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Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics,
and Cytoskeletal Interactions
*
□
S
Received for publication, November 4, 2009, and in revised form, January 6, 2010 Published, JBC Papers in Press, January 19, 2010, DOI 10.1074/jbc.M109.082008
Ruchi Bhalla-Gehi, Silvia Penuela, Jared M. Churko, Qing Shao, and Dale W. Laird
1
From the Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada
Pannexins (Panx) are a class of integral membrane proteins
that have been proposed to exhibit characteristics similar to
those of connexin family members. In this study, we utilized
Cx43-positive BICR-M1R
k
cells to stably express Panx1, Panx3,
or Panx1-green fluorescent protein (GFP) to assess their traf-
ficking, cell surface dynamics, and interplay with the cytoskel-
etal network. Expression of a Sar1 dominant negative mutant
revealed that endoplasmic reticulum to Golgi transport of
Panx1 and Panx3 was mediated via COPII-dependent vesicles.
Distinct from Cx43-GFP, fluorescence recovery after photo-
bleaching studies revealed that both Panx1-GFP and Panx3-
GFP remained highly mobile at the cell surface. Unlike Cx43,
Panx1-GFP exhibited no detectable interrelationship with
microtubules. Conversely, cytochalasin B-induced disruption of
microfilaments caused a severe loss of cell surface Panx1-GFP, a
reduction in the recoverable fraction of Panx1-GFP that
remained at the cell surface, and a decrease in Panx1-GFP vesic-
ular transport. Furthermore, co-immunoprecipitation and co-
sedimentation assays revealed actin as a novel binding partner
of Panx1. Collectively, we conclude that although Panx1 and
Panx3 share a common endoplasmic reticulum to Golgi secre-
tory pathway to Cx43, their ultimate cell surface residency
appears to be independent of cell contacts and the need for
intact microtubules. Importantly, Panx1 has an interaction with
actin microfilaments that regulates its cell surface localization
and mobility.
The pannexin family is a new class of integral membrane
glycoproteins that have been identified to share sequence
homology with the invertebrate gap junction proteins, innexins
(1). Unlike the connexin family that is composed of 21 members
(2), both the human and mouse genomes contain only three
pannexin-encoding genes (Panx1, Panx2, and Panx3) (1).
Although connexins and pannexins exhibit no sequence ho-
mology, pannexins are predicted to share similar topology with
connexins, which includes four transmembrane domains, two
extracellular loops, a cytoplasmic loop, and intracellular amino
and carboxyl termini (3–5). Our previous study has shown that
ectopically expressed Panx1 and Panx3 are capable of traffick-
ing to normal rat kidney (NRK)
2
cell surfaces; however, their
distribution profile at cell-cell interfaces is not typically clus-
tered or punctate as seen for Cx43 (5). Consistently, electron
micrographs of Panx1 overexpressing Madin-Darby canine
kidney cells also revealed dispersed Panx1 localization at the
plasma membrane with no evidence of gap junction plaques (4).
Cx43 has a relatively short half-life of ⬃1–3 h (6). As a result,
Cx43 subunits assembled into connexons within the trans-
Golgi network (7) are constantly being transported to and re-
moved from the plasma membrane (8). Recent reports
provide evidence that although Panx1 appears to form similar
hexameric channel units defined as pannexons (4), they exhibit
slower turnover dynamics in comparison with Cx43, as as-
sessed by the use of pharmacological blockers of protein syn-
thesis and protein trafficking (4, 5). Functionally, most studies
support the premise that pannexins, in particular Panx1, form
single membrane channels (4, 5, 9–12), whereas far less evi-
dence supports the notion that they also can form intercellular
channels (13, 14).
The delivery of Cx43 for the assembly of functional channels
at the cell surface has been extensively studied using fluores-
cent protein and epitope tags (15, 16). Cx43-GFP has been
shown to exhibit similar distribution and functional charac-
teristics as its untagged counterpart, when assessed by dye
permeability and electrical conductance (8, 17). Likewise, GFP
tagging of the carboxyl-terminal tail of Panx1 did not signifi-
cantly alter the localization profile of Panx1 in NRK cells (5).
However, a recent electrical conductance assessment provided
evidence that mouse Panx1-GFP exhibits reduced channel
function when expressed in human embryonic kidney (HEK)
293 cells (18), suggesting that its functional state is somewhat
impaired but not completely eliminated by the GFP tag. In
other studies, untagged and tetracysteine-tagged Panx1 exhib-
ited comparable capabilities in rescuing the trafficking of the
glycosylation-defective mutant of Panx1 in Madin-Darby
canine kidney cells (19). Collectively, these studies would sug-
gest that the trafficking and life cycle properties of Panx1 can be
assessed by GFP tagging approaches in conjunction with real-
time dynamic imaging.
It has been previously established through a number of stud-
ies using both untagged and GFP-tagged Cx43 that the delivery
and regeneration of Cx43 gap junction plaques is facilitated by
*This work was supported by a Canadian Institutes of Health Research oper-
ating grant (to D. W. L.)and a National Sciences and Engineering Council of
Canada studentship (to R. B.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–5 and Movies 1–5.
1
To whom correspondence should be addressed: Dept. of Anatomy and Cell
Biology, Dental Science Bldg., University of Western Ontario, London,
Ontario N6A 5C1, Canada. E-mail: Dale.Laird@schulich.uwo.ca.
2
The abbreviations used are: NRK, normal rat kidney; HEK, human embryonic
kidney; GFP, green fluorescent protein; BFA, brefeldin A; FRAP, fluores-
cence recovery after photobleaching; BSA, bovine serum albumin; PBS,
phosphate-buffered saline; ROI, region of interest; ER, endoplasmic reticu-
lum; C-tail, carboxyl-terminal tail.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 12, pp. 9147–9160, March 19, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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microtubules and requires a fully functional secretory pathway
(8, 20, 21). Other studies using rapid time lapse imaging of GFP-
and tetracysteine-tagged Cx43 have shown that Cx43 is deliv-
ered in 100–150-nm vesicles that coalesce laterally into the
preexisting gap junction plaques (15, 22). However, the delivery
of pannexins to the cell surface, their dynamic organization at
specific cell surface microdomains, and their dependence on an
intact cytoskeletal network have yet to be investigated.
The life cycle of Cx43 is governed, in part, by many direct and
indirect binding partners (23–25), whereas pannexin binding
partners are only beginning to be identified with some evidence
tosupportPanx1interactionwithaproteinsubunitofthevoltage-
dependent potassium channel (26) and regulatory cross-talk
with P2X7 receptors (9, 27). In the present study, we further
expand on a very limited number of pannexin-binding proteins
by identifying actin as a specific Panx1 binding protein.
EXPERIMENTAL PROCEDURES
Cell Culture and Reagents—BICR-M1R
k
cells originally de-
rived from a rat mammary tumor and HEK 293T, NRK, and
REK (rat keratinocyte) cells were cultured in high glucose Dul-
becco’s modified Eagle’s medium (Invitrogen), supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, 100
g/ml
streptomycin, and 2 mML-glutamine (all from Invitrogen). B16-
BL6 murine melanoma cells were kindly provided by Dr. Mou-
lay A. Alaoui-Jamali-(Department of Medicine and Oncology,
McGill University (Montreal, Canada)) and cultured as de-
scribed previously (28). Trypsin (0.25%, 1 mMEDTA), Opti-
MEM I medium, and Lipofectamine 2000 were purchased from
Invitrogen. Brefeldin A (BFA), nocodazole, and cytochalasin B
were purchased from Sigma.
Expression Constructs of Mouse Panx1, Panx2, and Panx3
and Engineering of the GST-Panx1 Carboxyl Domain—Un-
tagged and GFP-tagged expression constructs encoding full-
length mouse Panx1 and Panx3 were previously described (5),
and correspond to current NCBI reference sequence encoding
426 amino acids for Panx1 (NP_062355) and 392 amino acids
for Panx3 (NP_766042).
As previously described, Panx2 was originally cloned from
mouse brain, and the purified PCR product was inserted into
the EcoRI-SalI site of pEGFP-N1 vector (Clontech) to generate
the untagged Panx2 (29). The sequence was confirmed to
encode 677 amino acids according to RefSeq. Furthermore,
Panx2 cDNA was amplified using forward primer 5⬘-CCCAA-
GCTTATGCACCACCTC to create a HindIII site and 5⬘-GGCG-
ACCGGTCCAAACTCCACA to create an AgeI site at the 5⬘-
and 3⬘-ends of Panx2, respectively. PCR products and the
vector pEGFP-N1 (Clontech) were digested with HindIII and
AgeI and ligated, and clones were selected. GFP was fused in
frame to the carboxyl terminus of Panx2 with the addition of a
five-amino acid polylinker (GGACCGGTCGCCACC) and val-
idated by sequencing.
Panx1 carboxyl tail primers (forward, 5⬘-CTAGGATTCCG-
GCAGAAAACGGAC; reverse, 5⬘-CGAGTCGACTTAGCA-
GGACGGATT), with flanking sites for BamHI and SalI ampli-
fying the Panx1 carboxyl tail sequence (corresponding to amino
acids 299–426), were created. This sequence was ligated into
the pGEX-6P-3 GST vector and transformed into BL21 bacte-
ria. Batch purification using Sepharose 4B was performed as
described in the GST Gene Fusion System manual with some
modifications (GE Healthcare). A single BL21 clone trans-
formed with either GST alone or GST-Panx1 carboxyl tail was
grown to an A
600 nm
of 1 and was induced by the addition of 0.5
mMisopropyl

-D-1-thiogalactopyranoside. A total of 500 ml of
either GST or GST-Panx1 carboxyl tail bacterial culture was
shaken overnight at room temperature. The next day, purifica-
tion was performed using 400
l of 50% Sepharose slurry,
washed in PBS seven times, and stored at 4 °C for the co-sedi-
mentation assays.
Transfection and Engineering of Stable Cell Lines—DsRed-
tagged Sar1
WT
and Sar1
H79G
cDNA constructs were described
previously (8) and used for transfection into BICR-M1R
k
cells
engineered to express Panx1 or Panx3. Briefly, BICR-M1R
k
cells were grown overnight to 50 –70% confluence on glass cov-
erslips and transfected in Opti-MEM I medium containing 4
l
of Lipofectamine 2000 and 1
g of Panx1 or 3
g of Panx3
plasmid DNA together with 2
g of Sar1
WT
or Sar1
H79G
expres-
sion constructs.
The Panx1-GFP cDNA construct was described previously
(5) and used to transfect BICR-M1R
k
cells stably expressing
untagged Panx1 and B16-BL6 cells. Cells were grown overnight
to 50–70% confluence on 35-mm glass bottom dishes and
transfected in Opti-MEM I medium containing 1.5
l of Lipo-
fectamine 2000 and 1.5
g of Panx1-GFP plasmid DNA for 4 h
at 37 °C.
For Panx3 and Panx3-GFP co-transfections, BICR-M1R
k
cells were grown overnight to 20–30% confluence on 35-mm
glass bottom dishes and transfected in Opti-MEM I medium
containing 4
l of Lipofectamine 2000 and 3
g of Panx3
together with 1.5
g of Panx3-GFP plasmid DNA. Opti-MEM I
medium was replaced with complete culture medium 4 h after
transfection at 37 °C.
For Panx2 and Panx2-GFP expression, BICR-M1R
k
cells
grown overnight to 50–70% confluence on glass coverslips
were transfected in Opti-MEM I medium containing 3
lof
Lipofectamine 2000 and 1.5
g of Panx2 or 1.5
g of Panx2-
GFP plasmid DNA. For co-transfection of Panx2 with Panx2-
GFP, 0.75
g of each plasmid DNA was used in Opti-MEM I
medium containing 3
l of Lipofectamine 2000.
Full-length constructs encoding mouse Panx1, Panx1-GFP,
and Panx3 were inserted into the AP-2 retroviral vector and
transfected into the 293GPG packaging cell line as described
earlier by Qin et al. (30). Following transfection, replication-
defective retroviral supernatants were collected and filtered
through a 0.45-
m filter (Pall Gelman Laboratories, Ann
Arbor, MI). BICR-M1R
k
cells expressing Cx43-GFP were engi-
neered to stably express Panx1 and Panx3 by following a previ-
ously described protocol (30). Retrovirus encoding Panx1-GFP
was also used to stably express GFP-tagged Panx1 in BICR-
M1R
k
cells.
Treatments with Pharmacological Reagents—Panx1- and
Panx3-overexpressing BICR-M1R
k
cells were treated with 5
g/ml BFA for 19 h at 37 °C, and cell lysates were collected and
subjected to immunoblotting. For elucidating the role of
cytoskeletal elements in pannexin trafficking, Panx1-GFP-ex-
pressing cells were treated with 10
Mnocodazole or 2.5
g/ml
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cytochalasin B for 90 min at 37 °C and fixed for immunocyto-
chemistry. For fluorescence recovery after photobleaching
(FRAP) studies, Panx1-GFP expressing cells were pretreated
with cytoskeletal inhibitors for 90 min prior to imaging up to
3–4 h in presence of these same inhibitors.
Immunocytochemistry—Cells were immunolabeled as previ-
ously described (5). Briefly, cells grown on glass coverslips were
fixed using ice-cold 80% methanol and 20% acetone for 20 min
at 4 °C. Cytochalasin B-treated cells were fixed using 3.7%
formaldehyde for 30 min at room temperature and permeabi-
lized for 45 min in a 1% blocking solution (bovine serum albu-
min (BSA) (Sigma), containing 0.1% Triton X-100). Cells were
incubated with a 500-fold dilution of polyclonal anti-Cx43 anti-
body (Sigma), a 100-fold dilution of polyclonal anti-GPP130
antibody (Convance), polyclonal anti-Panx2 antibody (Zymed
Laboratories Inc., San Francisco, CA) or monoclonal anti-tubu-
lin antibody (Convance) for1hatroom temperature. Affinity-
purified polyclonal Panx1 and Panx3 antibodies were used at a
concentration of 2
g/ml. F-actin was localized using a 200-fold
dilution of rhodamine phalloidin (Invitrogen). Cells were incu-
bated in goat anti-rabbit antibody conjugated to Texas Red or
fluorescein isothiocyanate (1:100; Jackson Laboratories, West
Grove, PA) or a goat anti-mouse antibody conjugated to Texas
Red (1:100; Jackson Laboratory). Cells were rinsed with PBS,
and nuclei were stained with Hoechst 33342 and mounted.
Immunolabeled cells were imaged using a ⫻63 oil objective lens
mounted on a Zeiss LSM 510 META system (Zeiss, Toronto,
Canada).
Immunoblotting and Co-immunoprecipitation—Cell lysates
from BICR-M1R
k
cells transiently co-transfected with Sar1 and
Panx1 or Panx3 cDNA constructs, and BFA-treated cells were
collected using a lysis buffer containing 1% Triton X-100, 10
mMTris, 150 mMNaCl, 1 mMEDTA, 1 mMEGTA, 0.5% Non-
idet P-40, 100 mMsodium fluoride, and 100 mMsodium
orthovanadate and a protease inhibitor tablet (one tablet/10 ml
of buffer; Roche Applied Science), pH 7.4. Protein concentra-
tions were measured using a BCA protein determination kit
(Pierce). In total, 20–30
g of protein was resolved using 10%
SDS-PAGE and transferred to nitrocellulose membrane (Pall
Life Sciences, NY). Nitrocellulose membranes were blocked in
Licor blocking solution (Lincoln, NE) or 3% BSA solution and
probed overnight with polyclonal affinity-purified anti-Panx1
or anti-Panx3 antibodies (0.2
g/ml) at 4 °C. Monoclonal anti-

-actin antibody (1:5000; Sigma) was used to assess gel loading.
Detection of primary antibody binding was performed by using
mouse IgG IR dye 800 (Rockland Immunochemicals) and rabbit
IgG Alexa 680 (Invitrogen) with the Odyssey infrared imaging
system (Licor).
For co-immunoprecipitation experiments, 1 mg of protein
lysates from WT-, Panx1-, and Panx1-GFP-overexpressing
BICR-M1R
k
cells was incubated overnight at 4 °C in the lysis
buffer (1% Triton X-100, 10 mMTris, 150 mMNaCl, 1 mM
EDTA, 1 mMEGTA, 0.5% Nonidet P-40, 1 mMsodium fluo-
ride, and 1 mMsodium orthovanadate) containing 10
g/ml
anti-Panx1 antibody. The antibody complex was pulled down
with 30
l of precleaned protein A-Sepharose beads (in PBS) for
2 h on the rocker at 4 °C. The antibody-bead complex was cen-
trifuged at 4500 rpm at 4 °C for 2 min, and supernatant was
aspirated. Unbound nonspecific protein was separated from
bound proteins by washing three times with 500
l of lysis
buffer, and the bound complex was detached by boiling for 5
min in 30
lof2⫻Laemmli loading sample buffer containing

–mercaptoethanol. Samples were resolved by 10% SDS-PAGE
and transferred to nitrocellulose membranes, which were
probed with anti-Panx1 and anti-

-actin antibodies.
FRAP Analysis—To assess Panx1 or Panx3 dynamics at the
cell surface, BICR-M1R
k
cells expressing Panx1-GFP, alone
or in combination with the untagged Panx1, or Panx3-GFP
together with Panx3 were cultured on 35-mm glass bottom
dishes and subjected to FRAP. B16-BL6 cells expressing Panx1-
GFP were also subject to FRAP analysis. Rapid time lapse imag-
ing was performed on a Zeiss LSM 510 META system to quan-
tify the movement of Panx1-GFP into the bleached region of
interest (ROI), as described previously (16). Briefly, glass bot-
tom dishes were placed in an environmentally controlled cham-
ber, and ROIs representing the various plasma membrane
domains were selected and photobleached using scan iterations
at 488 nm with 100% laser strength. Images were acquired
⬃2–5 s apart for up to 60 s with 0.9% laser strength to avoid
further photobleaching. Fluorescence intensities within ROIs
were quantified as described previously (16). Briefly, fluores-
cence recovery was recorded before photobleaching, immedi-
ately upon completion of photobleaching (twas set to 0 s), and
postbleaching at the following time intervals: 15, 25, 50, and
60 s. Postbleach intensities were corrected and normalized for
any residual fluorescence, and the recoverable fraction of
Panx1-GFP or Panx3-GFP was calculated using the equation,
F
Nt
⫽(F
t
⫺F
0
)/(F
i
⫺F
0
) as previously described (31), where F
Nt
represents normalized fluorescence at a time point t;F
t
is fluo-
rescence intensity within ROI at tseconds postphotobleach; F
0
is fluorescence intensity upon photobleaching at t⫽0; and F
i
is
fluorescence intensity prior to photobleaching. All FRAP
experiments were repeated three times for each experimental
set, with each set containing multiple ROIs at t⫽15, 25, 50, and
60 s. F
Nt
values from replicates of each experimental set were
combined, and a non-linear regression analysis was per-
formed to obtain a curve of best fit using GraphPad Prism
software (San Diego, CA). To compare the mobility dynam-
ics of Panx1-GFP or Panx3-GFP in various plasma mem-
brane domains, a one-way analysis of variance followed by
Tukey’s multiple comparison tests were performed. For
comparisons between untreated and nocodazole- and
cytochalasin B-treated experimental sets, ttests were per-
formed using GraphPad Prism software.
Vesicle Movement—Panx1-GFP containing vesicles were
analyzed by measuring the total distance traveled of vesicles
that remained in confocal plane of focus for the duration of the
analysis. Vesicles of ⬃0.5–0.8
m in diameter were monitored
by 1.8-s interval image scans for a total period of 8.8 s (n⫽
18–20 over four independent repeats).
Actin Co-sedimentation Assays—Muscle actin (Cytoskeleton
Inc., Denver, CO) was resuspended in buffer (5 mMTris-HCl,
pH 8.0, 0.2 mMCaCl
2
) to a concentration of 1 mg/ml in 250
l
on ice for 30 min. 25
lof10⫻actin polymerization buffer (500
mMKCl, 20 mMMgCl
2
,10mMATP, 100 mMTris, pH 7.5) was
added to the monomeric actin and incubated at room temper-
Delivery and Cell Surface Dynamics of Panx1 and Panx3
MARCH 19, 2010•VOLUME 285• NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 9149
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ature for 1 h (F-actin stock at 23
M). Protein samples of BSA,
GST, Panx1 carboxyl terminal tail (C-tail), and GST-tagged
Panx1 C-tail were centrifuged at 150,000 ⫻gfor1hat4°C.
Supernatants were collected and placed on ice. For co-sedi-
mentation assays, a GST fusion protein containing the C-tail of
mouse Panx1 was used, either as a fusion protein after elution
from the Sepharose beads or after cleavage of the Panx1 C-tail
from the fusion protein using PreScission Protease (GE Health-
care) for 16 h at 4 °C, as per the manufacturer’s instructions.
Following the Cytoskeleton Inc. protocol for actin binding pro-
tein assays, 50-
l samples were prepared with F-actin alone,
GST protein with or without F-actin, BSA (negative control)
plus F-actin, and Panx1 C-tail or GST fusion protein with or
without F-actin. Samples were incubated at room temperature
for 30 min, followed by centrifugation at 150,000 ⫻gfor 1.5 h at
24 °C. Supernatants were carefully removed and mixed with 10
lof4⫻Laemmli reducing sample buffer. The pellets were
resuspended in 30
l of double-distilled water and mixed with
30
lof2⫻Laemmli buffer. Equal volumes of resuspended
pellets and supernatants were run in duplicate on 10% SDS-
PAGE. Gels containing samples were either stained overnight
with Sypro-Ruby Protein gel stain (Invitrogen) or transferred
onto nitrocellulose membranes using an iBlot apparatus for dry
transfer (Invitrogen). Sypro-Ruby-stained gels were destained
and exposed to UV for visualization of the major bands. Nitro-
cellulose membranes were incubated overnight with primary
anti-Panx1 antibodies, washed, and probed with Alexa-680
anti-rabbit secondary for detection of Panx1 bands with a LiCor
scanner as described previously (5).
Biotinylation Assays—BICR-M1R
k
cells grown on 100-mm
dishes were transiently transfected with 5–10
g of Panx3-
GFP-encoding cDNA constructs, and 48 h post-transfection,
cells expressing Panx3-GFP were subjected to biotinylation
treatment on ice as described previously (5). Cells were incu-
bated in PBS or with cold PBS containing EZ-link Sulfo-NHS-
LC-biotin (0.5 mg/ml; Pierce) for 20 min at 4 °C. Control and
biotin-treated cells were washed and incubated in 100 mMgly-
cine buffer for 15 min at 4 °C to quench the biotin. Cells were
then lysed with SDS lysis buffer (1% Triton X-100 and 0.1% SDS
in PBS), and protein concentrations were measured using a
BCA protein determination kit (Pierce). In total, 1000
gof
protein from control and biotin-treated cell lysates were rocked
overnight at 4 °C in the presence of 50
l of neutravidin-agarose
beads (Pierce). Beads were washed three times with immuno-
precipitation lysis buffer (150 mMNaCl, 10 mMTris-HCl, pH
7.4, 1 mMEDTA, 0.5% Nonidet P-40, and 1% Triton X-100)
containing 1 mMNaF and 1 mMNa
3
VO
4
and once with PBS
containing 1 mMNaF and 1 mMNa
3
VO
4
. The beads were air-
dried and resuspended in 50
lof2⫻Laemmli loading sam-
ple buffer containing

-mercaptoethanol before boiling for 5
min. As a lysate control, 40
g of total protein from control
and biotin samples was also resolved by SDS-PAGE and
transferred to nitrocellulose membranes for immunoblot-
ting with anti-Panx3 antibody. Glyceraldehyde-3-phosphate
dehydrogenase was used as a control to detect any unex-
pected biotin internalization.
RESULTS
We have previously shown that the Cx43-positive BICR-
M1R
k
(rat mammary tumor) cell line is an excellent reference
model for investigating dynamic delivery events and assembly
and turnover mechanisms of Cx43 (16, 32); therefore, we
designed our experimental approach to compare Panx1 and
Panx3 trafficking dynamics in Cx43-positive BICR-M1R
k
cells.
We engineered stable cells lines to ectopically express Panx1 or
Panx3. Western blots analysis revealed that wild-type BICR-
M1R
k
cells are negative for Panx1 and Panx3 (Fig. 1A), but
when engineered to express pannexins, Panx1 resolved as mul-
tiple species ranging from ⬃41 to 48 kDa, whereas Panx3 was
detected as a doublet at ⬃43 kDa (Fig. 1A). These multiple
pannexin species have previously been shown to be the result of
glycosylation (4, 5). Immunolabeling of both Panx1 and Panx3
revealed that both of these pannexins were capable of traffick-
ing and localizing in a relatively uniform pattern at the apposing
cell surface (Fig. 1B). In contrast, detectable Panx2 and Panx2-
GFP were mainly localized in the intracellular compartments of
not only BICR-M1R
k
cells but also HEK 293T, NRK, and rat
keratinocytes (supplemental Fig. 1). Not unexpected, the co-
expression of Panx2 with Panx2-GFP did not alter the distribu-
tion pattern of untagged or tagged Panx2. These studies indi-
cate that Panx2 has a unique distribution when compared with
Panx1 and Panx3 even when expressed in the same reference
cells.
Trafficking of Panx1 and Panx3 Is Mediated through Sar1-de-
pendent COPII Vesicles—It has been widely documented that
Sar1 (secretion-associated and Ras-related) GTPase is critical
for COPII (coat protein II) assembly and vesicular transport of
newly synthesized proteins from the endoplasmic reticulum
(ER) compartment (33, 34). Dominant negative GTP-bound
mutant Sar1
H79G
has previously been shown to block the ER-
FIGURE 1. Panx1 and Panx3 are capable of trafficking to the cell surface in
BICR-M1R
k
cells. BICR-M1R
k
cells were engineered to stably express Panx1 or
Panx3. Immunoblotting with affinity-purified antibodies (anti-Panx1 and
anti-Panx3) revealed multiple banding profiles of Panx1 (⬃41– 48 kDa) and
Panx3 (⬃41– 43 kDa) (A).

-Actin was used as a protein loading control (A).
Immunolabeling of Panx1 and Panx3 (B) identified that both pannexins traf-
ficked and localized to the cell surface (arrows). Nuclei are stained with
Hoechst 33342 (blue). Bars,10
m. Results shown are representative of three
independent experiments.
Delivery and Cell Surface Dynamics of Panx1 and Panx3
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Golgi transport of newly synthesized Cx43 in BICR-M1R
k
cells
(8). In order to determine if Panx1 and Panx3 follow the classi-
cal ER-Golgi secretory pathway mediated through a COPII-de-
pendent mechanism, we engineered BICR-M1R
k
cells to co-
express Panx1 or Panx3 along with Sar1
WT
or Sar1
H79G
.
Co-expression of Panx1 with Sar1
WT
revealed a typical uniform
distribution of Panx1 at the cell surface (Fig. 2A,arrows),
whereas Sar1
WT
was localized to the paranuclear region in an
intact Golgi-like compartment (Fig. 2, Aand B). In comparison,
expression of Sar1
H79G
resulted in fragmentation of a Golgi-like
compartment in the paranuclear region (Fig. 2, Aand B) and
retention of Panx1 and Panx3 in ER-like patterns with little
evidence of cell surface localization (Fig. 2, Aand B,arrow-
heads). In the same culture environments, BICR-M1R
k
cells
expressing Panx1 or Panx3 but not Sar1
H79G
revealed a uniform
cell surface labeling of Panx1 and Panx3 (Fig. 2, Aand B,insets,
arrows). Furthermore, as we previously reported (8), the distri-
bution of Cx43 gap junction plaque-like structures was not evi-
dent in the presence of Sar1
H79G
(data not shown).
Because Panx1 and Panx3 resolve as multiple bands (Figs. 1A
and 2C), we wanted to determine the effect of dominant-nega-
tive Sar1 on the different molecular species. When compared
with Panx1 alone or with Sar1
WT
, the presence of Sar1
H79G
revealed an accumulation of the intermediate species (previ-
ously demonstrated to be a high mannose glycosylation species
(4)), with a noticeable reduction in the most extensively glyco-
sylated species of Panx1 (Fig. 2C). Similarly, an accumulation of
lower molecular weight species (previously reported to be a
high mannose glycosylation species (29)) of the Panx3 doublet
was also revealed in the presence of Sar1
H79G
(Fig. 2C), suggest-
ing that the higher molecular weight species of Panx1 and
Panx3 is the consequence of additional post-translational proc-
essing that occurs upon exiting the ER. Consistently, long term
BFA treatment (19 h) (a pharmacological blocker known to
inhibit anterograde transport of proteins between the ER and
Golgi apparatus (35)) of Panx1- and Panx3-expressing BICR-
M1R
k
cells caused a detectable increase in the high mannose
species of Panx1 and Panx3, with a noticeable reduction in the
higher molecular weight species of both Panx1 and Panx3 (Fig.
2D). Collectively, these results suggest that both Panx1 and
Panx3 are co-translationally inserted into the ER and trans-
ported in a COPII-dependent mechanism to the Golgi appara-
tus, where they are substrates for further glycosylation and
editing.
GFP-tagged Panx1 Mimics the Distribution Profile of Un-
tagged Panx1 and Is Suitable to Investigate the Dynamic Distri-
bution of Panx1—In order to assess the dynamic properties of
Panx1, we first stably expressed Panx1-GFP in BICR-M1R
k
cells and evaluated its distribution profile with respect to
untagged Panx1. Immunofluorescent labeling revealed that
Panx1-GFP exhibited a cell surface distribution pattern (Fig.
3A,arrows) similar to that observed for untagged Panx1 (Fig.
3F,arrows), with a notable increase in intracellular fluorescent
signal (Fig. 3A). Clearly, the cell surface pattern for Panx1-GFP
was distinct from that observed for Cx43-GFP when expressed
in the same cell type (Fig. 3A,inset).
To assess the dynamic activity of Panx1 at the cell surface and
within intracellular compartments, we performed rapid time
lapse imaging on cells that expressed Panx1-GFP. Panx1-GFP
was not only visualized in a relatively uniform pattern at the cell
surface (Fig. 3A) but also as mobile bright fluorescent clusters
(Fig. 3, Band C). Rapid time lapse imaging revealed that these
clusters (suggestive of Panx1-GFP aggregates) were mobile at
the plasma membrane and in regions devoid of cell-cell con-
tacts (Fig. 3, Band C,filled arrows; see supplemental Movie 1).
In addition, Panx1-GFP was also found in distinct intracellular
vesicle-like structures that were highly mobile (Fig. 3D,unfilled
arrows, and supplemental Movie 1). Surprisingly, and distinct
from that observed for functional Cx43, Panx1-GFP was found
in dynamic finger-like projections indicative of membrane pro-
FIGURE 2. Trafficking of Panx1 and Panx3 was disrupted in the presence
of a dominant-negative Sar1 mutant. BICR-M1R
k
cells expressing Panx1 or
Panx3 together with Sar1
WT
or Sar1
H79G
were immunolabeled for Panx1 (A)or
Panx3 (B). Both Panx1 and Panx3 were capable of trafficking and localizing to
the cell surface in the presence of Sar1
WT
(Aand B,filled arrows). Expression of
Sar1
H79G
resulted in Panx1 and Panx3 being retained in an ER-like compart-
ment (Aand B,arrowheads); however, when cells expressed Panx1 or Panx3
without expressing Sar1
H79G
in the same cellular environment, both Panx1
and Panx3 trafficked to the plasma membrane (Aand B,insets,arrows). West-
ern blotting of Panx1 and Panx3 in the presence of Sar1
H79G
(C) or after long
term BFA treatment (19 h) (D) revealed an accumulation of the high mannose
species of Panx1 and Panx3, with a noticeable reduction in the higher molec-
ular weight glycosylation species (Cand D). Nuclei are stained with Hoechst
33342 (blue). Bars,10
m. Results shown are representative of three indepen-
dent experiments.
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trusion (Fig. 3E,arrowheads, and supplemental Movie 1). This
localization to membrane protrusion was also frequently evi-
dent in cells expressing untagged Panx1, suggesting that this
localization profile is not a consequence of the GFP tag (Fig. 3F,
arrowheads).
Panx1-GFP Is Highly Mobile at All Plasma Membrane Do-
mains as Revealed by FRAP Analysis—To analyze the dynamic
mobility characteristics of Panx1-GFP, we first assessed the
ability of Panx1-GFP to traffic and localize at three distinct
plasma membrane domains where there was no neighboring
cell (Fig. 4A,red arrow), the neighboring cell expressed Panx1-
GFP (Fig. 4A,blue arrow), or the neighboring cell was devoid of
Panx1-GFP (Fig. 4A,purple arrow). Once it was determined
that Panx1-GFP was not differentially distributed to any of
these cell surface domains (Fig. 4A), regions of Panx1-GFP
located at these distinct domains were selected and subjected to
FRAP analysis (Fig. 4, B–E). After initial photobleaching, within
2 s, there was a rapid movement of fluorescent molecules from
the outer edges into the photobleached area of all bleached cell
surface domains (Fig. 4, B–D,insets). FRAP curve analysis
revealed the mobile fluorescent fractions to be 45–60% at all
microdomains over a time course of only 60 s (Fig. 4E). More-
over, there was no significant difference in the total recovered
fraction of Panx1-GFP within any of the plasma membrane
domains, an observation that was similar to when we ana-
lyzed the percentage recovery of transiently transfected
Panx1-GFP (40–50%) in BICR-M1R
K
cells stably expressing
untagged Panx1 (supplemental Fig. 2), or B16-BL6 cells,
identified for the first time to express endogenous Panx1
(supplemental Fig. 3, Aand B).
A Subpopulation of GFP-tagged Panx3 Is Evident at the Cell
Surface When Expressed Alone or Co-expressed with Panx3 and
Reveals Dynamic Localization to the Membrane Protrusions—
We have previously reported that Panx3-GFP has a substantial
trafficking defect, causing it to be retained within the endoplas-
mic reticulum of NRK cells (5). However, when overexpressed
in BICR-M1R
k
cells, some evidence of Panx3-GFP localization
to the cell surface was detected (Fig. 5A,filled arrows), along
with the intracellular ER-like distribution of Panx3-GFP (Fig.
5A,unfilled arrows). To further confirm that a subpopulation of
Panx3-GFP can traffic and localize to the cell surface, we per-
formed biotinylation assays in live BICR-M1R
k
cells transiently
expressing Panx3-GFP. Incubation of Panx3-GFP-expressing
cells with biotin followed by pull-downs with neutravidin beads
revealed that the ⬃70-kDa Panx3-GFP indeed traffics to the cell
surface (supplemental Fig. 4). Glyceraldehyde-3-phosphate
dehydrogenase was used as a control to confirm that biotin was
not internalized (supplemental Fig. 4). It was interesting to note
that when Panx3-GFP was co-expressed with Panx3 in BICR-
M1R
k
cells, there was an apparent increase in the cell surface
population of Panx3-GFP (Fig. 5B,filled arrows), with some
expected intracellular distribution (Fig. 5B,unfilled arrows).
This finding suggests that Panx3-GFP may interact with Panx3
to facilitate its traffic to the plasma membrane.
To assess the dynamic activity of Panx3-GFP at the cell sur-
face and within intracellular compartments, we performed
rapid time-lapse imaging on cells co-expressing Panx3 and
Panx3-GFP. Panx3-GFP was localized at the cell surface in a
relatively uniform pattern and as bright clusters either
approaching or at the cell surface (Fig. 5C,filled arrows). In
addition, Panx3-GFP was also visualized in distinct vesicle-like
structures (Fig. 5C,unfilled arrows) and observed in cell surface
protrusions (Fig. 5D,arrows) that were also occasionally iden-
tified in cells expressing only Panx3 (Fig. 5E,arrows).
Panx3-GFP Is Highly Dynamic at All Cell Surface Domains—
We co-expressed Panx3 with Panx3-GFP and used GFP-tagged
Panx3 as a tracer to assess and quantify the mobile fraction in
various plasma membrane domains where the neighboring cell
surface was devoid of Panx3-GFP (Fig. 6A), the neighboring cell
expressed Panx3-GFP (Fig. 6Band supplemental Movie 2), or
there was no neighboring cell (Fig. 6C). FRAP analysis of
FIGURE 3. Panx1 was localized to multiple sites, compartments, and
microdomains. Immunolabeling of Panx1-GFP with an anti-Panx1 antibody
revealed its localization at the cell surface (A,filled arrows) in a pattern that
was distinct from Cx43-GFP (A,inset). Regions of interest from live BICR-M1R
k
cells expressing Panx1-GFP (B,red and blue rectangles) were chosen for live
imaging and imaged at t⫽0, 10, 50, 70, and 110 s (Cand D). Rapid time lapse
imaging revealed that Panx1-GFP is distributed primarily in a uniform pattern,
whereas mobile bright fluorescent clusters could be identified at the cell sur-
face (Band C,filled arrows) and within the cell (D,unfilled arrows). Panx1-GFP
was clearly localized to dynamic plasma membrane protrusions (E,arrow-
heads) that were evident in BICR-M1R
k
cells expressing untagged Panx1
(F,arrowheads). Nuclei in Aand Fare stained with Hoechst 33342 (blue). Bars,
10
m. Representative of three independent experiments.
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selected regions expressing Panx3-GFP revealed that within
60 s after photobleaching, there was a rapid reentry of GFP-
tagged Panx3 molecules into the photobleached area at all
microdomains (Fig. 6, A–C,insets),
and the mobile fraction was calcu-
lated to be ⬃30–40%. However, no
significant difference was noticed in
the total recovered fraction of Panx3-
GFP within any of the plasma mem-
brane domains (Fig. 6D).
Cell Surface Population of Panx1-
GFP and Panx3 Is Insensitive to
Nocodazole Treatment—It has been
documented that nocodazole dis-
ruption of microtubules impairs the
continuous trafficking and regener-
ation of Cx43-GFP at the cell sur-
face (8). To investigate if trafficking
and recovery of Panx1-GFP into the
photobleached area is dependent on
microtubules, Panx1-GFP-express-
ing BICR-M1R
k
cells were exposed
to nocodazole for 90 min. Nocoda-
zole treatment resulted in charac-
teristic morphological changes in
the cells from spindle-shaped to
more cuboidal with a notable dis-
ruption of the microtubule architec-
ture (Fig. 7, A–C). However, the dis-
tribution of Panx1-GFP (Fig. 7B)
and Panx3 (supplemental Fig. 5B)at
the cell surface remained relatively
unaffected by the nocodazole treat-
ment. Furthermore, FRAP studies
revealed that Panx1-GFP migrated
into the photobleached area simi-
larly in both untreated and nocoda-
zole-treated cells (Fig. 7, Cand D).
Interestingly, the inward progres-
sion of fluorescent recovery from
the edges of the photobleached area
toward the center was not detected
until 10 s postbleaching (Fig. 7C,
inset). Furthermore, FRAP analysis
of Panx1-GFP in the presence of
nocodazole treatment revealed no
significant difference in the recov-
ered fraction when compared with
the untreated cells (Fig. 7D). Thus,
nocodazole treatment does not
visually affect the cell surface local-
ization of Panx1-GFP or the dynam-
ics of recovery into the photo-
bleached area at the cell surface.
The Cell Surface Stability of
Panx1-GFP and Panx3 Is Sensi-
tive to Cytochalasin B Treatment,
whereas the Mobility of Panx1-GFP
Transport Vesicles Is Perturbed in the Absence of Intact
Microfilaments—We next wanted to assess the role of actin
microfilaments in stabilizing Panx1 and Panx3 at the cell sur-
FIGURE 4. Panx1-GFP is highly mobile at all plasma membrane locations. Panx1-GFP was localized to three
distinct plasma membrane domains of BICR-M1R
k
cells, as depicted by the schematic diagram (A). Fluorescent
images of Panx1-GFP were superimposed with DIC images to highlight the microenvironment surrounding the
cell being analyzed (A–D). Selected cell regions where Panx1-GFP was localized at the three distinct plasma
membrane domains were photobleached, and fluorescence recovery back into the photobleached areas was
assessed and normalized over 60 s (B–D). Panx1-GFP recovery within the photobleached area was not signifi-
cantly different (p⬎0.05) among all three domains (E). Bars,10
m. n⫽6 –9 per plasma membrane domain
collected from three independent experiments.
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face as well as evaluating their cell surface dynamic properties
and transport of Panx1-GFP. Treatment of cytochalasin B (90
min) caused a redistribution of F-actin from the cell periphery
(Fig. 8Aand supplemental Fig. 5A) to the paranuclear region
(Fig. 8Band supplemental Fig. 5A), with a subsequent change in
cell morphology. In cytochalasin B-treated cells, Panx1-GFP
and Panx3 were mainly localized in intracellular compartments
(Fig. 8Band supplemental Fig. 5A,arrowheads), whereas a
small population remained evident at the cell surface (Fig. 8B
and supplemental Fig. 5A, arrows). These findings suggest that
actin may play a crucial role in the cell surface stability of
Panx1-GFP and Panx3. FRAP analysis of the remaining and
detectable cell surface population of Panx1-GFP after cytocha-
lasin B treatment (supplemental Movie 3) revealed that Panx1-
GFP was mobile, but the recovery rate was slower and the
amount of the recovered fraction was significantly less (⬃15–
20%) when compared with the untreated cells (Fig. 8C). Fur-
thermore, rapid time lapse imaging on the same field of cells
visualized before (supplemental Movie 4) and after
(supplemental Movie 5) cytochalasin B treatment revealed that
Panx1-GFP-carrying vesicles were freely able to move and
travel in the untreated cells; however, this movement was
greatly perturbed when cells were treated with cytochalasin B
with a ⬃60% decrease in vesicle movement over a fixed interval
of time (8.8 s) (Fig. 8D). Collectively, these studies suggest that
microfilaments play a multifaceted role in pannexin stabiliza-
tion at the cell surface and vesicular transport.
F-actin Directly Binds Panx1 at the Carboxyl Terminus—To
further examine the possible interaction of Panx1 with actin,
lysates of wild-type BICR-M1R
k
cells engineered to express
Panx1 or Panx1-GFP were subjected to immunoprecipitation
of Panx1 prior to immunoblotting for Panx1 or

-actin. As
expected, multiple glycosylated species of Panx1 (resolved
below the IgG band) and Panx1-GFP were detected in the
immunoprecipitates and cell lysates of Panx1-overexpressing
cells but not wild-type BICR-M1R
k
cells (Fig. 9A,top). Interest-
ingly,

-actin was found to co-immunoprecipitate with Panx1
from both Panx1- and Panx1-GFP-expressing BICR-M1R
k
cells
(Fig. 9A,bottom). To further validate the interaction of actin
with Panx1, we conducted co-sedimentation assays where poly-
merized actin was mixed with GST fusion protein containing
the C-tail of Panx1. As observed by Sypro-stained gel, once
polymerized, F-actin typically sediments in the pellet fraction.
In the absence of polymerized actin, GST-Panx1 C-tail was
found in both the soluble and pellet fractions (Fig. 9B). How-
ever, when combined with F-actin, GST-Panx1 C-tail sedi-
ments preferentially in the pellet fraction (Fig. 9B). As a control,
GST alone did not sediment in the pellet fraction with or with-
out F-actin (Fig. 9B). Because the Panx1 C-tail fusion protein
detection at ⬃43 kDa was partially masked by actin, we cleaved
the C-tail of Panx1 from the GST and performed a similar co-
sedimentation assay. Further Panx1 immunoblots revealed the
Panx1 C-tail at ⬃15 kDa in the pellet fraction (Fig. 9C). These
results suggest that F-actin binds directly to the carboxyl termi-
nus of Panx1.
DISCUSSION
With the recent discovery of pannexins as a new family of
channel- or conduit-forming proteins, there has been a
growing interest in elucidating their biochemical properties,
life cycle, and cellular roles. Previous reports have linked
Panx1 channels to the release of ATP in neurons (36) and
astrocytes (37) and in cellular response to pathological
insults, such as initiation of inflammatory action (10, 38),
ischemia-induced death of neurons (39), and tumor suppres-
sion (13). Our understanding of Panx3 is even more rudi-
mentary. Although Panx3 has been shown to be a cell surface
glycoprotein that forms conduits capable of dye uptake (5,
40), its cellular function remains largely unknown. It was our
hypothesis that, being integral membrane proteins with
sequence relationships to invertebrate innexin gap junction
proteins, Panx1 and Panx3 would exhibit characteristics similar
to those of the well studied Cx43 gap junction protein in terms
of the secretory pathway governing their trafficking, dynamic
FIGURE 5. Delivery of Panx3-GFP to the cell surface. Wild-type BICR-M1R
k
cells engineered to express Panx3-GFP (A) or both Panx3 and Panx3-GFP (B)
were immunolabeled with anti-Panx3 antibody. Panx3-GFP was retained
mainly in an ER-like pattern (Aand B,unfilled arrows) with some evidence of a
cell surface distribution (Aand B,filled arrows), whereas co-expression of
Panx3 appeared to increase the cell surface population of Panx3-GFP (B).
Rapid time lapse imaging of live BICR-M1R
k
cells co-expressing Panx3-GFP
and Panx3 revealed that Panx3-GFP was distributed primarily in a uniform
pattern with notable mobile fluorescent clusters at the cell surface (C,filled
arrows) and within the cell (C,unfilled arrow). Localization of Panx3-GFP to
plasma membrane protrusions (D,arrows) was similar to that found in cells
expressing only Panx3 (E,arrows). Nuclei in A,B, and Eare stained with
Hoechst 33342 (blue). Bars,10
m. Results shown are representative of three
independent experiments.
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properties within the plasma membrane, and interplay with the
cytoskeletal network.
Trafficking of Panx1 and Panx3 to the Cell Surface—It has
been well established that Cx43 is co-translationally inserted
into the ER and oligomerizes into connexons in the trans-Golgi
network before trafficking to the plasma membrane (25). In the
case of Panx1, treatment with endoglycosidase H and peptide:
N-glycosidase F enzymes have revealed that the lower molecu-
lar weight species constitutes the core protein that gets glyco-
sylated to the high mannose form in the ER before further
glycosylation and editing in the Golgi and delivery to the cell
surface (4, 5). Although recent studies have extensively focused
on correlating glycosylation status with the delivery of Panx1 to
the cell surface (4, 19), molecular mechanisms underlying the
secretory pathway taken by Panx1 and Panx3 were largely
unknown. To address if the transport of Panx1 and Panx3
from the ER to the Golgi apparatus is mediated through
COPII vesicles, we transiently co-expressed Panx1 or Panx3
with a dominant-negative GTP-bound mutant Sar1
H79F
. Stabi-
lization of Sar1 in the dominant negative GTP-bound state has
been previously shown to efficiently
block COPII-mediated ER transport
of proteins to the cell surface (41).
Our data demonstrated that
Sar1
H79F
expression severely inhib-
ited the cell surface localization of
Panx1 and Panx3, thus suggesting
that COPII vesicular trafficking of
these pannexin family members is
required prior to their eventual
delivery to the cell surface. These
data also indicate that efficient GTP
hydrolysis of Sar1 is crucial for reg-
ulating their transport from the ER
compartment because restriction of
GTP hydrolysis of Sar1
H79G
to GDP
has been previously shown to arrest
the cargo-containing vesicles at the
ER exit sites (34). Consistent with
our findings, we previously showed
that Sar1 function was necessary for
delivery of Cx43 to the cell surface
(8). Our data support the premise
that the post-ER pool of Panx1 and
Panx3 is correlated with pannexin
processing to the highly complex
glycosylation species, whereas the
accumulation of the high mannose
intermediate species of Panx1 and
Panx3 is consistent with retention
of Panx1 and Panx3 in the ER. Sim-
ilar Sar1 GTPase dependence was
observed for K
ATP
channels, where
expression of dominant negative
mutants Sar1
H79G
(mutant incapa-
ble of hydrolyzing GTP) and
Sar1
T39N
(mutant restricted in its
ability to exchange GDP for GTP)
prevented proper channel processing and the cell surface
expression of the channel (42).
ER to Golgi transport of Panx1 and Panx3, as well as their
state of glycosylation, was further confirmed by the use of BFA,
which is known to inactivate Arf1, thereby inhibiting ER to
Golgi transport (43). Consistent with a previous study (19), we
noticed a dramatic increase in the high mannose form of Panx1
in BFA-treated cells. Interestingly, we also observed an accu-
mulation of the high mannose species of Panx3 in response to
BFA treatment, consistent with the inhibition of both Panx1
and Panx3 being delivered to the Golgi apparatus for further
processing. Vesicular trafficking of Panx1 and Panx3 would also
strongly suggest that, like Cx43, both proteins are integral
transmembrane proteins that get transported from ER mem-
branes in COPII vesicles.
Mobility Dynamics of Panx1 and Panx3—In our study, we
used Panx1-GFP and Panx3-GFP as tracer probes to elucidate
the distribution profile and cell surface dynamics of Panx1 and
Panx3. The distribution of Panx1-GFP at the plasma membrane
appeared to be uniform with occasional Panx1-enriched
FIGURE 6. Panx3-GFP is highly mobile at all plasma membrane domains. Panx3-GFP was localized to three
distinct plasma membrane domains of BICR-M1R
k
cells co-expressing Panx3 (A–C). Selected cell surface regions
containing Panx3-GFP were photobleached, and fluorescence recovery back into the photobleached areas
was assessed and normalized over the time course of 60 s. The percentage of Panx3-GFP recoverable fraction
was not found to be significantly different among all three plasma membrane domains examined (p⬎0.05)
(D). Bars,10
m. n⫽12–25 per plasma membrane domain collected from three independent experiments.
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domains, consistent with previous findings using the untagged
Panx1 (5). In contrast, Panx3-GFP alone revealed an increased
intracellular profile, as reported earlier (5), with some clear evi-
dence of its localization to the cell surface that was confirmed
by cell surface biotinylation assays. Since adequate delivery of
Panx3-GFP to the cell surface was required to investigate its
dynamics at the plasma membrane, we co-expressed it with
untagged Panx3 and found an increased cell surface expression
of Panx3-GFP. It is possible that Panx3 co-oligomerized with
Panx3-GFP to facilitate its delivery to the plasma membrane; if
this holds true, it would also suggest that having a GFP tag at the
carboxyl-terminal tail of Panx3 does not interfere with the
intermixing of Panx3 subunits. A similar mechanism has been
proposed for the cell surface rescue of a trafficking-defective,
glycosylation-deficient mutant of Panx1 when co-expressed
with either wild-type Panx1 or tet-
racysteine-tagged Panx1, further
indicating that tagging of pannexins
does not impair its ability to assem-
ble together with its untagged coun-
terpart (19).
In our study, delivery of both
Panx1-GFP and Panx3-GFP to the
cell surface appeared to occur at
multiple plasma membrane do-
mains via intracellular vesicle-like
structures that formed bright clus-
ters upon apparent fusion with the
plasma membrane. These clusters
were found to be quite mobile and
displaced laterally within the cell
surface membrane, thus supporting
a model of untargeted delivery of
Panx1 and Panx3 to all cell surface
microdomains. In contrast, Cx43-
GFP, typically known to localize in
punctate-like structures at the cell
surface, has been documented to
have both an arbitrary delivery to all
plasma membrane domains (8, 16)
as well as a preferred microtubule-
dependent delivery to adherens
junctions that reside in close prox-
imity to the preexisting gap junc-
tions (44). Interestingly, our data
support the premise that Panx1 and
Panx3 are enriched in membrane
protrusions at areas that are devoid
of contacting cells, a situation not
typically observed for Cx43 unless
non-functional Cx43 mutant stud-
ies are performed (16). Localization
of pannexins in the finger-like
membrane protrusions could sug-
gest that it may play a role in cell
migration because the process of
cell motility typically involves actin
polymerization and entails forma-
tion of fanlike or pointed projections (lamellipodium and filo-
podia, respectively) at the leading edge (45). Previously, the
absence of Panx1 from the leading edge of a corneal epithelium
wound in P
2
X
7
⫺/⫺
mice was correlated with delayed corneal
re-epithelialization and compromised wound healing (46).
Other channel-forming proteins, such as aquaporin-1, have
been implicated in increased cell migration by localizing to the
lamellipodia (47), whereas migration of lymphocytes (48) and
embryonic nerve cells (49) has been correlated with voltage-de-
pendent K
⫹
channels, thus supporting the role of channel-
forming proteins in regulating cell motility.
Our FRAP assessments of Panx1 and Panx3 mobility identi-
fied that, similar to Cx43 (16), lateral movement of Panx1-GFP
and Panx3-GFP occurred from the outer edge to the center of
the photobleached areas. It is notable that although the recov-
FIGURE 7. The cell surface population of Panx1-GFP is insensitive to nocodazole treatment. Untreated (A)
or nocodazole-treated (B) Panx1-GFP-expressing BICR-M1R
k
cells were immunolabeled for tubulin. As
expected, nocodazole treatment collapsed tubulin into paranuclear regions (Aand B); however, the distribu-
tion profile of Panx1-GFP at the cell surface and in the intracellular compartments (Aand B) remained relatively
unchanged with collapsed tubulin (B). FRAP analysis in presence of nocodazole revealed that Panx1-GFP was
able to recover into the photobleached area, and the percentage of recoverable fraction was not significantly
different from the untreated cells (Cand D). Bars,10
m. n⫽5–10 per plasma membrane domains, data
collected over four independent repeats.
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ery of fluorescence into the photobleached area is probably the
result of lateral movement of pannexins due to the time course
being examined, there is also probably a contribution from
newly delivery fluorescent protein-tagged pannexins to the cell
surface. The relative rate of Panx1-GFP and Panx3-GFP recov-
ery into the photobleached area was quite comparable in any of
the examined plasma membrane microdomains, thus suggest-
ing that the assembly state of these pannexin family members
remains relatively unchanged with respect to its subcellular
location within the plasma membrane. In our study, Panx1-
GFP (and to a slightly lesser extent Panx3-GFP) exhibited over
twice the mobility of Cx43-GFP molecules, which are typically
arranged in gap junction-like clus-
ters (16). Slow recovery of Cx43-
GFP was also reported in HeLa cells
(50). Given the relatively uniform
cell surface distribution of Panx1
and Panx3 that appear to be untar-
geted to specific microdomains, we
speculate that these pannexins are
not likely to be packaged into dense
crystalline-like structures as re-
ported for Cx43 (51). Thus, the dis-
tribution and mobility of these
pannexins are more in line with
other channels and receptors, such
as Na
⫹
channels (52) and acetylcho-
line receptors (53).
The percentage of fluorescence
recovery after photobleaching for
both Panx1-GFP and Panx3-GFP
(representing the mobile fraction)
reached a plateau between 40 and
60%, with the remaining compo-
nent representing the immobile
fraction. Typically, the size of the
immobile fraction is dependent on
the nature of the protein and the
membrane microenvironment being
assessed. For instance, the immobile
fraction of sodium channels ranges
from ⬃10% in the cell body to ⬃40%
in the neurite terminals (52). Like-
wise, the mobile fraction of glycine
receptors ranges from ⬃50% in the
neuronal cell body to ⬃70% in the
processes (54). In addition, the vis-
cosity of the membrane microdo-
main (55), tethering of proteins with
scaffolds/binding partners, and
interaction with the cytoskeleton
can all contribute to the size of the
immobile fraction (56).
Cytoskeletal Dependence of
Pannexin Trafficking and Mobility—
In order to assess the role of the
cytoskeleton in pannexin trafficking
and cell surface mobility dynamics,
we used nocodazole and cytochalasin B to disrupt microtubules
and microfilaments, respectively. Nocodazole-induced disrup-
tion of microtubules did not significantly alter the cell surface
distribution of either Panx3 or Panx1-GFP, which may not be
totally unexpected given the predicted long half-life of Panx1 (4,
5). This finding is quite distinct from Cx43, where enhanced
growth of gap junctions (20) and Cx43 molecular movement
into the photobleached gap junctions was minimal in nocoda-
zole-treated cells (8), suggesting that Cx43 is much more de-
pendent on microtubules than Panx1. In contrast, disruption of
microfilaments revealed concomitant accumulation of paranu-
clear Panx1-GFP and Panx3 with collapsed actin microfila-
FIGURE 8. Effect of cytochalasin B on Panx1-GFP. Untreated (A) or cytochalasin B-treated (B) Panx1-GFP-
expressing BICR-M1R
k
cells were labeled with phalloidin for F-actin localization. As expected, cytochalasin B
caused the redistribution of F-actin from the cell surface (A) to the paranuclear region (B). The collapse of F-actin
microfilaments coincided with the intracellular accumulation of Panx1-GFP (B,arrowheads), whereas a small
population of Panx1-GFP remained evident at the cell surface (B,arrows). FRAP analysis in the presence of
cytochalasin B treatment revealed that the cell surface population of Panx1-GFP was significantly impaired
from entering the photobleached area (p⬍0.05) (C)n⫽3. Quantification of the total distance traveled by
Panx1-GFP carrying vesicles within the same field of cells analyzed before and after the cytochalasin B treat-
ment indicated a significant (p⬍0.05) reduction in vesicle mobility in cytochalasin B-treated cells (D). Bars,10
m. Results shown are representative of five independent experiments.
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ments, whereas only a subpopulation of Panx1-GFP or Panx3
remained at the cell surface. The longer turnover dynamics of
pannexins and the rapid intracellular accumulation upon short
term cytochalasin B treatment support the premise that actin
provides stability to the cell surface population of Panx3 and
Panx1. On the other hand, Cx43 gap junctions have been doc-
umented to remain considerably more independent to the
assembly state of microfilaments (57). Mobility assessment of
FIGURE 9. F-actin binds Panx1 at the carboxyl terminus. Wild type (WT) or Panx1- or Panx1-GFP-expressing BICR-M1R
k
cells were lysed and subjected to
immunoprecipitation (IP) for Panx1 prior to immunoblotting (IB) the immunoprecipitates and cell lysates for Panx1 or

-actin.

-Actin co-immunoprecipitated
with Panx1 and Panx1-GFP (A). Monomeric actin was polymerized into F-actin, incubated with either GST fusion protein containing the carboxyl-terminal tail
of Panx1 (B) or the carboxyl-terminal tail of Panx1 alone (C) and separated into supernatant or pellet fractions (denoted by Sand P, respectively) prior to
immunoblotting for Panx1. Panx1 was found to co-sediment with F-actin in the pellet fractions (Band C). Parallel gels were stained with Sypro gel stain, and BSA
and GST were used as controls in the co-sedimentation assays. Results shown are representative of three independent experiments.
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the remaining cytochalasin B-insensitive subpopulation of
Panx1-GFP at the cell surface revealed a smaller mobile fraction
of ⬃35% in the absence of intact microfilaments. This surpris-
ing finding was somewhat distinct from Na,K-ATPase and reg-
gie-1/flotillin-2, where the disruption of microfilaments caused
an increase in the mobile fraction (58, 59). The smaller mobile
fraction of Panx1-GFP noticed in our study may be explained by
the fact that transport vesicles carrying Panx1-GFP either to or
from the plasma membrane were less mobile, and this decrease
may mechanistically account for the reduced fluorescent recov-
ery into the photobleached area. The relative speeds of Panx1-
GFP-carrying vesicles in untreated cells (⬃0.2
m/s) and
cytochalasin B-treated cells (⬃0.07
m/s) were also quite dif-
ferent. Although credited to trafficking on microtubules tracks,
vesicles containing Cx43-GFP showed comparable average
speeds of ⬃0.5
m/s in untreated HeLa cells (21).
Interaction of Panx1 with Actin—Given the finding that actin
microfilaments regulated the distribution and cell surface
mobility of Panx1, we speculated that a direct interaction
between Panx1 and actin may exist. Because we were best
equipped to address this question for Panx1, we first demon-
strated that actin does in fact co-immunoprecipitate with
Panx1. To further assess 1) if Panx1 binds to monomeric or
filamentous actin, 2) if Panx1 interaction to actin is direct, and
3) which domain of Panx1 might be responsible for interaction,
we conducted a co-sedimentation assay with a GST-Panx1
C-tail fusion protein. Here we show that Panx1 sediments pref-
erentially in the F-actin fraction, the interaction appears to be
direct, and it is the carboxyl-terminal tail of Panx1 that seems
responsible for interacting with actin. Comparatively, actin
binding to Cx43 is thought to be facilitated via its interaction
with zonula occludens-1, which is a known Cx43 binding part-
ner (60). Future studies will be needed to elucidate the Panx1
motif responsible for actin binding at the C-tail and whether
actin also binds to Panx3.
In summary, trafficking and assembly of pannexins is a pre-
cisely regulated process. Our study is the first to report that
Panx1 and Panx3 transport is dependent on Sar1-mediated
COPII vesicles, cell surface Panx1 and Panx3 have dynamic
mobility properties, and Panx1 directly interacts with F-actin.
Acknowledgment—We acknowledge Jamie Simek for providing exper-
tise in optimizing the FRAP technique.
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Delivery and Cell Surface Dynamics of Panx1 and Panx3
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Churko, Qing Shao and Dale W. Laird
Ruchi Bhalla-Gehi, Silvia Penuela, Jared M.
Interactions
Surface Dynamics, and Cytoskeletal
Pannexin1 and Pannexin3 Delivery, Cell
Cell Biology:
doi: 10.1074/jbc.M109.082008 originally published online January 10, 2010
2010, 285:9147-9160.J. Biol. Chem.
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