1 Assembly, Maturation, and Turnover of KATP Channel Subunits Ana Crane and Lydia Aguilar ...

32 Pages·2003·0.41 MB·English

JBC Papers in Press. Published on December 29, 2003 as Manuscript M311079200
Assembly, Maturation, and Turnover of K Channel Subunits
ATP

Ana Crane1 and Lydia Aguilar-Bryan2

Departments of Medicine2 and Molecular and Cell Biology1
Baylor College of Medicine
Houston, TX
77030

D
o
w
n
lo
ad
e
d
To whom correspondence should be addressed. fro
m
h
[email protected] ttp
://w
w
w
[phone] .jb
c
.o
rg
b/
y
g
u
e
s
t o
n
D
e
c
e
m
b
e
r 2
6
, 2
0
1
8
1
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Running title: Turnover of K channels
ATP

Acknowledgements: This work was funded by NIH grant DK57671 (LAB). We thank Dr.
Joseph Bryan for valuable discussions about the manuscript and Maria Janecki for help with
plasmid constructions.

D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://w
w
w
.jb
c
.o
rg
b/
y
g
u
e
s
t o
n
D
e
c
e
m
b
e
r 2
6
, 2
0
1
8
2
ATP-sensitive K+, or K channels are comprised of K 6.x and SUR subunits which
ATP IR
assemble as octamers, (K /SUR) . The assembly pathway is unknown. Pulse-labeling
IR 4
studies show that when K 6.2 is expressed individually, its turnover is biphasic, ~60% is
IR
lost with t ~36 minutes. The remainder converts to a long-lived species (t ~26 hrs) with a
½ ½
estimated half-time of 1.2 hours. Expressed alone, SUR1 has a long half-life, ~25.5 hrs.
When K 6.2 and SUR1 are co-expressed, they associate rapidly and the fast degradation of
IR
K 6.2 is eliminated. Based on changes in the glycosylation state of SUR1, the half-time for
IR
the maturation of K channels, including completion of assembly, transit to the Golgi,
ATP
and glycosylation, is approximately 2.2 hours. Estimation of the turnover rates of mature,
fully glycosylated SUR1 associated with K 6.2, and of K 6.2 associated with myc-tagged
IR IR
SUR1 gave similar values for the half-life of K channels, a mean value of ~7.3 hours. D
ATP o
w
n
KATP channel subunits in INS-1 β-cells displayed qualitatively similar kinetics. The results loa
d
e
d
imply the octameric channels are stable. Two mutations, KIR6.2 W91R and SUR1 ∆F1388, fro
m
identified in patients with the severe form of familial hyperinsulinism, profoundly alter the http
://w
rate of K 6.2 and SUR1 turnover, respectively. Both mutant subunits associate with their w
IR w
.jb
respective partners, but dissociate freely and degrade rapidly. The data support models of c
.o
rg
channel formation in which K 6.2/SUR1 heteromers assemble functional channels and are b/
IR y
g
u
inconsistent with models where SUR1 can only assemble with K 6.2 tetramers. es
IR t o
n
D
e
c
e
m
b
e
r 2
6
, 2
0
1
8
3
The rules that govern the maturation and assembly of oligomeric membrane proteins, particularly
ion channels, are not well understood. Glycosylation, ER retention and exit mechanisms, and
timing are implicated as important factors restricting trafficking to fully assembled complexes
(1-7). Multi-subunit ion channels provide excellent models to study these issues. ATP-sensitive
K+, or K , channels are large, octameric, (K 6.x/SUR) , complexes (2,8,9) composed of two
ATP IR 4
disparate subunits, potassium inward rectifiers, K 6.1 (KCNJ8) or K 6.2 (KCNJ11), that form
IR IR
the pore of the channel, and sulfonylurea receptors, SUR1 (ABCC8) or SUR2 (ABCC9), ATP-
binding cassette (ABC) proteins which activate the pore and regulate its gating. Glycosylation
takes place at two residues within SUR (7,10) and is required for efficient surface expression
implying quality control via the glycoprotein-ER-associated degradation (GERAD) system (11).
D
In addition, dibasic ER retention motifs, specifically an –RKR– motif, have been identified in o
w
n
lo
both K and SUR subunits. Their removal or mutation results in loss of the requirement of the a
IR d
e
d
partner subunit for correct trafficking (3,12). The retention mechanism is poorly understood. A fro
m
h
recent report indicates that the dibasic motif can interact with COPI proteins resulting in ttp
://w
retention ((13,14), reviewed in (15)). Binding with COP1 complexes can be antagonized by 14- w
w
.jb
3-3 isoforms to facilitate ER exit and the interaction between dimeric 14-3-3 and the –RKR– c.o
rg
motif in K 6.2 has been reported to be enhanced by assembly of K subunits (14). by/
IR IR g
u
e
s
Reduced or loss-of-function mutations in K 6.2 and SUR1 are known to account for ~50% of t o
IR n D
e
the mild and severe cases of hyperinsulinemic hypoglycemia (HI) (16,17). Nonsense and splice ce
m
b
e
site mutations that truncate SUR1 result in loss of functional channels at the cell surface in r 2
6
, 2
patient β-cells and retention of truncated SUR1 in the ER (4,18). Missense mutations have been 01
8
identified that result in loss of stimulation by MgADP (19-22) and other mutations are reported
to affect trafficking to the cell surface as a consequence of improper folding or possibly by
altering interactions with the ER retention motif(s) (23-25). One general suggestion has been
that retention in the ER provides additional time for completion of channel assembly. K 6.2
IR
subunits lacking the –RKR– motif can assemble channels in the absence of an SUR, and these
will reach the surface indicating SUR is not obligatory for assembly of the channel pore. The
assembly pathway is unknown, and at what stage SUR interacts with K is a matter of
IR
conjecture. Similarly, the lifetimes of the individual subunits, of mutant subunits, and of the
channel complex have not been reported.
4
We have used 35S-met/cys pulse labeling methods to determine the lifetimes of SUR1 and K 6.2
IR
alone, to examine the effect of two severe HI mutations on subunit lifetime, and to follow the
maturation of channels by monitoring changes in the glycosylation state of SUR1. The results
show that when SUR1 is expressed in the absence of the inward rectifier it is long-lived, in
contrast for example, to CFTR which has a half-life of ~30 minutes in the ER (26,27). When
K 6.2 is expressed in the absence of SUR1, a fraction (~60%) degrades rapidly, but the
IR
remainder is converted to a long-lived species. When K 6.2 and SUR1 are co-expressed,
IR
complexes form quickly and the rapid degradation of K is eliminated. In the complex SUR1
IR
undergoes a time-dependent change in its glycosylation state as the channel transits to the cell
surface. Two loss-of-function mutations, ∆F1388 in SUR1, and W91R in K 6.2, both
IR
drastically decrease subunit lifetime irrespective of whether they are co-assembled with their D
o
w
n
partner subunit. lo
a
d
e
d
Experimental Procedures fro
m
h
ttp
Molecular Biology & Plasmid Construction - KIR6.2 and SUR1 proteins were modified by ://w
w
w
addition of extracellular myc tags. The details of construction of the hamster pECE SUR1, .jb
c
.o
pECE SUR1myc and human pECE KIR6.2 have been described (2,4,28). brg/
y
g
Cell Culture and Transient Transfection - COSm6 cells were cultured in Dulbecco’s modified ue
s
t o
n
Eagle’s medium (DMEM), 4.5g/L glucose, supplemented with 10% fetal bovine serum. D
e
c
e
Approximately 7 x106 cells were transfected with 8 µg of the pECE SUR1 and/or 1 µg of the mb
e
r 2
pECE KIR6.2 plasmids by electroporation (BioRad Laboratories, Inc., Hercules, CA) following 6, 2
0
1
8
the manufacturer’s directions (950 µF, 0.220V in 0.4 mm cuvettes in RPMI medium
supplemented with 10% FBS and 1.25% DMSO). The average efficiency of transfection was 7-
10%, estimated by co-transfection with a GFP-marker plasmid. There was no significant
difference in transfection efficiency when multiple plasmids were used. Pulse-chase experiments
were carried out 18 hours after transfection.
INS-1 β-cells, line 832/13 (29) (a kind gift from Dr. Christopher Newgard), were grown in
RPMI-1640 with 11 mM D-glucose, supplemented with 10% fetal bovine serum, 100 U/ml
penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate,
and 50 µM β-mercaptoethanol.
5
Pulse Chase and Immunoprecipitation Protocol - Cells were transfected and seeded in 6 well
plates in duplicate, at 70-80% confluence. The following day cells were incubated for 2 hr in L-
methionine/L-cysteine free media (Invitrogen Life Technology, CA) at 37oC with 5% CO .
2
Subsequently, cells were labeled for 60 minutes with a mixture of [35S]-methionine/cysteine (200
µCi/ml; EaseTagTM mix, PerkinElmer, Boston MA) then incubated with pre-warmed DMEM
supplemented with 5mM unlabeled L-methionine/L-cysteine. At the indicated times, wells were
washed twice with cold PBS, collected, then lysed, on a rotating wheel, for 6 hours at 20oC in 1
ml of 1% digitonin (Sigma Chemical Corp, St. Louis, MO) in PBS plus cysteine and serine
protease inhibitors (Boheringer Mannheim Biochemicals, Indianapolis, IN). Lysates were
clarified at 9000xg for 15 minutes at 4oC. The supernatants were pre-absorbed for 1 hour with
protein-G plus, then incubated with anti-myc or anti-K 6.2 (N-18) antibodies (Santa Cruz D
IR o
w
Biotechnology, Santa Cruz, CA) overnight at 20oC followed by a 3 hour incubation with protein- nlo
a
d
e
d
G agarose (Santa Cruz Biotechnology, Santa Cruz, CA). The agarose beads were washed five fro
m
times with modified lysis buffer containing 0.1% digitonin. A modified 2x loading sample http
buffer (2.5 % SDS, 0.1M DTT, 0.06 M Tris Base, 20% glycerol, 0.008M EDTA. pH 6.8) was ://ww
w
added to the beads. Proteins were separated on 7.5% or 10% SDS-polyacrylamide gels (30). .jbc
.o
rg
Gels were stained, fixed in 50% methanol, 10% acetic acid, then treated with Fluorenhance b/
y
g
u
(Amersham Biosciences, Piscataway, NJ) for 30 minutes, dried, and exposed to either X-ray film es
t o
n
(Hyperfilm (Kodak Chemical Corp, Rochester, NY) at 80oC for 16-24 hours, or put on an D
e
c
e
intensifying screen for quantification with a StormTM phosphorimager (Amersham Biosciences, mb
e
r 2
6
Piscataway, NJ). , 2
0
1
8
Note on Sample Preparation - The mobility of SUR1 on SDS-polyacrylamide gels is dependent
on its glycosylation state; the immature or ‘core’ glycosylated form displays an apparent
molecular mass of ~140 kDa and the mature, fully glycosylated form a mass of ~150-170 kDa
(1,2). Since maturation of membrane glycoproteins occurs in the medial Golgi apparatus
(reviewed in (31)), the mobility difference is a marker for trafficking of SUR1 (4). During the
course of these experiments we observed that inclusion of the usual heating step (95oC, 5
minutes) in the preparation of samples for SDS-PAGE resulted in aggregation. The left panel of
Fig. 1 shows the effect of heating solubilized immunoprecipitates containing SUR1myc. The
right panel shows a similar effect for fully glycosylated, myc-tagged SUR1 present when the
SUR and K subunits are co-expressed. Additionally, the lower right panel confirms an earlier
IR
6
report on the reduced recovery of K 6.2 following heating (32). The appearance of the heated,
IR
myc-tagged SUR1 samples is not affected by treatment with endoglycosidases (data not shown).
The samples in Fig.1 were identified by photolabeling with 125I-azido glibenclamide (2), but
equivalent results were obtained using [35S]-met/cys labeled subunits. To avoid aggregation,
samples were not subject to heating before SDS-PAGE.
Data Analysis – The relative intensities of bands were determined using a phosphorimager. The
data are plotted as intensity values, or as the fraction remaining obtained by dividing by the zero
time value. We have assumed homogenous compartments and that degradation of a given
molecule is a random event. Single exponential functions were fit to the data, with the exception
of K 6.2 expressed in the absence of SUR where two exponentials were used. The results are
IR
D
expressed as the half-life for a given species. The half-life values can be converted to the mean o
w
n
lo
residence times for a molecule in a compartment by dividing by 0.693. We used a simple two a
d
e
d
compartment model in Modelmaker 4 (Cherwell Scientific, Ltd., Oxford, UK) to simulate the fro
m
h
conversion of monomeric KIR6.2 to the tetramer and estimate the half-time for the assembly. All ttp://w
other fitting was done using the non-linear routines in Origin Pro (OriginLabs, Corp., w
w
.jb
Northampton, MA). The results are given as means ± SD. c.o
rg
b/
y
Results g
u
e
s
t o
Free KATP channel subunits turnover with different kinetics - SUR1 is a member of the ABC n D
e
c
e
superfamily, specifically the ABCC subfamily. CFTR, another ABCC protein, whose turnover m
b
e
r 2
and trafficking has been examined in some detail degrades rapidly in the ER, a t ~30 minutes 6
½ , 2
0
1
(26,27), with only 20-40% estimated to leave the ER. The mature, fully glycosylated CFTR at 8
the cell surface is longer lived, reported t values of 7.5-to-16 hours (26,27). P-glycoprotein
½
(MDR1), another ABC protein, has reported half-lives >24 hours, while mutants have been
described with t values of ~3 hours (33-35). 35S-met/cys pulse-chase experiments with COSm6
½
cells transfected with myc-tagged SUR1, in the absence of K 6.2, show there is no conversion to
IR
the mature species and that the immature receptor turns over slowly in the ER (Fig. 2A). A
single exponential decay fits the data points reasonably well giving an estimated half-life of
~25.5 ± 4.4 hours (n = 6) for SUR1 subunits in the ER in the absence of the inward rectifier.
The turnover of K 6.2 expressed alone is markedly different that SUR1. Turnover is biphasic
IR
with approximately 60% of the 35S-met/cys labeled subunits being rapidly degraded with a half-
7
life ~35.7 ± 11.8 minutes. The remaining 40% turns over slowly with a half-life ~26.1 ± 8.2
hours (n = 3). K 6.2 can assemble functional tetrameric pores in the absence of SUR which will
IR
reach the cell surface if their ER retention sequences are altered or deleted (36-38), thus we
propose that the biphasic decay of the K 6.2 reflects a difference in the rates of degradation of
IR
unassembled subunits vs assembled pores. We used this two-state assumption and the estimated
degradation rates to model the turnover-assembly process. The estimated half-life for conversion
of a K 6.2 monomer into a stable species, assumed to be a tetramer, is ~1.2 hours. The solid
IR
line in Fig. 2B was calculated using these parameters.
Maturation of K channels - The 35S-met/cys pulse-chase protocol followed by
ATP
immunoprecipitation with either anti-K 6.2 or anti-myc antibodies was used to assess the
IR
D
assembly and maturation of K channels in COSm6 cells co-transfected with both SUR1 and o
ATP w
n
lo
K 6.2. In principle, using two antibodies should provide information on the assembly of SUR1 a
IR d
e
d
with KIR6.2 complexes and vice-versa. The results using the anti-KIR antibodies are shown in from
h
Fig. 3A. Co-expression with SUR1 markedly affects the turnover of KIR6.2. The rapid decay, ttp://w
evident in Fig. 2B when K 6.2 is expressed alone, is missing. The amount of K 6.2 in the w
IR IR w
.jb
immunoprecipitates increases over the first 60 minutes then decays with a half-life of ~8.5 ± 4 c.o
rg
hours providing one measure of channel life-time. The result implies SUR1 must rapidly by/
g
u
e
associate with and stabilize the early assembly intermediates (Fig. 3A). This interpretation is st o
n
D
supported by the observation that K 6.2 and SUR1 co-immunoprecipitate at the earliest time e
IR ce
m
b
point, increasing in amount for ~2 hours (compare first bands in Fig. 3A vs 3B). In addition, e
r 2
6
K 6.2/SUR1 complexes were detectable after brief (10 minute) pulse-labeling periods (data not , 2
IR 0
1
8
shown). As shown in Fig. 3B, there is essentially no detectable fully glycosylated SUR1 present
at the earliest times, but the mature receptor becomes evident within 2 hours, peaking at
approximately 10 hours. Since the immunoprecipitation was carried out with anti-K 6.2
IR
antibodies, SUR1 must be associated with K 6.2. The appearance of the mature receptor as the
IR
core species disappears is consistent with a precursor-product relationship. The estimated half-
life for conversion to mature SUR1 is approximately 2.2 ± 0.14 hours, based on fitting a single
exponential (dotted line in Fig. 3B) to the disappearance of the immature form of the receptor
beginning with the 2 hour time point. The estimated half-life of the channel complex, based on
fitting a single exponential to the mature SUR1 data, beginning with the 10 hour time point, is
approximately 5.9 ± 1 hours. This is in reasonable agreement with the ~8.5 ± 4 hour estimate for
8
K 6.2 since the K number will include contributions from any subunits not assembled with
IR IR
SUR1.
The results for co-immunoprecipitation of myc-tagged SUR1 and K 6.2 with anti-myc
IR
antibodies are shown in Fig. 4. Consistent with the anti-K 6.2 immunoprecipitation result (Fig.
IR
3A), K 6.2 does not turnover over rapidly when associated with SUR1. There is a progressive
IR
increase in the amount of K 6.2 precipitating with SUR1, with a peak at ~5 hours, consistent
IR
with slow sequential assembly. The estimated channel half-life, based on the lifetime for K 6.2
IR
associated with SUR1, is 7.8 ± 1.1 hours. The precursor-product relation between immature and
mature SUR1 is apparent with nearly the same timing (half-life for conversion of core-to-
complex SUR1 is approximately 2.6 ± 0.6 hr). The estimated channel half-life, based on fitting a
D
single exponential to the mature SUR1 data, is 6.9 ± 0.2 hours. The ratio of immature SUR1-to- o
w
n
K 6.2 at the end of the 35S-met/cys pulse (t = 0) is higher than that observed for loa
IR d
e
d
immunoprecipitation with anti-KIR6.2 (9.5 vs 2.9, respectively) implying SUR1 is in excess from
h
under these transfection conditions. This idea is supported by comparison of the later time points ttp
://w
in the SUR1 intensity data in Fig. 3B vs 4B. The maturation of SUR1, marked by conversion to w
w
.jb
the mature form, is essentially complete in less than 20 hours for SUR1 associated with KIR6.2 c.org
(Fig. 3B), while the immature form remains readily detectable after 30 hours in the anti-myc by/
g
u
e
immunoprecipitates (Fig. 4B). The result is consistent with there being non-KIR6.2 associated st o
n
D
SUR1 in the ER which turns over slowly as shown in Fig. 1A. e
c
e
m
b
e
Increased rates of turnover for two KATP channel mutants - A number of mutations in either r 26
, 2
SUR1 or KIR6.2 are known to cause familial hyperinsulinism (10,39,40) and several have been 018
shown to affect trafficking reportedly as a consequence of improper folding. We have used the
35S-met/cys-pulse-chase-immunoprecipitation protocol to examine the turnover of two mutant
subunits, SUR1 ∆F1388 and K 6.2 W91R, expressed alone or with their respective partner. The
IR
∆F1388 mutation is in the second nucleotide binding domain (NBD) of SUR1 and has been
identified as a common cause of familial hyperinsulinism in Ashkenazi populations (41). The
W91R mutation substitutes an arginine for the second in a pair of tryptophan residues near the
top of K 6.2 (10). In KcsA, this tryptophan pair is in the pore helix and is thought to contribute
IR
significantly to the stability of the tetramer (42). Both mutations are associated with severe
forms of hyperinsulinism.
9
Fig. 5 compares the turnover of K 6.2 W91R vs wildtype K 6.2 subunits. K 6.2 W91R is
IR IR IR
degraded rapidly in either the presence or absence of SUR1. The conversion to a long-lived
species is missing in the mutant. Co-immunoprecipitation experiments (see inset) show that
K 6.2 W91R subunits do associate with SUR1, but this association does not appear to slow
IR
degradation significantly. The estimated half-lives for the K 6.2 W91R subunits in the absence
IR
and presence of SUR1 are 26.4 ± 2.4 minutes (n = 2) vs 34.0 ± 2.6 minutes (n = 2). These values
are not significantly different from those determined for the fastest component observed when
wildtype K 6.2 is expressed in the absence of SUR1 (35.7 ± 11.8 minutes; Fig. 2b). The results
IR
are consistent with the hypothesis that the W91R subunits are unable to assemble into stable
tetramers and turnover rapidly as free monomers. To support this idea, we expressed myc-
tagged KIR6.2 with either KIR6.2 W91R or wildtype KIR6.2, and then determined the levels of Do
w
n
W91R vs wildtype KIR6.2 in anti-myc immunoprecipitates (Fig. 6). The myc tag does not loa
d
e
substantially alter the properties of KIR6.2/SUR1 channels, but has a greater molecular mass and d fro
m
serves to identify the ‘carrier’ subunits in the immunoprecipitates. K 6.2 W91R is degraded h
IR ttp
more rapidly than wildtype K 6.2 being nearly undetectable after 5 hours, while the turnover of ://w
IR w
w
wildtype K 6.2 parallels that of the myc-tagged K 6.2 subunits (Fig. 6). .jb
IR IR c.o
rg
b/
The turnover of SUR1 ∆F1388 is dramatically faster than the wildtype receptor (Fig. 7). The y
g
u
e
s
half-lives for SUR1 ∆F1388 vs wildtype SUR1, expressed alone, are 3 ± 0.4 (n = 2) vs 25.5 ± 4.4 t o
n
D
e
(n = 3) hours, respectively. The SUR1 ∆F1388 subunits are able to assemble with K 6.2 (Fig. c
IR em
b
e
7B), but this association does not affect turnover of the mutant receptor significantly. We see no r 2
6
, 2
mature, fully glycosylated mutant receptors, consistent with their not leaving the ER. The 0
1
8
estimated half-life for the K 6.2/SUR1 ∆F1388 complex is 2.1 ± 0.6 hr, not significantly
IR
different than the mutant receptor alone. The association with SUR1 ∆F1388 does significantly
slow the degradation of K 6.2 (Fig. 7B). We assume there is a constantly changing mixture of
IR
SUR1/K 6.2 complexes in this experiment, but have fit a single exponential function, t = 5.6 ±
IR ½
2.3 hours, to the data for purposes of comparison. It is noteworthy that both
immunoprecipitations show enrichment of K 6.2 subunits vs SUR1 ∆F1388 at later times
IR
consistent with dissociation of ∆F1388 from the complexes and subsequent degradation.
Turnover of K channel subunits in the INS-1 β-cell line – The kinetics of turnover and
ATP
maturation of K channel subunits in INS-1 β-cells, determined by immunoprecipitation with
ATP
10