From the Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received for publication, October 2, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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We demonstrate that the C-terminal truncation of
hIK1 results in a loss of functional channels. This could be caused by
either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among
these possibilities, a hemagglutinin epitope was inserted into the
extracellular loop between transmembrane domains S3 and S4. Surface
expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp
techniques. Although deletion of the last 14 amino acids of hIK1
(L414STOP) had no effect on plasma membrane expression and function,
deletion of the last 26 amino acids (K402STOP) resulted in a complete
loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu406 (L399A/L406A) completely
abrogated membrane localization. Additional mutations within the heptad
repeat (L385A/L392A, L392A/L406A) or of the a positions
(I396A/L403A) resulted in a near-complete loss of membrane-localized
channel. In contrast, mutating individual leucines did not compromise
channel trafficking or function. Both membrane localization and
function of L399A/L406A could be partially restored by incubation at
27 °C. Co-immunoprecipitation studies demonstrated that leucine
zipper mutations do not compromise multimer formation. In contrast, we
demonstrated that the leucine zipper region of hIK1 is capable of
co-assembly and that this is dependent upon an intact leucine zipper.
Finally, this leucine zipper is conserved in another member of the gene
family, SK3. However, mutation of the leucine zipper in SK3 had
no effect on plasma membrane localization or function. In conclusion,
we demonstrate that the C-terminal leucine zipper is critical to
facilitate correct folding and plasma membrane trafficking of hIK1,
whereas this function is not conserved in other gene family members.
Intermediate conductance, Ca2+-activated
K+ channels (IK)1
are known to play crucial roles in a wide array of physiological
processes, including transepithelial ion transport (1-3), T-cell
activation (4-6), volume regulation (7), cell growth and
differentiation (8, 9), and regulation of vascular tone (10). The human IK channel (hIK1/hSK4) underlying these physiological processes was
recently cloned (11, 12). Indeed, hIK1 has been shown to possess all
the biophysical and pharmacological hallmarks of the endogenously
expressed channel, including (i) slight inward rectification in
symmetrical K+, having a chord conductance of ~35
pS at The second messenger-dependent regulation of hIK1 has been
extensively investigated at the molecular level during the past several
years. That is to say both IK and SK channels are known to
constitutively bind calmodulin and both have
Ca2+-independent and Ca2+-dependent
calmodulin binding domains within the first 100 amino acids of the
C-terminal tail (13, 16, 17). Indeed, it is the binding of
Ca2+ to EF hands 1 and 2 of calmodulin that results in the
Ca2+-dependent gating of these channels (17).
More recently, the crystal structure of calmodulin bound to the
proximal C terminus of rSK2 was determined, demonstrating that
calmodulin cross-links two C-terminal tails in the presence of
Ca2+ (18). We demonstrated recently (19) that the IK, but
not the SK, channel was activated by ATP-dependent
phosphorylation and that this was independent of consensus kinase
phosphorylation sites on hIK1. We further mapped the domain for this
kinase-dependent regulation to a 14-amino acid region
(Arg355-Met368) overlapping the
Ca2+-dependent calmodulin-binding domain,
suggesting this may be the site of additional protein-protein
interactions (20). Similarly, PKC has been shown to acutely regulate
IK1, and this is independent of consensus PKC phosphorylation sites
(21).
In contrast to these results on the second messenger regulation of IK1,
the molecular motifs required for tetramerization of hIK1 in the
endoplasmic reticulum and the sorting motifs required for correct
plasma membrane localization have been little studied. We demonstrated
previously that truncation of the 427-amino acid hIK1 at
Lys402 resulted in the complete loss of channel function,
as assessed by patch clamp techniques (20). This loss of function could result from either a failure of hIK1 to traffic to the plasma membrane
or the expression of non-functional channels. To delineate among these
possibilities an HA epitope was inserted into the extracellular loop
between transmembrane domains S3 and S4 such that cell surface
expression could be monitored by immunofluorescence (IF) and cell
surface immunoprecipitation (CS-IP) techniques, whereas the function of
hIK1 could be measured using the whole-cell patch clamp technique. We
demonstrate that a C-terminal leucine zipper, distal to the
calmodulin-binding domain, is required for the trafficking of hIK1 to
the plasma membrane and that mutations of the leucine zipper alter the
assembly of the distal C-terminal tail of hIK1. In contrast, this
conserved leucine zipper is not required for the correct trafficking of
another gene family member, SK3, to the plasma membrane.
Molecular Biology--
pBF plasmid containing the cDNAs for
full-length hIK1 and rSK3 were kindly provided by J. P. Adelman
(Vollum Institute, Oregon Health Sciences University). These cDNAs
were subcloned into pcDNA3.1(+) (Invitrogen) using the
EcoRI and XhoI restriction sites. A hemagglutinin (HA; YPYDVPDYA) epitope was inserted into hIK1 (HA-hIK1) between Gly132 and Ala133, i.e. the
extracellular loop between transmembrane domains S3 and S4, by
sequential overlap extension PCR using plasmid-specific primers in
conjunction with the primers: forward,
5'-TATCCGTACGACGTGCCCGACTACGCCGCGCCGCTGACCTCCCCGCAG-3'; reverse,
5'-GGCGTAGTCGGGCACGTCGTACGGATACCCTAAATCCTGCACGCACGGCGGG-3', where the HA epitope is highlighted in boldface. The PCR product was
subcloned into pcDNA3.1(+) using EcoRI and
XhoI restriction sites. All mutations in HA-hIK1, L414STOP,
K402STOP, L378A/L385A (ZIP1,2), L385A/L392A (ZIP2,3), L392A/L406A
(ZIP3,5), L399A/L406A (ZIP4,5A), L399P/L406F (ZIP4,5P), I396A/L403A
(A-POS), L378A (ZIP1), L385A (ZIP2), L392A (ZIP3), L399A (ZIP4), L406A
(ZIP5), and L409A/L410A as well as rSK3 (L660A/L667A; L667A/L674A),
were generated using the QuikChangeTM site-directed
mutagenesis strategy developed by Stratagene, La Jolla, CA. hIK1 was
tagged with a C-terminal myc epitope (EQKLISEEDL) through
PCR amplification of hIK1 in pcDNA3.1(+) and then subcloned in-frame into pcDNA4/myc (Invitrogen) by utilizing
EcoRI and BamHI restriction sites.
Addition of either the Xpress (DLYDDDDK) or V5 (GKPIPNPLLGLDST) epitope
tag to the last 59 amino acids of the C terminus of hIK1
(Val369-Lys427; C59) were generated by PCR
amplification of the C59 fragment and subcloning into either
pcDNA4/HisMax for Xpress or pcDNA3.1-V5/His-TOPO for V5
(Invitrogen). In total, 45 amino acids were added during the generation
of the V5-C59 construct, whereas 35 amino acids were added in
generating the XP-C59 construct, thereby allowing separation via
SDS-PAGE.
The fidelity of all constructs utilized in this study was confirmed by
sequencing (ABI PRISM 377 automated sequencer, University of
Pittsburgh) and subsequent sequence alignment (NCBI BLAST) with hIK1
(GenBankTM accession number AF022150) or rSK3
(GenBankTM accession number U69884).
Cell Culture--
Human embryonic kidney (HEK293) cells were
obtained from the American Type Culture Collection (Manassas, VA) and
cultured in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin in a humidified 5% CO2, 95%
O2 incubator at 37 °C. Cells were transfected using
LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were generated for all constructs by
subjecting cells to antibiotic selection (1 mg/ml G418) 48 h
post-transfection. Selection was typically complete within 14 days
post-transfection. Following selection the concentration of G418 was
reduced to 0.2 mg/ml. Note that clonal cell lines were not subsequently
selected from this stable population of cells in order to avoid clonal variation.
Electrophysiology--
During whole-cell patch clamp recording,
the bath contained (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES (pH adjusted to
7.4 with NaOH). The pipette solution contained (in mM) 144 KCl, 7.6 CaCl2, 5.6 MgCl2, 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH) to achieve an intracellular free
Ca2+ concentration of 300 nM. For whole-cell
recording of SK channels, the bath contained (in mM) 164.5 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES (pH adjusted
to 7.4 with KOH). The pipette solution contained (in mM)
135 KCl, 8.7 CaCl2, 2 MgCl2, 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH) to achieve an intracellular free
Ca2+ concentration of 1 µM. All experiments
were performed at room temperature. Currents were recorded using a List
EPC-7 amplifier (Medical Systems, Greenvale, NY). Electrodes were
fabricated from thin-walled borosilicate glass (World Precision
Instruments, Sarasota, FL), pulled on a vertical puller (Narishige,
Long Island, NY) and had a resistance of 1-4 megohms. Following
establishment of the whole-cell configuration, voltage steps were
applied from a holding potential of Antibodies and Immunofluorescence Labeling--
To detect
HA-hIK1 and rSK3 in immunofluorescence (IF), immunoprecipitation (IP),
and immunoblotting (IB), experiments antibodies were obtained from the
following sources (dilutions used are indicated): V5 and Xpress (XP)
(1:5,000; Invitrogen), polyclonal HA (1:150) and monoclonal (1:1,000)
HA (HA.11, Covance, Richmond, CA), c-myc (clone 9E10,
1:1,000; Roche Molecular Biochemicals), and polyclonal rSK3 antibody
directed against residues 2-21 of human SK3 (1:1,000; Chemicon
International, Temecula, CA). Secondary antibodies were obtained from
various sources as follows: biotin-conjugated goat anti-mouse IgG
(1:200; Molecular Probes, Eugene, OR), streptavidin conjugated to Alexa
Fluor® 488 (1:500; Molecular Probes), Cy3.18-conjugated goat
anti-mouse IgG (1:3,000; Amersham Biosciences), HRP-conjugated goat
anti-mouse IgG (1:2,000; Kirkegaard & Perry Laboratories, Gaithersburg,
MD), and HRP-conjugated goat anti-rabbit IgG (1:2,000; BD Biosciences).
For IF labeling, HEK293 stable cell lines were grown on
poly-L-lysine (Sigma)-coated glass coverslips for 24 h
prior to labeling. For detection of cell surface HA-hIK1, the cells
were washed in ice-cold PBS, blocked in 1% BSA (3 × 5 min)
followed by goat serum (10% for 20 min). HA-hIK1 was then labeled
sequentially by incubating in 1° (primary) monoclonal HA
antibody (1:1,000; 90 min) and 2° (secondary) biotin-conjugated goat
anti-mouse IgG (1:200; 90 min) followed by streptavidin conjugated to
Alexa-488 (1:500) for 90 min. Each labeling step was followed by 3-5
washes with 1% BSA (5 min each) to remove unbound Ab. All steps were
performed at 4 °C to prevent endocytosis of the channel. Following
cell surface labeling, the cells were again washed in ice-cold PBS,
fixed with 2% paraformaldehyde/PBS, permeabilized with 0.1% Triton
X-100/2% paraformaldehyde/PBS, blocked with 1% BSA and 10% goat
serum as above, and then intracellular localized HA-hIK1 was labeled
sequentially with 1° monoclonal HA antibody (1:1,000; 90 min),
and 2° Cy3.18 conjugated goat anti-mouse IgG (1:3,000) antibody.
Finally, nuclei were labeled with Hoechst 33258 (Sigma). This approach
allowed us to detect both cell surface and intracellular HA-hIK1 in the same cells. Cells were then subjected to laser confocal microscopy using a Leica TCSNT 3 laser 4 PMT system. To ensure maximal X-Y spatial
resolution, sections were scanned at 1024 × 1024 pixels, using
sequential 2-color image collection to minimize cross-talk between the
channels imaged. All images shown in a single figure were scanned on
the same day using identical settings. The images were then imported
into Adobe Photoshop, combined into a single figure, and RGB
brightness/contrast adjusted identically for all panels.
Immunoprecipitation (IP)--
C59 constructs were translated
from cDNA in the presence of [35S]methionine using
the TNT T7-Coupled Reticulocyte Lysate System (Promega, Madison, WI).
Aliquots of the translation reaction were diluted 10-fold in IP buffer
(50 mM HEPES, pH 7.4, 150 mM NaCl, 1% v/v
Triton X-100, 1 mM EDTA containing complete EDTA-free
protease inhibitor mixture mix), and pre-cleared with protein G
(Omnisorb; Calbiochem). Samples were then incubated with either anti-V5
or anti-XP (1:5,000 dilution) antibodies, and immune complexes were precipitated by incubation with protein G. Following extensive washing,
the protein G pellets were solubilized in Laemmli sample buffer and
heated to 37 °C for 5 min prior to removal of aggregates by
centrifugation. Samples were resolved by SDS-PAGE (10% gel), and the
gels were dried and subjected to autoradiography.
For co-immunoprecipitation of HA- and Myc-tagged hIK1 constructs,
HEK293 cells were transiently transfected in 60-mm dishes using
LipofectAMINE 2000 and 5 µg of each plasmid (total, 10 µg of DNA
and 20 µl of lipid). When only a single construct was transfected (HA
or Myc), empty pcDNA3.1(+) was included (5 µg) to keep the final
concentration of plasmid and lipid the same in all dishes. 18-24 h
post-transfection, cells were washed three times with ice-cold PBS and
then lysed with IP buffer. Protein concentrations were determined and
normalized to achieve equivalent loading. Crude lysates were then
pre-cleared with protein A-Sepharose beads (Sigma) and incubated with
rabbit polyclonal anti-HA antibodies. Immune complexes were
precipitated with protein A-Sepharose beads, followed by sequential
washes in IP buffer containing 500, 300, and 150 mM (2×)
NaCl, supplemented with 1× radioimmunoprecipitation assay (RIPA)
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1% v/v Triton X-100, 1% w/v sodium deoxycholate, and 0.1% w/v SDS).
After the final wash, the pellet was resuspended in Laemmli sample
buffer, and proteins were resolved by SDS-PAGE (12% gel) and
transferred to nitrocellulose for immunoblot analysis as described below.
For CS-IP, cells were grown to confluence in a 100-mm dish and then
washed in ice-cold PBS, blocked in 1% BSA/PBS, and labeled with
polyclonal HA.11 Ab (1:500) for 90 min at 4 °C. Unbound Ab was
removed by extensive washing in 1% BSA followed by washes in PBS. As
above, all steps were performed at 4 °C to prevent endocytosis of
the channel and/or Ab. The cells were then lysed, and protein
concentrations were normalized and the immune complexes directly
subjected to IP as detailed above. Following transfer to
nitrocellulose, an IB was performed using monoclonal HA Ab (1:1,000) as
detailed below. In addition to the IP, 15 µg of protein was set aside
following cell lysis for an IB. In this way, we were able to confirm
similar levels of protein expression in cells failing to correctly
traffic HA-hIK1 to the cell surface.
Immunoblot Analysis--
HEK293 cells were grown to confluence,
lysed with IP buffer, separated by SDS-PAGE, and transferred to
nitrocellulose. Blots were blocked for 1 h at room temperature
using TBS-blocking solution containing 5% w/v milk powder, 0.1% (v/v)
Tween 20, 0.005% (v/v) antifoam A. Subsequently, blots were incubated
in 1° Ab (mouse monoclonal HA, 1:1,000; mouse monoclonal
myc, 1:1,000; or rabbit polyclonal rSK3, 1:2,000) for 1 h at room temperature and extensively washed (TBS-blocking solution
deficient in milk powder) followed by incubation in 2° Ab (1:2,000;
HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-rabbit
IgG). The blot was then extensively washed, and detection was performed
using West Pico Chemiluminescent Substrate (Pierce).
Proteinase K Digestion--
Proteinase K digestion was performed
following methods described previously (22). Briefly, HEK cells stably
transfected with either wild-type or ZIP3,4 rSK3 were washed with
ice-cold PBS and incubated with 150 mM NaCl, 2 mM CaCl2, 10 mM HEPES, pH 7.4, with
or without 200 µg/ml proteinase K (Sigma) at 37 °C for 30 min.
Proteinase K digestion was quenched by adding ice-cold PBS containing 6 mM phenylmethylsulfonyl fluoride and 25 mM
EDTA. This treatment was followed by three washes in ice-cold PBS.
Cleared lysates were prepared and analyzed by immunoblotting, as
described above.
Chemicals--
All chemicals were obtained from Sigma, unless
otherwise stated. DCEBIO was synthesized in the laboratory of R. J. Bridges (University of Pittsburgh), as described previously (15).
Both DCEBIO and clotrimazole were made as 10,000-fold stock solutions in Me2SO. Complete EDTA-free protease inhibitor
mixture mix was obtained from Roche Molecular Biochemicals.
Statistics--
All data are presented as means ± S.E.,
where n indicates the number of experiments. Statistical
analysis was performed using a Student's t test. A value of
p < 0.05 was considered statistically significant and
is reported. All IF labeling and protein biochemical experiments were
carried out a minimum of 3 times on each construct to ensure the
voracity of our results.
We previously demonstrated that truncation of hIK1 at
Lys402 resulted in a complete loss of functional channels
at the cell surface (20). This result could be caused by either a
failure of channels to correctly traffic to the plasma membrane or from
channels that traffic normally but are non-functional. To distinguish
between these possibilities, an HA epitope was inserted into the
extracellular loop between S3 and S4 (see "Experimental
Procedures") such that cell surface expression could be evaluated by
IF and CS-IP techniques, whereas function was assessed by the
whole-cell patch clamp technique. Initially, we confirmed that HA-hIK1
could be detected at the cell surface by IF. As shown in Fig.
1A, wild-type HA-hIK1 is highly expressed at the cell surface (green) as well as
being expressed intracellularly (red), as expected. Cells
expressing hIK1 (no HA epitope) exhibited no fluorescence when labeled
using an identical protocol (data not shown). We next determined
whether insertion of the HA epitope had any effect on the biophysical and pharmacological characteristics of hIK1 as assessed by the excised,
inside-out patch clamp technique. We and others demonstrated previously
that hIK1 is activated by the pharmacological agent, DCEBIO (15), and
inhibited by clotrimazole (2, 4, 7, 11). In excised patch clamp
studies, DCEBIO activated HA-hIK1 with an apparent
Ks of 3.8 ± 0.8 µM
(n = 3), whereas clotrimazole inhibited HA-hIK1 with an
apparent Ki of 65 ± 5 nM
(n = 3), values similar to those reported previously for hIK1 (2, 4, 7, 11, 15). In addition, insertion of the HA epitope
had no significant effect on either the single channel I-V relationship
of the channel (chord conductance of 31 ± 1 pS at
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 mV and 10 pS at +100 mV; (ii) time- and voltage-independent
gating kinetics; (iii) activation by Ca2+, ATP, 1-EBIO, and
DCEBIO in excised, inside-out patches; and (iv) inhibition by
both extracellular charybdotoxin and clotrimazole (2, 4, 11-15). hIK1
is a member of the KCNN gene family, having 40-42% identity at
the amino acid level with the other members of the gene family,
including the small conductance, apamin-sensitive, Ca2+-activated K+ channels (SK1-3) (11, 12).
These channels are structurally similar to the voltage-gated family of
K+ channels, possessing 6 transmembrane domains (S1-S6), a
single pore region, and cytoplasmic N- and C-terminal tails.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 mV at 250-ms pulses every
2 s from
100 to +80 mV in 20-mV increments to generate a
current-voltage (I-V) relationship. Current was sampled at steady state
(50 ms) for the purpose of evaluating current density. Similar I-Vs
were generated following stimulation with DCEBIO (15) (10 µM) and inhibition with clotrimazole (CLT; 3 µM) or apamin (30 nM) for hIK1 or rSK3
channels, respectively. The pharmacological opener, DCEBIO, was
utilized to eliminate any variability that could be caused by
artificially raising Ca2+ in the presence of pipette EGTA.
Current density (pA/pF) was calculated by dividing the difference
between DCEBIO-stimulated and CLT- or apamin-blocked current (pA) by
the cell capacitance (pF) at a voltage of
20 or +40 mV for hIK1 and
rSK3 channels, respectively. Data analysis was performed using pCLAMP
(version 5.5, Axon Instruments, Foster City, CA). For all HA-hIK1
constructs, whole-cell current densities were obtained on at least 10 cells to account for any variation in expression across the stable cell lines.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 mV
and 11 ± 1 pS at +100 mV, n = 4) or
Ca2+-dependent gating (Ks = 831 ± 29 nM, Hill coefficient of 2.1;
n = 3), as these are values similar to what we and
others have reported previously (7, 11, 12, 14, 23, 24) for
endogenously and heterologously expressed hIK1. In total, these results
indicate that insertion of an HA epitope into hIK1 did not affect
channel function. Thus, we utilized this construct to define the role
of the cytoplasmic C terminus in the cell surface expression of
hIK1.
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Fig. 1.
C-terminal truncations compromise
surface expression of HA epitope-tagged hIK1 stably transfected into
HEK cells. Top panel, cells were sequentially incubated
with 1° (HA), 2° (G M-biotin), and 3° (tertiary)
(streptavidin-Alexa 488) antibodies at 4 °C to label plasma membrane
hIK1 (green). The cells were then fixed in 2%
paraformaldehyde/PBS and permeabilized with 0.1% Triton X-100/PBS to
label intracellular channel protein (red). A,
HA-hIK1, (B) L414STOP, and (C) K402STOP.
D, illustrative examples of DCEBIO-stimulated whole-cell
currents elicited by applying voltage pulses from
100 to +80 mV in
20-mV increments for 250 ms every 2 s, from a holding potential of
60 mV. E, current density (pA/pF) for each construct at
20 mV (mean ± S.E.; number of experiments is indicated in
parentheses). hIK1 currents that were significantly
different from wild-type hIK1 are indicated (*, p < 0.05, Student's t test). Control denotes
untransfected HEK cells.
Based on the observation that the HA epitope did not alter the function
of hIK1 channel activity in excised patches, we confirmed HA-hIK1 could
be activated by the pharmacological opener, DCEBIO (10 µM), and inhibited by clotrimazole (3 µM)
in whole-cell patch clamp studies. Following establishment of the
whole-cell configuration, current averaged 224.0 ± 38.5 pA at
20 mV, indicative of very few active channels (Fig. 1E).
DCEBIO increased the whole-cell current to 5,013.4 ± 597.0 pA,
and this was inhibited by clotrimazole to 930.5 ± 176.4 (n = 19). As the average capacitance for these 19 cells
was 26.6 ± 1.4 pF, this yielded an average current density of
166.6 ± 19.4 pA/pF, as shown in Fig. 1E. Furthermore,
as seen in Fig. 1D, HA-hIK1 displays no significant time- or
voltage-dependent activation during whole-cell recording as
reported previously for hIK1 (11, 12).
Truncation of hIK1 C Terminus Compromises Cell Surface Expression-- We demonstrated previously (20) that functional hIK1 channels could be recorded in excised, inside-out patches following truncation at Leu414, whereas truncation at Lys402 resulted in a complete loss of channel function. Thus, we initially introduced these stop mutations into HA-hIK1 to determine whether K402STOP and L414STOP traffic to the plasma membrane. As shown in Fig. 1, in contrast to HA-hIK1 (A), K402STOP (C) failed to express at the cell surface as assessed by IF (no green), although intracellular channel is prevalent in an intracellular compartment (red). In contrast, deletion of the last 14 amino acids (L414STOP) did not abrogate cell surface expression of the channel (Fig. 1B). To provide a quantitative estimate of cell surface expression of these truncated channels, we utilized the whole-cell patch clamp technique. Representative whole-cell recordings for HA-hIK1, K402STOP, and L414STOP are shown in Fig. 1D with average current density data shown in Fig. 1E. Whereas HA-hIK1 was highly expressed (166.6 ± 19.4 pA/pF, n = 19), K402STOP failed to express functional channels at the cell surface (0.6 ± 0.2 pA/pF, n = 12). In contrast to this complete loss of functional expression, L414STOP resulted in a more modest decrease in current density (95.8 ± 20.8 pA/pF, n = 17). These data suggest that amino acid residues within the distal C terminus, between Lys402 and Leu414, are critical for the correct trafficking of hIK1 to the plasma membrane.
Mutation of the C-terminal Leucine Zipper Abrogates
Membrane Trafficking of hIK1--
Sequence gazing of the amino acids
between Lys402 and Leu414 reveals two potential
structural motifs that may be required for correct trafficking of hIK1:
(i) a di-leucine motif (Leu409/Leu410), and
(ii) the terminal leucine (Leu406) of a
proposed leucine zipper motif. The last 59 amino acids of the hIK1 C
terminus (Val369-Lys427), encompassing the
entire leucine zipper, are shown in Fig.
2. Di-leucine motifs are known to play a
critical role in both Golgi exit as well as endocytic recycling of a
wide range of proteins (25), including ion channels (26), whereas
leucine zippers are important in protein-protein interactions (27).
Therefore, we used site-directed mutagenesis to define the role of the
di-leucine and leucine zipper motifs in the trafficking of hIK1. As
shown in Fig. 3, mutation of
Leu409/Leu410 to alanines (DI-LEU)
did not prevent trafficking of HA-hIK1 to the cell surface. Whole-cell
patch clamp analysis confirmed functional expression of di-leucine at
the cell surface (Fig. 4B),
although the current density was significantly reduced (87.0 ± 26.1 pA/pF; n = 18) compared with HA-hIK1. Although
these results suggest this di-leucine motif may play some role in the
trafficking of hIK1, they cannot explain the complete loss of surface
expression observed following truncation at Lys402 (Fig.
1).
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To assess the role of the leucine zipper in the trafficking of HA-hIK1, we initially mutated the 4th (Leu399) and 5th (Leu406) positions in the leucine zipper to proline (L399P) and phenylalanine (L406F). This mutation resulted in a complete abrogation of plasma membrane expression, as assessed by both IF (Fig. 3A, ZIP4,5) and cell surface immunoprecipitation (CS-IP, Fig. 3B). However, as shown in Fig. 3, this loss of function was not due to a loss of protein, as shown by both the IF labeling of intracellular channel (Fig. 3A) as well as the similar levels of protein upon immunoblot (Fig. 3B). Note that hIK1 runs as a doublet under the conditions utilized in our immunoblot and immunoprecipitation studies. Although the reasons for this micro-heterogeneity are unclear, it is a consistent finding for all of the constructs studied (Fig. 3B) and is also observed following in vitro translation in the presence of [35S]methionine (data not shown). We confirmed the role of this C-terminal leucine zipper in the trafficking of HA-hIK1 by introducing additional double mutations in the 2nd and 3rd leucines (L385A/L392A; ZIP2,3), the 3rd and 5th leucines (L392A/L406A; ZIP3,5), and the 1st and 2nd leucines (L378A/L385A; ZIP1,2). As shown in Fig. 3, ZIP2,3 and ZIP3,5 failed to traffic to the plasma membrane, although the channel was expressed intracellularly. In contrast, the ZIP1,2 mutation was clearly expressed at the cell surface as assessed by both IF (not shown) and CS-IP (Fig. 3B).
In coiled-coil domains, the hydrophobic pocket is determined by both the leucines in the d position, as well as hydrophobic amino acids in the a position (27). Thus, we determined whether the a position amino acids corresponding to Leu399 and Leu406 would similarly affect channel localization. Therefore, we mutated Ile396 and Leu403 to alanines (a-POS). As was true with the ZIP4,5 mutation, mutation of I396A/L403A resulted in a channel that failed to traffic to the plasma membrane as determined by both IF (Fig. 3A) and CS-IP (Fig. 3B). In an additional series of studies, we determined whether individual Leu/Ala mutations in the leucine zipper would affect trafficking of HA-hIK1. As shown in Fig. 3, A and B, mutating each of the leucine residues within the leucine zipper individually failed to affect surface expression of HA-hIK1.
To confirm the functional expression of these leucines zipper mutations, we utilized the whole-cell patch clamp technique to determine current densities for each of these constructs. As shown in Fig. 4, ZIP4,5P failed to express functional channels at the cell surface (0.2 ± 0.1 pA/pF, n = 14), consistent with our IF and CS-IP data. As this mutation (L399P/L406F) would be expected to cause a significant alteration in protein structure, we also generated an alanine-substituted mutant (L399A/L406A; ZIP4,5A). As shown in Fig. 4B, this resulted in a similar reduction in current density (4.8 ± 2.4 pA/pF, n = 10). Current density measurements confirmed that the ZIP2,3, ZIP3,5 and the a position (a-POS) mutations were expressed at very low levels at the plasma membrane (ZIP2,3 = 18.4 ± 7.0 pA/pF, n = 16; ZIP3,5 = 1.1 ± 0.6 pA/pF, n = 14; a-POS; 13.0 ± 5.2 pA/pF, n = 12) (Fig. 4B), confirming the critical role of the leucine zipper in the trafficking of HA-hIK1. However, similar to our IF and CS-IP measurements, the current densities measured following mutation of L378A/L385A (ZIP1,2; 126.4 ± 22.1 pA/pF, n = 29; Fig. 4B) or the individual amino acids comprising the leucine zipper did not compromise functional cell surface expression (ZIP1, 153.8 ± 24.9 pA/pF, n = 16; ZIP2, 181.3 ± 23.5 pA/pF, n = 15; ZIP3, 179.2 ± 17.8 pA/pF, n = 15; ZIP4, 219.6 ± 30.4 pA/pF, n = 16; ZIP5, 195.5 ± 24.5 pA/pF, n = 13). These results indicate that, in contrast to double mutations in the leucine zipper, single alanine substitution mutations are insufficient to alter cell surface expression of hIK1.
Rescue of a Leucine Zipper Mutant by Lowering
Temperature--
Misfolded proteins are often retained in the
endoplasmic reticulum. Our results indicate that hIK1 is retained
within an intracellular compartment when the leucine zipper is mutated
(Fig. 3A). Recent studies demonstrate that misfolded
proteins, including cystic fibrosis transmembrane conductance
regulator (28) and the HERG K+ channel (29-32), will
escape the endoplasmic reticulum if the cells are incubated at reduced
temperatures. Therefore, we examined the effect of reducing the
incubation temperature of cells from 37 to 27 °C on plasma membrane
expression and function of ZIP4,5A-HA-hIK1. As shown in Fig.
5A, reducing the temperature
to 27 °C resulted in a partial restoration of plasma membrane
expression of Zip4,5A-HA-hIK1 (green, right
panel). Note that we did not label the intracellular channel
following incubation at 27 °C in order to highlight expression at
the cell surface. This partial correction of cell surface expression was confirmed by CS-IP measurements. As shown in Fig. 5B,
Zip4,5A-HA-hIK1 was not detected at the cell surface when grown at
37 °C, although protein expression was not compromised
(IB in Fig. 5B). However, growing the cells at
27 °C for 24 h resulted in a partial restoration of cell
surface expression. Whole-cell patch clamp studies (Fig. 5,
C and D) confirmed that, following incubation at
27 °C for 24 h, DCEBIO stimulated a significant CLT-sensitive
whole-cell current in ZIP4,5A-HA-hIK1 expressing cells compared with
cells grown at 37 °C (27 °C, 46.9 ± 13.5 pA/pF,
n = 17; 37 °C, 4.8 ± 2.4 pA/pF,
n = 10). Note that this channel displayed normal
macroscopic gating kinetics following correction of the trafficking
defect by incubation at 27 °C suggesting this leucine zipper does
not play a fundamental role in channel gating.
|
Mutation of the C-terminal Leucine Zipper Does Not Affect Assembly
of hIK1 Subunits--
The inability of HA-hIK1 to correctly traffic to
the plasma membrane following mutation of the C-terminal leucine zipper
could be caused by either a gross structural change, thereby precluding tetramer formation, or by a more subtle effect that affects only the
distal C terminus of hIK1. To investigate the role of the C-terminal
leucine zipper in tetramer formation, we generated a myc-tagged hIK1
and performed co-immunoprecipitation experiments. HA-hIK1 and myc-hIK1
were transiently transfected either alone or in combination into HEK293
cells, immunoprecipitated using anti- HA Ab, separated by SDS-PAGE,
and immunoblotted with anti-myc Ab. As shown in Fig.
6, we were able to
co-immunoprecipitate myc-hIK1 with an HA antibody (3rd
lane), confirming assembly of hIK1 into minimally dimers and
likely tetramers. Following mutation of the C-terminal leucine zipper
in the HA-hIK1 backbone (ZIP4,5A-hIK1), a co-immunoprecipitate pulled
down quantitatively similar amounts of Myc-tagged wild-type hIK1
(5th lane), suggesting that a heterotetrameric complex
between wild-type and mutated subunits assembles correctly. Finally,
when the leucine zipper was mutated in both myc-hIK1 and HA-hIK1,
quantitatively similar amounts were also detected on
co-immunoprecipitates (6th lane) demonstrating that mutation of the C-terminal leucine zipper does not affect channel
tetramerization.
|
Assembly of the Distal C Terminus of hIK1 Is Dependent Upon an
Intact Leucine Zipper--
Our co-immunoprecipitation studies
demonstrate that the C-terminal leucine zipper of hIK1 is not required
for channel tetramerization. Thus, we considered the possibility that
leucine zipper mutations might modify more subtle sub-domain
interactions within the C terminus. To address this question we
utilized a differentially epitope-tagged (either V5 or Xpress) 59-amino
acid C-terminal domain of hIK1 (Fig. 2;
Val369-Lys427; C59), encompassing the leucine
zipper, but not the calmodulin-binding domain, to examine the role of
the leucine zipper in C-terminal self-assembly. As shown in Fig.
7A, following in
vitro translation in the presence of [35S]methionine
and IP, the V5 and Xpress (XP) epitope-tagged constructs run at
different apparent molecular masses of ~11 and 10 kDa, respectively
(lanes 1 and 4), thereby allowing co-assembly to be evaluated via IP. Upon co-translation of wild-type V5 and XP constructs, V5 and XP antibodies pulled down products corresponding to
both V5-C59 and XP-C59 (lanes 2 and 3,
respectively), demonstrating co-assembly of these C-terminal domains.
However, when the Xpress epitope-tagged C-terminal leucine zipper was
mutated (L399A/L406A; XP-ZIP4,5), co-assembly with V5-C59 was abrogated
(lanes 6 and 7), demonstrating a critical role
for the leucine zipper in C-terminal self-assembly. Interestingly,
mutation of either L399A (XP-ZIP4; lanes 8 and 9)
or L406A (XP-ZIP5; lanes 10 and 11) alone was not sufficient to disrupt co-assembly of this C-terminal domain. Thus, our
results with the C terminus of hIK1 exactly mirror our results on
full-length hIK1, i.e. double leucine zipper mutations are required to disrupt assembly/trafficking. The specificity of these antibodies was confirmed by demonstrating that V5 Ab failed to pull
down XP-C59 (lane 12) and XP Ab failed to pull
down V5-C59 (lane 13) when either V5- or XP-C59 was
expressed alone. Furthermore, as shown in Fig. 7B,
co-assembly of epitope-tagged C59 fragments occurred only when the
fragments were translated together (co; 1st and
3rd lanes) and not when the fragments had been synthesized separately and then mixed afterward (mix; 2nd and
4th lanes).
|
The C-terminal Leucine Zipper Is Not Required for Membrane
Trafficking of rSK3--
hIK1 belongs to a gene family, KCNN,
containing three additional members, the small conductance,
apamin-sensitive, Ca2+-dependent K+
channels, SK1-3. Whereas hIK1 shares only 40-42% identity with the
SK channels, the C-terminal leucine zipper is conserved in SK1 and SK3
(the 2nd leucine position is replaced by phenylalanine in SK2). Thus,
we determined whether the function of this leucine zipper as a
trafficking determinant was conserved in rSK3. For these studies we
mutated the 3rd and 4th leucines (L660A/L667A) of the C-terminal
leucine zipper in rSK3 to alanines. Although the leucine zipper is
conserved between hIK1 and rSK3, it is out of register by a single
heptad repeat when sequence alignments are compared. Thus, the 3rd and
4th leucines of rSK3 correspond to the 4th and 5th leucines of hIK1.
Cell surface expression and function were determined by proteinase K
digestion and whole-cell patch clamp studies, respectively. Stable cell
lines expressing either rSK3 or the mutant rSK3-ZIP3,4 were treated
with proteinase K, a nonspecific serine protease that when applied
externally cleaves peptide bonds adjacent to the carboxylic group of
aliphatic and aromatic amino acids. Cell lysates prepared from
proteinase K-treated and control cells were then analyzed by SDS-PAGE
and immunoblotting using a commercially available rSK3 antibody. As shown in Fig. 8A, lysates
prepared from untreated cells expressing wild-type rSK3 (lane
1) and mutant rSK3-ZIP3,4 (lane 3) exhibit a single
product with an apparent molecular mass of ~80 kDa, representative of
full-length rSK3. Enzymatic digestion with externally applied proteinase K eliminated the bulk of the 80-kDa form and induced the
appearance of a novel lower molecular mass product of ~50 kDa in both
wild-type rSK3- (lane 2) and rSK3-ZIP3,4 (lane
4)-expressing cells. This lower molecular mass product is
indicative of proteolytic digestion of rSK3. These data demonstrate
that both wild-type rSK3 and mutant rSK3-ZIP3,4 are expressed
predominantly on the cell surface. Functional expression of this
mutated rSK3 channel was confirmed utilizing whole-cell patch clamp
current density measurements. As shown in Fig. 8B, mutation
of ZIP3,4 in rSK3 did not significantly affect current density
(rSK3 = 26.8 ± 9.9 pA/pF, n = 4;
rSK3-ZIP3,4 = 19.7 ± 7.6 pA/pF, n = 3). To
further confirm that an intact leucine zipper is not required for
efficient trafficking of rSK3, we introduced the additional double
mutations, L667A/L674A (rSK3-ZIP4,5) and L653A/L660A (rSK3-ZIP2,3), as
these are the same mutations, with regard to leucine zipper position, made in hIK1. Cell surface expression was evaluated by proteinase K
digestion. As shown in Fig. 8C, similar to the ZIP3,4
mutation, these additional leucine zipper mutations in rSK3 did not
abrogate cell surface expression. Collectively these data suggest that, in contrast to hIK1, the C-terminal leucine zipper of rSK3 is not
critical for correct trafficking to the plasma membrane.
|
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DISCUSSION |
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Members of the KCNN gene family, including SK1-3 and IK1 are, as a family, widely expressed in both brain and peripheral tissues where they play critical roles in a host of physiological processes. However, the molecular motifs involved in assembly and trafficking of these channels to the plasma membrane, both in general and to specific subdomains (e.g. the basolateral membrane of epithelial cells), have been little evaluated. To begin to address these questions we inserted an HA epitope into the extracellular domain between S3 and S4 of hIK1. We demonstrate that this epitope insertion can be utilized for the detection of cell surface-localized hIK1 by both IF and cell surface IP techniques while having no effect on either the single channel properties (single channel conductance, Ca2+ dependence), macroscopic gating kinetics, as assessed by whole-cell patch clamp, or pharmacological regulation (activated by DCEBIO, inhibited by clotrimazole) of hIK1. As such, we have utilized this construct to define the role of the distal C terminus in the trafficking of hIK1 to the plasma membrane.
Members of the KCNN gene family share a conserved leucine zipper motif in their distal C termini. Previous studies (33-35) have demonstrated a role for leucine zippers in the voltage-dependent gating of K+ and Ca2+ channels. In contrast to these voltage-gated channels, mutation of the C-terminal leucine zipper in hIK1 had no apparent effect on macroscopic channel gating, i.e. following correction of the trafficking defect by incubating the cells at 27 °C, the resultant DCEBIO-induced current displayed apparently normal gating characteristics (Fig. 5). Also, we demonstrate that an intact leucine zipper is not required for the assembly of hIK1; mutation of the C-terminal leucine zipper in all four subunits of hIK1 does not diminish their ability to assemble, as assessed by co-immunoprecipitation studies (Fig. 6). Rather, we demonstrate that an intact leucine zipper is required for the correct trafficking of hIK1 to the plasma membrane (Fig. 3), suggesting a more subtle folding defect as opposed to a complete loss of subunit assembly. The ability to correct this apparent folding defect by growing the cells at 27 °C (Fig. 5) also argues for a more subtle folding effect. Our results are similar to data previously reported (36) for the GABAB receptor where the coiled-coil interaction of receptor subunit C termini stabilizes the active conformation but are not necessary for functional dimerization.
Although our studies have focused on the distal C terminus of hIK1, Joiner et al. (37) recently demonstrated that the assembly of calmodulin with the proximal C-terminal CAMBD is required for the targeting of hIK1 to the plasma membrane and that this was due to an enhanced assembly of hIK1 into tetramers in the presence of calmodulin. It was further demonstrated that the overexpression of the distal C terminus of hIK1, including the leucine zipper, inhibited the cell surface expression of full-length hIK1 (37). Here we demonstrate that the distal C terminus of hIK1 self-assembles in a leucine zipper-dependent manner and that this is required for the correct targeting of hIK1 to the plasma membrane.
The small conductance Ca2+-activated K+ (SK) channels, SK1-3, share 42-44% identity with hIK1. Whereas the majority of this conservation occurs in the backbone (S1-S6) region of the channels, the C termini also demonstrate regions of high homology. Indeed, each of these channels possesses a CAMBD in their proximal C termini as well as a conserved leucine zipper in their distal C termini. Despite this conservation of a leucine zipper in SK1, SK3, and hIK1, our results demonstrate a lack of conservation in function for this motif, i.e. whereas mutation of the leucine zipper of hIK1 results in a dramatic loss of cell surface localized channel (Fig. 3), a similar mutation in SK3 had no effect on cell surface expression (Fig. 8). In this regard, it is interesting to note that Xia et al. (16) demonstrated that truncation of rSK2, distal to the CAMBD, did not affect expression of functional channels. In total, these results suggest that the distal C terminus in general, and the leucine zipper in particular, does not play a critical structural role in the SK members of the KCNN gene family, whereas it is absolutely required for the more distantly related IK gene family member.
Whereas our results clearly point to a role for the leucine zipper in the trafficking of hIK1, we further demonstrate that the most proximal leucine (Leu378, ZIP1) can be mutated, either alone or in combination with Leu385 (ZIP1,2), with no deleterious effects on channel trafficking (Figs. 3 and 4). Interestingly, distinct studies have demonstrated that the CAMBD of IK and SK channels extends 95-98 amino acids from the S6 transmembrane domain (amino acids R287 to N384 in IK1) with the Ca2+-dependent binding domain being at the distal end of this motif (13, 17). Thus, Leu378 would be predicted to overlap with the Ca2+-dependent CAMBD such that it may not be accessible for protein-protein interactions.
We demonstrate that the distal C terminus of hIK1 (C59, Fig. 2)
co-assembles in a leucine zipper-dependent manner (Fig. 7). Similar to the trafficking of full-length HA-hIK1, the co-assembly of
C59 is only disrupted by the introduction of a double mutation (ZIP4,5), whereas single mutations (ZIP4 or ZIP5) have no effect on the
assembly process. These results suggest that, in full-length hIK1, the
distal C terminus self-assembles and that this is required for correct
trafficking of the channel. Schumacher et al. (18) recently
used x-ray crystallography to identify the structure of calmodulin
bound to the proximal C terminus CAMBD of the rSK2 channel (18).
Unfortunately, this crystal structure consists of only the CAMBD; it
does not incorporate the C-terminal leucine zipper of rSK2. Therefore,
it will be of particular interest to obtain crystals of the entire C
terminus for both IK and SK channels and to determine the alignment of
the leucine zipper -helices within the context of the
CAMBD-Ca2+-calmodulin complex so that we can envisage how
mutations within the zipper compromise membrane trafficking of IK but
not the SK channels.
Whereas the C-terminal leucine zipper of hIK1 may associate with itself there are at least two alternative possibilities that should be considered. First, leucine zippers are known to be involved in protein-protein interactions (27). Therefore, the C-terminal leucine zipper in hIK1 may be required for interactions with additional proteins necessary for the trafficking of the channel to the plasma membrane. In this regard, if the SK channels do not share these protein-protein interactions then mutation of the leucine zipper in these channels would not have the same effect on channel expression. A second possibility is that the C-terminal leucine zipper interacts with another domain within hIK1 itself. For example, hIK1 has a second potential leucine zipper extending from the cytosolic NH3 terminus into the first transmembrane domain (Leu18/Leu25/Leu32/Leu39), a domain not conserved in SK1-3. Thus, the NH3 and C termini of hIK1 may assemble in order to form a channel that can traffic efficiently to the plasma membrane. This association of cytosolic domains is known to be important in the proper assembly and trafficking of other ion channels (38).
In conclusion, we demonstrate that the trafficking of hIK1 is dependent
upon an intact C-terminal leucine zipper. Although this leucine zipper
is not required for channel tetramerization, it appears to be crucial
for the assembly of the C terminus of hIK1 into a trafficking competent
conformation. Interestingly, whereas this motif is conserved across the
KCNN gene family it is not functionally conserved, suggesting a
clear divergence in the structural requirements for the correct folding
and trafficking of IK and SK gene family members.
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FOOTNOTES |
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* This work was supported by the Lazaro J. Mandel Young Investigator award from the American Physiological Society (to D. C. D.), National Institutes of Health Grant DK54941 (to D. C. D.), and the Pennsylvania/Delaware Affiliate of the American Heart Association Grant 0120544U (to C. A. 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.
To whom correspondence should be addressed: Dept. of Cell Biology
and Physiology, University of Pittsburgh School of Medicine, S312 BST,
3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8755; Fax:
412-648-8330; E-mail: dd2@pitt.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M210072200
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ABBREVIATIONS |
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The abbreviations used are: IK, Ca2+-activated K+ channels; hIK1/hSK4, human IK channel; HA, hemagglutinin; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Ab, antibody; CAMBD, calmodulin binding-domain; HRP, horseradish peroxidase; IF, immunofluorescence; CS-IP, cell surface immunoprecipitation; CLT, clotrimazole; IP, immunoprecipitation; IB, immunoblotting; a-POS, a position; DCEBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one.
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