent lines of evidence suggest the
putative adapter protein is not a typical AKAP that targets PKAc to
several other ion channels (21-23). First, in electrophysiological assays concentrations of ht31 peptide (33) that disrupt PKA/AKAP interactions at other ion channels (21-23) had no significant effect on channel regulation by cAMP. Second, co-IP of PKAc with the channel
was observed both with anti-PKAc antibodies that IP holoenzyme as well
as with antibodies that IP PKAc subunits not associated with their
regulatory subunits. Finally, PKAc pull-down was observed in the
presence of saturating concentrations of cAMP that would dissociate
PKAc from its regulatory subunits.
In contrast to the requirement for an adapter protein to target PKAc
with mammalian BK channels reported here PKAc binds directly to the C
terminus of Drosophila BK channels (3, 19). BK channels from
Caenorhabditis elegans and Drosophila, in
contrast to mammalian BK channels, display amino acid substitutions at
the 3rd and 5th d positions with phenylalanine and arginine
residues, respectively. Thus, whether PKA can also be targeted to dSlo
via the putative LZ1 domain in addition to direct PKA binding to more
C-terminal domains in dSlo remains to be elucidated.
The mammalian LZ1 domain is located after residues important for the
proposed fixed interface (resulting from helices Because the PKAc signaling complex can interact with a fusion protein
encompassing the LZ1 domain but not incorporating residues within the
core RCK domain essential for tetramerization (26-28), our data would
suggest that each subunit or subunit dimer of the BK channel tetramer
may interact with its own PKAc signaling complex. Furthermore, the
interaction of LZ1 with the PKAc signaling complex is not promiscuous
as PKAc failed to co-immunoprecipitate with fusion proteins spanning
other putative LZ domains, including LZ2. Whether, other potential BK
channel LZ motifs play an important role in BK channel targeting with
other signaling proteins or channel assembly per se remains
to be elucidated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits of BK channels in all mammalian tissues (9). Phenotypic variation in
native BK channels results from extensive alternative exon splicing of
the
-subunit (6, 9, 10) as well as through interaction with
regulatory
-subunits and accessory proteins (11-13). The BK channel
-subunit is a target for regulation by multiple protein kinases and
protein phosphatases (3, 14-18), and several protein kinases have been
reported to co-immunoprecipitate with mammalian BK channels (3, 16,
17). Although several consensus phosphorylation sites have been
identified by mutagenesis within the intracellular C-terminal domain,
BK channel
-subunits do not contain previously identified
protein-protein interaction domains. Thus, the molecular basis for
protein kinase or phosphatase targeting to mammalian BK channels is
essentially unknown. In Drosophila, the catalytic subunit,
but not the holoenzyme, of cAMP-dependent protein kinase
(PKA)1 binds directly to the
intracellular C terminus (3, 19) of the channel. Although PKA
co-immunoprecipitates with mammalian BK channels in a splice
variant-independent manner, the mechanism of complex assembly is
unknown (17).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E strand (26-28) starting at
amino acid Gln-489 and extending to amino acid Ile-616 (Fig. 5a). LZ1489-616 thus does not include core RCK
domain residues essential for tetramerization (28). The
ZERO580-984 and STREX580-984 thioredoxin
fusion proteins were designed to span LZ2 and to include the mammalian
STREX alternative site of splicing as well as the conserved PKA
consensus site at S899 (Fig. 5a). The starting amino acid in
the respective construct was Val-580 and terminating in amino acid
Ala-984. ZERO580-984 and STREX580-984 are,
thus, identical apart from the addition of 59 amino acids of the mouse
STREX (25) insert in STREX 580-984 (Fig. 5a:
amino acid numbering has been retained as for ZERO for consistency) and
do not contain LZ1. All constructs were verified by DNA sequencing.
1 Zeocin (Invitrogen) or 0.8 mg ml
1
Geneticin (Invitrogen) as appropriate.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Putative LZ motifs in mammalian BK
channels. a, schematic representation of the location of
putative LZ domains (LZ1 and LZ2) in the intracellular C terminus of
the mouse BK channel pore forming -subunit. The regulator of
potassium channel conductance (RCK) domain, alternative site of
splicing for STREX (a 59-amino acid insert at this site), and known
protein kinase A (PKA) consensus phosphorylation sites are
indicated. The amino acid sequence (single-letter code) of the mouse
(GenBankTM accession number GI:347144) LZ1 and LZ2 motifs
are indicated with the a and d positions of each
heptad repeat in bold. The underline indicates
the respective LZ1 or LZ2-competing peptides used in these studies (the
LZ1 peptide also included a N-terminal aspartate (Asp (D)) residue to
improve solubility; see "Materials and Methods"). Italicized
residues indicate leucine residues mutated to alanine in the
non-competing peptides or mutant channel constructs. Amino acid
numbering is as for the murine mbr5 BK channel variant (accession
number GI:347144) with starting methionine M1ELEH.
b, sequence alignment of corresponding LZ1 domains in other
species demonstrating homology in vertebrates. Numbers
indicate the first a position residue from the following
accession numbers: mouse (GI:347144); human (GI:507922); rat
(GI:4972782); bovine (GI:15822581); dog (GI:1127824); chicken
(1907289); Xenopus (14582152); Drosophila
(GI:158469); C. elegans (GI:16755827); MthK,
M. thermoautotrophicum calcium-activated K channel
(GI:2622639).
G helix and also contributes to an extended linker region
between the
G helix and
G strand (26-28). However, in MthK the
extended linker is absent, and the mammalian LZ1 heptad repeats are not conserved in MthK or other prokaryotic RCK domains (Fig.
1a), suggesting LZ1 and the linker region play an additional
role in mammalian BK channels.
5.1 ± 14.7%, n = 8, Fig. 2). LZ1-competing peptide also blocked cAMP
stimulation of the untagged channels (the mean change in activity was
1.9 ± 3.6%, n = 3). However, cAMP regulation
of ZERO channels was unaffected by the alanine-substituted
LZ1-competing peptide (the mean activation was 56.8 ± 14.6%,
n = 4) or LZ2-competing peptide (the mean activation
was 57.9 ± 8.9%, n = 4). Although the effect of
the LZ1 peptide is specific, relatively high concentrations of peptide
are required, LZ1 peptide concentrations <5 µM were largely ineffective (not shown). Thus, to confirm the requirement for a
LZ1 domain in ZERO channel regulation by endogenous PKA, we
mutated the third and fourth d position LZ1 leucine residues to alanine (ZEROL530A/L537A). This mutation had no
significant effect on the half-maximal voltage required for channel
activation (V0.5) under the assay conditions;
V0.5 for the respective HA-tagged channels in
the presence of 0.2 µM free calcium and Mg-ATP were 86 ± 7mV (n = 4) (ZERO) and 90 ± 10 mV
(n = 3) (ZEROL530A/L537A). Importantly,
ZEROL530A/L537A channels were unaffected by application of
cAMP to their intracellular face (mean change in activity was 2.5 ± 5.6%, n = 6) (Fig. 2b).
View larger version (25K):
[in a new window]
Fig. 2.
LZ1 is required for PKA activation of ZERO
channels. Representative single channel traces and corresponding
diary plots of channel activity from isolated inside-out patches from
HEK293 cells expressing wild type HA-tagged mouse ZERO channels (ZERO)
(a) or the alanine-substituted leucine zipper (LZ1) mutant
channels (ZEROL530A/L537A) before (control) and
10 min after (+cAMP) application of 1 mM cAMP to
the intracellular face of the patch (b).
NP0, (number of functional channels × open
probability of channel). c, summary of the effect of
cAMP application to the intracellular face of patches from ZERO
channels in the absence (ZERO, n = 8) or presence of 80 µM corresponding leucine zipper-competing peptides
(LZ1, n = 6; Ala-LZ1, n = 4; LZ2,
n = 4) as well as for the ZEROL530A/L537A
channels (n = 8). All data are expressed as the
percentage of change to pretreatment BK channel activity measured at
+40 mV in the presence of 0.2 µM
[Ca2+]i and 2 mM Mg-ATP as described
under "Materials and Methods." Mean ± S.E. **,
p < 0.01 compared with ZERO group.
56.1 ± 11.2%, n = 6) by endogenous PKA upon application of
cAMP to the intracellular face of excised inside-out patches from
HEK293 in the presence of Mg-ATP (Fig.
3). No significant cAMP inhibition of
STREX channel activity was observed in the presence of 25-80
µM LZ 1-competing peptide (mean % change in activity was
5.8 ± 8.9%, n = 10, Fig. 3). A similar LZ1
peptide block of PKA inhibition of the untagged channel was also
observed (mean % change in activity was 9.6 ± 3.9%,
n = 3). However, robust PKA-mediated inhibition of
STREX was observed in the presence of 80 µM
alanine-substituted LZ1 peptide (Ala-LZ1; mean inhibition
61.5 ± 6.2%, n = 7) or 80 µM competing
peptide corresponding to the more C-terminal putative LZ2 domain (mean
% change in activity was
61.6 ± 9.3%, n = 6).
To further address the requirement for a functional LZ1 domain for PKA
action, the third and fourth d position LZ1 leucine residues
(Leu-530 and Leu-537) in the HA-tagged STREX channel were mutated to
alanine and expressed in HEK293 cells. Although the half-maximal
voltage for activation of STREX channels is shifted to more negative
potentials than for ZERO channels (25), the LZ1 mutant STREX channels
(STREXL530A/L537A) expressed in HEK293 cells displayed no
significant shift in their half-maximal activation voltage compared
with wild type STREX channels under the assay conditions used. In the
presence of 0.2 µM free calcium and Mg-ATP, the
respective V0.5 was 47 ± 8 mV, n = 4 (STREX) and 52 ± 5 mV, n = 4 (STREXL530A/L537A). Importantly, application of cAMP to the
intracellular face of patches expressing STREXL530A/L537A
had no significant effect on channel activity (mean change in channel
activity was 14.6 ± 6.9%, n = 6).
View larger version (29K):
[in a new window]
Fig. 3.
LZ1 is required for PKA inhibition of STREX
channels. Representative single channel traces and corresponding
diary plots of channel activity from isolated inside-out patches from
HEK293 cells expressing wild type HA-tagged mouse STREX channels
(STREX) (a) or the alanine-substituted leucine zipper (LZ1)
mutant channels (STREXL530A/L537A) before
(control) and 10 min after (+cAMP) application of
1 mM cAMP to the intracellular face of the patch
(b). NP0, (number of functional
channels × open probability of channel). c, summary
of the effect of cAMP application to the intracellular face of patches
from STREX channels in the absence (STREX, n = 6) or
presence of 25-80 µM corresponding leucine zipper
competing peptides (LZ1, n = 10; Ala-LZ1,
n = 7; LZ2, n = 8) as well as for the
STREXL530A/L537A channels (n = 6). All data
are expressed as the percentage of change to pretreatment BK channel
activity measured at +40 mV in the presence of 0.2 µM
[Ca2+]i and 2 mM Mg-ATP as described
under "Materials and Methods." Mean ± S.E. **,
p < 0.01 compared with STREX group.
57.2 ± 2.2%,
n = 4). Taken together these data suggest that LZ1 is
required to target endogenous PKAc to BK channel splice variants and
that targeting or regulation is not dependent upon a typical protein
kinase A-anchoring protein.
View larger version (28K):
[in a new window]
Fig. 4.
PKAc co-immunoprecipitation with BK channel
splice variants requires a functional LZ1. a, representative
Western blots of co-IPs from HEK293 cells expressing HA-tagged STREX or
ZERO channels and their respective LZ1 mutants
(STREXL530A/L537A and ZEROL530A/L537A). IPs
were performed using a mouse monoclonal anti-HA antibody, and
immunoprecipitates were probed with either rabbit anti-HA antibody or
sheep anti-PKAc antibody. b, representative Western blots of
co-IPs of PKAc with HA-tagged BK channel variants using a sheep
anti-PKAc antibody to IP PKAc that is not associated with its
regulatory subunits (see "Materials and Methods"). Cell lysates
were probed with the same anti-PKAc antibody, and channels were
detected in the IPs using a rabbit anti-HA antibody. The STREX
S4A:S899A construct is identical to STREX, except that the
STREX insert (S4) and conserved (S899) consensus PKA phosphorylation
serine residues are mutated to alanine. Detection was by enhanced
chemiluminescence with purified PKAc run as a positive control for PKAc
blots. Apparent molecular masses in SDS-PAGE gels were: PKAc, ~45
kDa; HA-tagged BK channels, ~128 kDa.
View larger version (21K):
[in a new window]
Fig. 5.
LZ1 is required for channel association with
a PKAc signaling complex. a, schematic illustrating the
thioredoxin fusion constructs used in pull-down assays. Fusion proteins
expressed an N-terminal thioredoxin fusion with a C-terminal V5 and
hexahistidine epitope tag. Amino acid numbering is as in Fig. 1.
b, representative Western blots from IP assays using the
thioredoxin fusion proteins of the LZ1 domain
(LZ1489-616) and its corresponding alanine mutant
(mLZ1489-616) as bait. IPs using an anti-V5 antibody were
undertaken in the presence (input) of purified PKAc or
solubilized rat brain extracts, and precipitates were separated by
SDS-PAGE and blotted for PKAc. c, representative Western
blots from IP assays using the thioredoxin fusion proteins of the
intracellular C terminus containing the LZ2 domain, conserved PKA
consensus site (Ser-899) with either the ZERO
(ZERO580-984) or STREX (STREX580-984) variant
at the STREX alternative site of splicing. IPs were performed as above
using purified PKAc (top panel) or solubilized rat brain
extracts (lower panel) as prey, and precipitates were
separated by SDS-PAGE and blotted for PKAc. Detection was by enhanced
chemiluminescence. The apparent molecular mass of PKAc in SDS-PAGE gels
was ~45 kDa.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
D and
E in the
crystal structure of the calcium-activated potassium channel (MthK)
from M. thermoautotrophicum) as well as for salt bridge
formation in the RCK (or tetramerization) domain of mammalian BK
channels (26-28). Comparison of the mammalian BK channel sequence with
residues in the RCK domain of MthK channels suggests that LZ1 would
contribute to the
G helix as well as the extended linker region
between the
G helix and
G strand. Thus, LZ1 would be predicted to
contribute to the RCK variable subdomain that protrudes from the gating
ring (26). The
G helix and variable subdomain in MthK together
contribute to a flexible interface between RCK domains (26). However,
both the mammalian LZ1 heptad repeats and extended linker is absent in
MthK and other prokaryotic RCK domains, suggesting LZ1 plays an
important additional role in mammalian BK channels (26-28). Because
the LZ1 motif is highly conserved in vertebrates and is not disrupted
by known sites of alternative splicing in mammals (6), LZ1 may thus
contribute to both a flexible interface as well as a domain required
for targeting of a PKAc signaling complex with the channel. Whether LZ1
is the actual interaction interface or is required to target the PKAc
signaling complex to downstream regions within the 127-amino acid
LZ1489-616 fusion protein (i.e. that would be
predicted to form the majority of the variable flexible domain) remains to be elucidated. Importantly, LZ1-competing peptides or mutagenesis of
the third and fourth d position LZ1 leucine residues to
alanine had no significant effect on the half-maximal voltage required
for activation of STREX or ZERO channels at free calcium concentrations
of <1 µM used in this study. Recently, mutagenesis of
methionine Met-540 to isoleucine, which lies within the last heptad
repeat of the LZ1 motif, was shown to reduce murine BK channel calcium
sensitivity at calcium concentrations of 10 µM and above
(35). Because the BK channel RCK domain contributes to calcium
sensitivity (35, 36), the M540I mutation may disrupt the LZ1 motif and
modify intrinsic calcium binding sites within the RCK or other
C-terminal region of the channel. Alternatively the M540I mutation may
displace an extrinsic calcium sensor that interacts with the RCK in a
LZ1-dependent manner.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Roger Clegg (Hannah Research Institute) for the generous gift of the sheep anti-PKAc polyclonal antibody and Dr. Yi Zhou and Professor Irwin B Levitan for the generous gift of a mouse BK channel clone containing a C-terminal HA epitope. We thank members of the Membrane Biology Group for useful discussions.
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FOOTNOTES |
---|
* This work was supported by Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom and Wellcome Trust grants (to M. J. S.).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.
Contributed equally to this work.
§ A University of Edinburgh Medical School Crichton Scholar.
¶ Recipient of a Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom Committee studentship.
** To whom correspondence should be addressed: Membrane Biology Group, Division of Biomedical Sciences, Hugh Robson Bldg., University of Edinburgh, Edinburgh, Scotland, UK, EH8 9XD. Tel.: 44-131-650-3253; Fax: 44-131-650-6527; E-mail: mike.shipston@ed.ac.uk.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211661200
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ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; PKAc, catalytic subunit of PKA; LZ, leucine zipper; AKAP, protein kinase A-anchoring protein; HA, hemagglutinin; IP, immuno- precipitation.
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