Change in permeant size selectivity
by phosphorylation of connexin 43
gap-junctional hemichannels by PKC
Xiaoyong Bao*, Sung Chang Lee†, Luis Reuss*, and Guillermo A. Altenberg†‡§
*Department of Neuroscience and Cell Biology,†Division of Nephrology and Hypertension, Department of Internal Medicine, and‡Sealy Center
for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0437
Edited by Michael V. L. Bennett, Albert Einstein College of Medicine, Bronx, NY, and approved January 8, 2007 (received for review April 18, 2006)
Gap-junctional channels, permeable to large hydrophilic solutes of
up to Mr? 1,000, are responsible for cell-to-cell communication.
Phosphorylation of connexin 43 (Cx43) by PKC abolishes the
permeability of gap-junctional channels and hemichannels to large
on a methodology to produce purified hemichannels of controlled
subunit composition and apply it to the generation of hemichan-
nels with variable number of PKC-phosphorylated subunits. The
subunit composition was determined by luminescence resonance
energy transfer. We show that all Cx43 subunits in the hemichan-
nel hexamer have to be phosphorylated to abolish sucrose (Mr342)
permeability. We also show that the hemichannel pores with all
subunits phosphorylated by PKC have a sizable diameter, allowing
for permeation of the small hydrophilic solute ethyleneglycol (Mr
62). These results indicate that phosphorylation of Cx43 by PKC
affects dye transfer between cells without consistent effects on
luminescence energy transfer ? membrane protein ? transport
of two connexin hexamers (hemichannels or connexons), one
from each of the neighboring cells (1–3). Gap-junctional chan-
nels are permeable to large hydrophilic solutes of up to Mr?
1,000, depending on their isoform composition (1, 4). Connexin
43 (Cx43) is expressed in cells from organs such as brain,
myocardium, and kidney, and in vascular endothelial cells, where
it mediates cell-to-cell communication, a process essential for
development and organ function (5–7). Uncoupled hemichan-
nels have been shown to exist in several cell types (see below).
Activation of these large nonselective Cx43 hemichannels, e.g.,
during ischemia, may overwhelm the normal membrane-
transport mechanisms and alter intracellular composition, con-
tributing to cell injury. This notion is supported by data on
cardiomyocytes (8, 9), astrocytes (10), and renal proximal tubule
cells (11) that show Cx43 hemichannel activation by ATP
depletion. One possibility is that ATP depletion activates the
Cx43 hemichannels by decreasing their phosphorylation state
(12), although other possibilities have been proposed (13).
Activation of PKC decreases cell-to-cell movement of hydro-
philic permeability probes, such as fluorescent dyes (dye cou-
pling), while electrical communication between cells persists
(reviewed in ref. 14). These direct effects are independent of the
effects of phosphorylation on trafficking, assembly, and/or deg-
radation (15). Single-channel studies have shown that stimula-
tion of PKC decreases the frequency of the dominant (?100 pS)
conductance state, favoring a lower conductance state (?50 pS)
of Cx43 gap-junctional channels (reviewed in ref. 14). This
change is likely to occur concomitantly with an increase in open
probability (16), which explains the lack of consistent effects of
changes in PKC activity on cell-to-cell electrical communication
(14, 16–18). We hypothesize that the dissociation of the effects
ap-junctional channels are responsible for cell-to-cell com-
munication (1). They are formed by head-to-head docking
on ‘‘large’’ and ‘‘small’’ solute permeability is the consequence
hemichannels by PKC-mediated phosphorylation. To test this
hypothesis, we studied the transport of hydrophilic solutes of
different sizes through hemichannels formed by Cx43 either fully
dephosphorylated (Cx43-dP) or fully PKC-phosphorylated
Results and Discussion
Phosphorylation of All Six Cx43 Subunits by PKC Produces a Partial
Reduction of the Effective Cross-Sectional Area of the Hemichannel
Pore. For the transport studies we measured retention of per-
meability probes after gel filtration of proteoliposomes pre-
loaded with radiolabeled probes (19). The methodology (probe-
retention studies) and experimental system (reconstituted
purified hemichannels) have been described (18, 19), and addi-
tional validation is presented in supporting information (SI) Text
and SI Fig. 6. We used sucrose as the main permeability probe
because its molecular mass is in the lower side of the range of the
masses of second messengers of functional significance that
permeate gap-junctional channels and hemichannels. Phosphor-
ylation by PKC of all six Cx43 subunits at Ser-368 abolishes
sucrose permeability of reconstituted hemichannels (18). The
absence of sucrose transport through Cx43-P hemichannels was
that the maltose permeability is abolished by PKC-mediated
phosphorylation, as expected, because both disaccharides have
the same molecular weight (Mr342). The smaller probe ethyl-
eneglycol (Mr62), however, was permeant through hemichan-
nels formed by either Cx43-dP or Cx43-P, indicating that a
hemichannel pore of significant size remains when all six Cx43
molecules are phosphorylated by PKC. In other words, phos-
phorylation of all of the subunits by PKC produces a partial
decrease of the cross-sectional area of the Cx43 hemichannel
pore, i.e., the hemichannels remain permeable to ethyleneglycol.
Although an accurate estimation of the minimum hydrophilic
pore size of Cx43-P hemichannels is complicated by the potential
interaction of the permeability probes with pore-lining side
chains (20), the hydrodynamic diameter of ethyleneglycol is 4.4
Author contributions: X.B. and S.C.L. contributed equally to this work; L.R. and G.A.A.
designed research; X.B., S.C.L., and G.A.A. performed research; X.B., S.C.L., L.R., and G.A.A.
analyzed data; and L.R. and G.A.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Freely available online through the PNAS open access option.
Abbreviations: Cx43, connexin 43; Cx43-dP, dephosphorylated Cx43; Cx43-P, PKC-
phosphorylated Cx43; decylmaltoside, n-decyl-?-D-maltopyranoside; DTPA, diethylene-
triaminepentaacetate; EMCH, ?-maleimidocaproic acid hydrazide; LRET, luminescence
resonance energy transfer.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
March 20, 2007 ?
vol. 104 ?
no. 12 ?
Å (21), i.e., significantly larger than that of hydrated K?and Cl?
(?3.3 Å) (22), the main ions carrying currents through gap-
junctional channels. Therefore, our results explain why activa-
tion of PKC reduces dye transfer (17, 18), but has no substantial
effect on cell-to-cell gap-junctional currents (14). The differen-
tial modulation of Cx43 permeability by PKC-mediated phos-
phorylation could reduce fluxes of organic hydrophilic solutes
such as ATP, cAMP, IP3, and NAD?(300–700 Da molecular
mass) without major effects on small-ion fluxes and electric
As mentioned in the Introduction, stimulation of PKC de-
creases the frequency of the dominant (?100 pS) conductance
state of Cx43 gap-junctional channels, favoring a lower conduc-
tance state (?50 pS) (reviewed in ref. 14). It seems possible that
these lower conductance channels are formed by the fully
phosphorylated hemichannels permeable to ethyleneglycol that
are described here. However, the level of Cx43 phosphorylation
in the cells is uncertain, and it is important to point out that there
is no simple correlation between the single-hemichannel con-
ductance and the permeability to large hydrophilic solutes of
hemichannels formed by different connexin isoforms (1, 23).
Given the observation that hemichannels formed by Cx43-dP are
sucrose-permeable, whereas those formed by Cx43-P are not, we
investigated how many Cx43 subunits in a hemichannel must be
phosphorylated to abolish sucrose permeability. To this end, we
generated hemichannels with a known number of PKC-
phosphorylated subunits (by mixing purified Cx43-dP and
Cx43-P in different proportions) and measured their sucrose
permeability. Because the generation of hemichannels of con-
trolled composition was attempted in vitro, it was critical to
demonstrate that there is exchange between solubilized Cx43
subunits and that the composition of the reconstituted
hemichannels is that expected from the ratio of Cx43-dP and
Cx43-P in the mixture. Previous studies (24–27) have shown
solubilization of gap-junctional plaques into hemichannels, but
compared with our studies there were differences in the solu-
bilization conditions and/or isoforms studied. One possibility to
explain the apparent discrepancies is the observation that the
outcome of the solubilization depends on the nature of the
membranes (28). When Cx32 synthesized in vitro in the presence
of microsomes was solubilized with dodecylmaltoside, it ap-
peared mostly as monomers, whereas supplementation with
Golgi membranes increased the amount of solubilized hexamers
significantly (28). This effect was not observed for Cx26 (28),
indicating that it is isoform-dependent. Insect cells have mem-
branes of unusual properties and composition (29), which may
explain why solubilization of Cx43 with several detergents yields
monomers (at low concentrations), which can assemble as
hexamers (at higher concentrations) that display subunit ex-
change. In the present study, we specifically looked at the
possibility of exchange of subunits in solubilized hemichannels.
Definitive evidence that exchange occurs is presented below
[gel-filtration experiments, luminescence resonance energy
transfer (LRET) experiments, sucrose-transport studies]. It is
important to mention that this subunit exchange occurs when the
subunits are solubilized, and that we do not have evidence for
exchange once the purified hemichannels are reconstituted.
One potential concern is that the purified hemichannels
generated from solubilized subunits differ significantly from
native hemichannels in cell membranes. This concern cannot be
ruled out, but we have evidence for significant functional and
structural similarities between the native and purified hemichan-
nels: (i) The permeability properties of native and purified Cx43
hemichannels are internally consistent, and phosphorylation of
Ser-368 by PKC abolishes large-solute permeability in both
purified and native hemichannels (17, 18). (ii) The residue at
position 161 of transmembrane helix 3 is a pore-lining residue in
native and purified hemichannels (SI Text). (iii) The face of
transmembrane helix 3 that lines the pore is the same in native
and purified hemichannels, as determined by the substituted Cys
accessibility method (unpublished observations). (iv) The dis-
tances between homologous residues of transmembrane helix 3
structure of Cx43 gap-junctional channels [unpublished mea-
surements using LRET, based on the same principles recently
described for a study of K?channels (30)].
to an EGFP at the C-terminal end (Cx43-EGFP) forms hexamers,
as assessed by gel filtration. The first indication of a dynamic
oligomerization process was suggested by additional gel filtration
after sample dilution, Cx43-EGFP elutes as monomers (data not
shown). To demonstrate subunit exchange, we studied n-decyl-?-
D-maltopyranoside (decylmaltoside)-solubilized hemichannels by
using gel filtration chromatography. Because the difference in
molecular size between Cx43-dP and Cx43-P is very small for
detection by gel filtration, we mixed Cx43 with Cx43-EGFP. The
?26-kDa EGFP increases the molecular mass significantly, allow-
ing for detection of mixed oligomers. We have previously shown
that the regulation of Cx43 by PKC-mediated phosphorylation is
not affected by EGFP fusion (18). Fig. 2 shows that mixing of
hemichannels formed by Cx43 with those formed by Cx43-EGFP
yields hemichannels containing both Cx43 and Cx43-EGFP. The
quantitative molecular mass analysis of the major peaks is consis-
tent with Cx43 hexamers (Fig. 2, black traces), Cx43-EGFP hex-
amers (Fig. 2, green traces), and ‘‘mixed’’ Cx43/Cx43-EGFP hex-
amers (Fig. 2, red traces). The detergent bound to the hexamers
(assessed from the difference between the calculated molecular
mass and that expected from six connexins) seems to be ?5–10%
of the hexamer weight. Clearly, the Cx43:Cx43-EGFP mix localizes
mostly to a peak that elutes between the hexamers of Cx43 and
Cx43-EGFP. The peak of the hexamer mix is only slightly wider
than those of the Cx43 and Cx43-EGFP hexamers and does not
permeability to hydrophilic solutes. The proteoliposomes were loaded with
radiolabeled probes, and the percent retention of the permeability probes
Cx43 fully phosphorylated by PKC (all six Ser-368 residues phosphorylated,
Cx43-P). Values were normalized to the amount of probe retained by lipo-
somes formed by Cx43-P (statistically indistinguishable from the value mea-
sured in liposomes without hemichannels, 112 ? 9%, n ? 7), after subtraction
of the background measured in DMSO-permeabilized liposomes. Ethyleneg-
of four to seven experiments.*, P ? 0.05 compared with proteoliposomes
containing Cx43-dP hemichannels.
Effects of PKC-mediated phosphorylation of hemichannels on their
www.pnas.org?cgi?doi?10.1073?pnas.0603154104Bao et al.
extend from the Cx43-EGFP to the Cx43 peaks, indicating that it
corresponds to mixed Cx43:Cx43-EGFP hemichannels and not to
separate Cx43 and Cx43-EGFP that coexist without connexin
exchange. Additional evidence for subunit exchange was obtained
by LRET and sucrose-transport studies and is described later.
The gel-filtration experiments indicate that there is a dynamic
equilibrium between subunits in solubilized hemichannels. In
three independent experiments, the peak of the Cx43-
EGFP:Cx43 mix ran between the Cx43 and the Cx43-EGFP
hexamers peaks, but its relative location differed slightly from
experiment to experiment, preventing the accurate determina-
tion of the hemichannel composition (e.g., discrimination be-
tween hemichannels containing three from those containing five
Cx43-EGFP subunits was not possible).
Analysis of Hemichannel Subunit Composition by LRET. To improve
the analysis of mixed hemichannels, we developed a method
based on LRET, using the rare element Tb3?, characterized by
a long lifetime emission, as donor (31) and fluorescein as
acceptor. We measured LRET between Cx43 subunits labeled
with either Tb3?or fluorescein to determine the composition of
hemichannels based on the number of acceptor-labeled mono-
connexin molecular weight and has been used in a few studies of
membrane proteins to assess intersubunit and intramolecular
distances (32–35). LRET has many advantages for our experi-
ments when compared with traditional FRET (31, 32). As shown
later and in SI Text, these advantages translate in very low
background, high signal-to-noise ratio, and independence of the
labeling stoichiometry (32).
For our experiments, we mixed fluorescein-labeled, Tb3?-
labeled and unlabeled Cx43 in different proportions. The Cx43
mixtures had a low proportion of Tb3?-labeled Cx43 (0.5 mol per
hemichannel), so most hemichannels had zero or one Tb3?-
labeled Cx43 subunit, and only a small percentage (?8%;
calculated from the binomial distribution) had more than one
donor-label subunit. Under these conditions, and considering
that essentially all subunits are assembled as functional
hemichannels (see Materials and Methods and ref. 18), the
amplitude of fluorescein emission caused by energy transfer
from Tb3?(sensitized fluorescein emission with long lifetime)
depends on the number of acceptors (fluorescein-labeled Cx43
per hemichannel) and can be used to determine the subunit
composition of the hemichannels. This is because all LRET
under the conditions of our experiments occurs between sub-
units in a hemichannel. There is no intramolecular LRET
because subunits are labeled with donor or acceptor separately,
before mixing. Because of the long distances involved (?1,000
Å) and the high dependence of energy transfer on the distance
(inversely proportional to the sixth power of the distance),
significant LRET between hemichannels in solution or
hemichannels in different liposomes is not expected. The ab-
sence of interhemichannel LRET was demonstrated by measur-
ing the time course of the increase in sensitized emission upon
mixing two populations of detergent-solubilized hemichannels,
one labeled with Tb3?and the other one labeled with fluores-
cein. From such experiments, a halftime of subunit exchange of
?14 min was measured, with essentially no signal immediately
after mixing. Also, there was no significant LRET when two sets
of proteoliposomes, one containing donor-labeled hemichannels
and another one containing acceptor-labeled hemichannels,
were mixed (data not shown).
WT Cx43 contains nine Cys of which at least four are likely to
form intramolecular disulfide bonds. Under the labeling condi-
stoichiometry of labeling was 3.2 ? 0.1 fluorescein molecules
(n ? 4) or 3.1 ? 0.1 Tb3?(n ? 3) per Cx43. These findings
suggest that only 1/3 of the Cys in WT Cx43 are accessible to the
labeling reagents. In a given hemichannel containing a donor-
donors in the subunit and each of the acceptors in an acceptor-
labeled subunit. Therefore, there will be nine energy-transfer
processes between the donor subunit and each acceptor-labeled
subunit. We use total energy-transfer data, without deconvolut-
ing the individual transfers (which depend on the distances
between each donor-acceptor pair). Thus, each labeled subunit
in a hemichannel can be treated as a donor or acceptor, with the
sensitized emission between a donor-labeled and an acceptor-
labeled subunit as the sum of the energy transfer arising from the
nine donor–acceptor pairs.
In experiments with WT Cx43 hemichannels in which the label-
a decrease in the magnitude of the signal was observed, but the
subunits was not affected (data not shown). Additional data
showing independence of the methodology on the labeling stoichi-
ometry and the absence of significant background (measured on
Cys-less Cx43 hemichannels) are presented in SI Fig. 6. Connexin
labeling and the LRET data were also independent of whether
Cx43 was dephosphorylated or PKC-phosphorylated (data not
shown). Therefore, the assembly of detergent-solubilized Cx43 into
previous observations (18).
Because the energy transfer depends on the sixth power of the
distance between donor and acceptor, the largest fraction of the
total sensitized emission is the result of energy transfer between
the Tb3?-labeled subunit and the closest fluorescein-labeled
subunits. However, considering a Fo ¨rster distance R0 for the
Tb3?–fluorescein pair of ?45 Å, and distances between subunits
in a hemichannel ?100 Å, even the acceptor-labeled subunit
diametrically opposed to the donor-labeled subunit contributes
significantly to the total energy transfer in a hemichannel. In
summary, all acceptor-labeled subunits contribute to the sensi-
tized emission, and therefore the signal is proportional to the
number of labeled subunits in the hemichannel.
Fig. 3A shows that, as expected, the steady-state fluorescence
Cx43 and Cx43-EGFP gently mixed overnight at a 4:2 molar ratio, at 4°C, were
kDa, labeled 4) or thyroglobulin (669 kDa, labeled 1), catalase (232 kDa,
labeled 3), and aldolase (158 kDa, labeled 4) were mixed with Cx43 and
Cx43-EGFP, respectively, before injection into the FPLC system. Absorbance
was measured at 280 nm (A280) and normalized to the hexamer peak values.
Gel-filtration chromatography of hemichannel mixtures. Purified
Bao et al.
March 20, 2007 ?
vol. 104 ?
no. 12 ?
increases with the content of fluorescein-labeled Cx43 per
hemichannel. The important result is that when the emission is
measured in a gated mode (without recording during the first 60
?s after a 1-ns nitrogen laser pulse), the fluorescein emission
caused by direct excitation is very low (Fig. 3B, black trace). This
finding is expected from the short duration of the excitation
pulse (1 ns) and the short lifetime of the fluorescein excited state
(4–5 ns). In addition, the lack of signal from scattering of the
excitation pulse and the sample autofluorescence, caused by the
short excitation pulse and nanosecond lifetimes of native flu-
orophores, combine to contribute to a low emission background.
Fig. 3B (blue trace) shows that lanthanide emission is in sharp
peaks with interposed dark regions (31), and therefore measur-
ing at wavelengths where the donor does not emit eliminates the
luminescence of the lanthanide complex itself. The red trace in
Fig. 3B shows an emission with a peak at ?520 nm that
corresponds to sensitized emission from fluorescein, i.e., emis-
sion resulting from LRET from Tb3?, as opposed to direct
fluorescein excitation. As mentioned above, the latter is essen-
tially absent in the sample containing only fluorescein-labeled
Cx43 (Fig. 3B, black trace). Therefore, the sensitized emission
from fluorescein is easily isolated by using a bandpass emission
filter (Fig. 4A), with negligible background fluorescence (Figs.
3B and 4A, black traces).
As expected from energy transfer, sensitized fluorescein emis-
sion increased (Fig. 4A) as a function of the number of acceptors
donor, which in LRET translates in the decreased sensitized
emission, clearly apparent in Fig. 4B (see Inset for a direct
comparison). Fig. 4C shows that the sensitized fluorescein
emission is proportional to the number of fluorescein-labeled
Cx43 per hemichannel. The LRET-based method has several
advantages compared with gel filtration for the determination of
the composition of purified hemichannels. First, it is more
accurate because it allows for discrimination between hemichan-
nels containing ?1 acceptor-labeled subunits (only the differ-
ence between hemichannels with two vs. three acceptor-labeled
subunits was not statistically significant). This discrimination is
clearly superior to that achievable with gel filtration. Second, the
measurements can be carried out easily in either proteolipo-
somes or solution. This is very important in the case of mem-
brane proteins because it allows for parallel determinations of
oligomerization and function.
Phosphorylation by PKC of All Cx43 Hemichannel Subunits Is Needed
to Abolish Sucrose Permeability. We next asked how many subunits
must be phosphorylated by PKC to abolish hemichannel sucrose
permeability. To accomplish this aim, we performed experiments
with liposomes containing an average of 0.8 hemichannels and
determined the percentage of sucrose-impermeable liposomes. If
there is random exchange of connexin subunits, the distribution of
e.g., for a 3/3 mixture the most frequent hemichannels will contain
decreasing frequencies of 2/4 and 4/2 (?23% each), 1/5 and 5/1
(?9% each), and 0/6 and 6/0 (?2% each). Proteoliposomes recon-
stituted with Cx43-dP hemichannels or hemichannels from Cx43-
dP/Cx43-P mixtures at ratios of 5/1, 4/2, or 3/3 did not display
significant sucrose retention (Fig. 5). Sucrose retention by proteo-
liposomes reconstituted with a 3/3 Cx43-dP/Cx43-P ratio was 6 ?
11% of the value in sucrose-impermeable proteoliposomes con-
taining Cx43-P hemichannels (Fig. 5), although the percentage of
liposomes with hemichannels containing three to six Cx43-P sub-
units was ?66% according to the binomial distribution. This result
indicates that the presence of three Cx43-P subunits per hemichan-
nel yields sucrose-permeable hemichannels. Fig. 5 shows that at
hemichannels. Purified WT Cx43 solubilized in 0.3% decylmaltoside was la-
beled with either fluorescein maleimide or Tb3?-DTPA-cs124-EMCH, by incu-
bation for 2 h at 4°C with a 10-fold molar excess of the thiol reagents.
Unreacted labels were removed by gel filtration, and unlabeled and labeled
proteins were mixed in varying proportions and incubated for at least 2 h
before analysis. (A) Steady-state fluorescence emission spectra (excitation at
490 nm). Data are normalized to the peak value of fully fluorescein-labeled
present during Cx43 labeling there is no significant LRET between free Tb3?
and free fluorescein after gel filtration. Data were normalized to the peak
value of 1 Tb/5 fluorescein-labeled preparation. Traces are representative
from four independent experiments.
LRET between Tb3?- and fluorescein-labeled Cx43 subunits in
(red trace) lifetimes. Data in A and B correspond to typical traces that were obtained by using Cx43 solubilized in decylmaltoside at a concentration ?1.5 mg/ml.
(C) Sensitized fluorescence emission as a function of the average number of fluorescein-labeled connexins per hemichannel. Data were normalized to the peak
value of 1 Tb/5.5 fluorescein-labeled preparation and are presented as means ? SEM of seven to nine experiments. Cx43 hemichannels were reconstituted at
a ratio of 0.8 hemichannels per liposome (20 ?g protein per measurement). Similar results were obtained in detergent-solubilized Cx43 at ?1.5 mg/ml (data not
shown), where essentially all Cx43 subunits form hemichannels (see Fig. 2). All values are statistically different from the previous one (P ? 0.001), except for that
at the 3/3 ratio. For additional details see Fig. 3.
Determination of hemichannel composition. (A) Sensitized fluorescence emission (60-?s delay after pulse), normalized as described in Fig. 3B. (B) Time
www.pnas.org?cgi?doi?10.1073?pnas.0603154104Bao et al.
inversely proportional to the hemichannel content of Cx43-dP, but
even for an average of five Cx43-P per hemichannel, sucrose
retention was much less than that observed with proteoliposomes
containing six Cx43-P hemichannels. For the Cx43-dP/Cx43-P
reconstitution ratio of 1/5, only ?30% of the proteoliposomes were
sucrose-impermeable, although ?83% of the Cx43 is Cx43-P in the
detergent mixture. If five Cx43-Ps per hemichannel were sufficient
to render the hemichannels sucrose-impermeable, then the ex-
pected sucrose-retention value would be ?74%, the sum of the
proteoliposomes containing five (?40%) and six (?34%) Cx43-P
subunits, according to the binomial distribution. This value is
but is similar to the percentage of proteoliposomes containing six
Cx43-P subunits (?34%). These results strongly suggest that all six
hemichannel subunits must be phosphorylated to abolish sucrose
It is important to consider that the equilibration time of
transport probes such as sucrose across liposomes containing
Cx43-dP hemichannels (1 ms) is orders of magnitude shorter
than the transit of the liposomes through the gel-filtration
column. Therefore, our measurements provide steady-state in-
rates, needed to analyze detailed permeability changes (18).
Therefore, our results cannot rule out that partial phosphory-
lation of the Cx43 hemichannels decreases sucrose permeability,
but show that complete phosphorylation is needed to abolish
Although dynamic exchange of subunits between detergent-
solubilized hemichannels was directly demonstrated by the gel-
filtration and LRET experiments, the sucrose permeability
studies confirmed this important observation. For example, in
the absence of subunit exchange, 50% and 83% of the proteo-
liposomes with hemichannels reconstituted from 3/3 and 1/5
Cx43-dP/Cx43-P ratios would contain Cx43-P hemichannels
impermeable to sucrose, respectively. However, the values mea-
sured were not statistically different from zero and ?30%,
respectively (Fig. 5), which is incompatible with the absence of
subunit exchange, and strongly suggest that hemichannels are
reconstituted in the liposomes according to their composition in
In this work, we described a method to generate hemichannels of
controlled subunit composition by mixing detergent-solubilized
connexins and show that PKC-mediated phosphorylation of all
hydrophilic solutes. Our results strongly suggest that for PKC to
abolish permeability to larger hydrophilic solutes all six Cx43
hemichannel subunits must be phosphorylated.
The assessment of hemichannel composition using LRET and
the ability to generate hemichannels of controlled subunit
composition should be useful for studying disease-causing con-
nexin mutants and assessing the effects of the isoform compo-
sition of hemichannels (heteromeric hemichannels) on perme-
ability properties. One of the major advantages of LRET is that
it can be used not only with proteins in solution, but also with
lipid-reconstituted proteins. This property of LRET is essential
for structural-functional studies of membrane proteins.
Materials and Methods
Cx43 Expression, Purification, Dephosphorylation, PKC-Mediated
Phosphorylation, and Reconstitution. Details on the generation of
recombinant baculoviruses and protein expression in High-Five
insect cells have been described (18). In these studies, we used
Cx43 and Cx43-EGFP (18). Protein purification was performed
essentially as described (18), based on the affinity of a 6-His
C-terminal tag to Ni2?, but membrane solubilization and puri-
fication were carried out in decylmaltoside instead of n-octyl-
?-D-glucopyranoside (octylglucoside). Membranes were solubi-
lized with 1% decylmaltoside in 2 M NaCl, 10 mM EDTA, 10
mM DTT, 10 mM PMSF, and 10 mM glycine/NaOH, pH 10, at
a protein concentration ?2 mg/ml. During purification and
subsequent steps, decylmaltoside concentration was 0.3%. To
test the function of purified Cx43, we reconstituted Cx43-dP (see
below) in ?100-nm-diameter unilamellar liposomes (18) at
average ratios of 0.8 or 2.3 hemichannels/liposome. Under these
are predicted from the Poisson distribution to be permeable to
sucrose, respectively. The experimental values, measured using
a rapid filtration assay (18), were 66 ? 5 (n ? 4) and 90 ? 5%
(n ? 7) for the 0.8 or 2.3 hemichannels/liposome average ratios,
respectively. These data indicate that most, if not all, of the
reconstituted hemichannels are functional, and that the fraction
of purified Cx43 that can form functional hemichannels is higher
than that observed when octylglucoside was the detergent (18).
The analysis of the oligomeric state of Cx43 and Cx43-EGFP
showed that in decylmaltoside, at protein concentrations ?1
mg/ml, essentially all Cx43 and Cx43-EGFP molecules form hex-
amers (see Fig. 2), whereas under similar conditions in
octylglucoside, 20–25% of the protein remained as monomers
(18). Based on these observations and data by others (24), we
mediated phosphorylation, we first treated purified Cx43 solu-
bilized in decylmaltoside with immobilized alkaline phosphatase
cells (18). Then, we phosphorylated a fraction of the dephos-
phorylated connexins with PKC, using conditions that result in
phosphorylation of all of the Ser-368 residues (1 mol of Ser-368
phosphorylated per mol of Cx43) (18).
LRET Experiments. For the LRET experiments, purified WT Cx43
solubilized in 0.3% decylmaltoside was divided in three aliquots.
One remained unlabeled and the others were labeled with either
fluorescein maleimide (Invitrogen, Carlsbad, CA) or Tb3?-DTPA-
cs124-EMCH (DTPA, diethylenetriaminepentaacetate; EMCH,
?-maleimidocaproic acid hydrazide), by incubation for 2 h at 4°C
with a 10-fold molar excess of the thiol reagents. Details on the
nel on sucrose permeability. Effects of varying Cx34-dP/Cx43-P average ratios
radiolabeled probe are shown. Values were normalized as described in Fig. 1.
The average number of hemichannels per liposome was 0.8, and data are
long dash), ?3 (gray, solid), ?4 (blue), ?5 (red), or 6 (black). The lines were
obtained by joining with spline lines the values, calculated from the binomial
distribution, for each Cx43-dP/Cx43-P mixture.
Bao et al.
March 20, 2007 ?
vol. 104 ?
no. 12 ?
propertiesandsynthesis(performedintheOrganicChemistryCore Download full-text
Laboratory at the University of Texas Medical Branch) of Tb3?-
DTPA-cs124-EMCH have been published (36). DTPA-cs124-
EMCH contains carbostyril 124 as an ‘‘antenna’’ that absorbs the
incident light from a nitrogen laser source and transfers it to the
Tb3?, which by itself displays a weak absorbance. The chelator in
DTPA-cs124-EMCH, binds the lanthanide tightly and shields it
from the quenching effects of water. The thiol-selective maleimide
group from EMCH allows for protein labeling. After protein
labeling, the unreacted compounds were removed by gel filtration,
and unlabeled and labeled proteins were mixed in varying propor-
tions and incubated for at least 2 h before the experiments. This
time was sufficient for essentially complete subunit exchange
halftime is ?14 min). In some experiments, the mixtures were
reconstituted into liposomes for the LRET measurements. The
samples were analyzed in 100-?l quartz cuvettes. Excitation at 337
nm was carried out with a 1.45-MW pulsed nitrogen laser (GL-
3300; Photon Technology International, Birmingham, NJ; 1-ns
pulses at 10 Hz). Luminescence spectra were recorded on a TM11
phosphorescence lifetime system (Photon Technology Interna-
tional) with an R928 photomultiplier detector (Hamamatsu,
Bridgewater, NJ). For most measurements, the emission mono-
chromator was removed and replaced by band-pass filters to
increase light throughput. The bandpass filters for fluorescein
Omega Optical (Brattleboro, VT). Steady-state fluorescence was
measured on a fluorolog-2 spectrofluorometer (SPEX Industries,
Hydrophilic Solute Transport. For the transport experiments using
purified Cx43 and mutants, radiolabeled probes were loaded
by extrusion during the production of liposomes or proteoli-
posomes (18). After loading, the samples were run through a
gel filtration column to remove the extraliposomal probe, and
the radiolabel retained by the proteoliposomes was deter-
mined by liquid scintillation counting. The radiolabels used
were14C-sucrose and14C-maltose (Amersham Biosciences, St.
Louis, MO) and14C-ethyleneglycol (American Radiolabeled
Chemicals, Piscataway, NJ). Background from probe trapped
into compartments inaccessible for transport was assessed by
permeabilization with 0.1% DMSO as described (19).
Statistics. Data are presented as means ? SEM, and statistically
significant differences were assessed by Student’s t test for paired
or unpaired data, or one-way ANOVA, as appropriate.
was supported in part by National Institutes of Health Grants GM068586
and R21DC007150, American Heart Association Texas Affiliate Grant
0455115Y, and a grant from the John Sealy Memorial Fund for Bio-
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