From the Thomas C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218
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ABSTRACT |
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Connexin channels mediate molecular communication
between cells. However, positive identification of biological ligands
that directly and noncovalently modulate their activity has been
elusive. This study demonstrates a high affinity inhibition of connexin channels by the purine cyclic monophosphates cAMP and cGMP. Purified homomeric connexin-32 and heteromeric connexin-32/connexin-26 channels
were inhibited by exposure to nanomolar levels of the nucleotides prior
to incorporation into membranes. Access to the site of action, or
affinity for the nucleotides, was greatly reduced following
incorporation of the connexin channels into membranes, where inhibition
required millimolar concentrations of the nucleotides. The high
affinity inhibition did not occur with similar concentrations of AMP,
ADP, ATP, cTMP, or cCMP. This is the first report of a direct ligand
effect on connexin channel function. The high affinity and specificity
of the inhibition suggest a biological role in control of connexin
channels and also may lead to the application of affinity reagents to
study of connexin channel structure-function.
The gating of connexin channels by ligands is a key
uncharacterized element of intercellular signaling. Connexin channels mediate intercellular molecular signaling that is important in developmental and physiological contexts (1-3). However, the biological ligands that directly regulate connexin channel activity have been difficult to positively identify. Direct action on connexin channels is difficult to establish due to the intercellular channel structure and the cytoplasmic location of channel modulatory sites.
Junctional coupling between cells can be reduced by exposure of cells
to various compounds (reviewed in Refs. 4-6). In some cases, it is
known that the effects on junctional conductance are not due to direct
action of the applied compounds on the connexin channels, but to
cytoplasmic mediators (e.g. kinases, cf. Ref. 7).
However, in most cases the mechanism of action, and whether it is
direct or indirect, is not known. The most well documented direct
modulators of connexin channels are voltage (cf. Refs. 8-11) and phosphorylation (7, 12, 13), which can each alter substate
occupancy of certain connexins. However, voltage is not likely to be a
physiologically important regulator except in specific locations in
excitable tissues. Cytoplasmic acidification causes most cells to
uncouple, but this may not be a direct effect (14-16).
The identification of ligands of connexin channels is important for
understanding the physiology of intercellular signaling. It is also
important because it can lead to the use of affinity reagents to
explore the structure-function of connexins. The biophysical study of
connexin channels has been hindered by the absence of specific affinity
reagents or toxins. Such agents (e.g. tetrodotoxin and
charybdotoxin) have been vitally important in elucidating molecular
mechanisms of other channels (17, 18)
The studies reported here utilize connexin channels composed of
connexin-32 (Cx32)1 and
connexin-26 (Cx26) purified from native tissues by immunoaffinity chromatography. These two connexins have a high degree of sequence identity (62%) and are often found in the same cells and in the same
junctional plaques (19-23). Their close association implies a
functional and/or structural relationship in situ.
Compounds that alter junctional coupling when applied to cells were
tested for efficacy in a reconstituted system. A high affinity,
modulatory action of purine cyclic monophosphates, but not other
nucleotides, on connexin channel activity was discovered. The chemical
specificity and nanomolar affinity suggest a biological role.
Preliminary reports of this work have appeared in abstract form
(24-27).
Materials--
Egg phosphatidylcholine, bovine
phosphatidylserine, azolectin (soybean
L-phosphatidylcholine), and lissamine rhodamine B-labeled phosphatidylethanolamine were purchased from Avanti Polar Lipids. Tween
20, nitro blue tetrazolium, and diisopropylfluorophosphate were
obtained from Sigma. N-Octyl-D-glucopyranoside
(octylglucoside) was from Calbiochem. Bio-Gel (A-0.5 m; exclusion
limit, 500,000 Da) was purchased from Bio-Rad. Alkaline
phosphatase-conjugated goat anti-mouse IgG and
5-bromo-4-chloro-3-indolyl-phosphate were purchased from Boehringer
Mannheim Biochemicals. CNBr-activated Sepharose beads were obtained
from Amersham Pharmacia Biotech and Immobilon-P transfer membrane from
Millipore. Rats and mice were obtained from Taconic. Use and care of
animals was according to institutional guidelines.
Immunopurification of Connexin Proteins--
Connexin was
affinity-purified from an octylglucoside-solubilized crude membrane
fraction of rat or mouse liver using a monoclonal antibody against Cx32
as described in Refs. 28 and 29, with the modification that 5 mM EGTA was included in the homogenization and phosphate buffers.
Gel Electrophoresis, Protein Blots, and Immunoblots--
Samples
of liposomes for gel electrophoresis were prepared by extracting lipids
in absolute methanol and washing and concentrating the protein on a
30-kDa cutoff filter cartridge (Ultrafree, Millipore Corp.). Gel
electrophoresis, blotting, and staining of blots were carried out as
described in Ref. 29.
Antibodies--
The monoclonal antibody (M12.13) used in the
immunoaffinity purification and for specific staining of connexin-32 on
Western blots is directed against a cytoplasmic domain of Cx32
(30).
Reconstitution of Purified Connexin into Unilamellar Phospholipid
Liposomes--
Liposome formation and protein incorporation followed
the protocol of Mimms et al. (31) as modified by Harris
et al. (32) and Rhee et al. (28), and summarized
in the companion paper (33). To ensure that the potential modulatory
agents were available without dilution to the connexin until liposome
formation, a 4 ml volume of buffer containing the agents was loaded
onto the gel-filtration column before the liposome-forming solution was loaded.
Transport-specific Fractionation (TSF)--
The procedure used
to fractionate liposomes into two populations based on sucrose
permeability is described and fully characterized in Harris et
al. (32, 34) and Rhee et al. (28) and summarized in the
companion paper (33). Liposomes containing functional channels are
separated from liposomes without functional channels by TSF achieved by
centrifugation through an iso-osmotic density gradient formed by urea
and sucrose solutions. Equilibration of extraliposomal and
intraliposomal osmolytes is rapid (milliseconds for these 900-Å
diameter liposomes). Therefore, even a channel that opens only
infrequently for brief times will mediate full exchange of osmolytes
and cause liposome movement to the characteristic lower position. The
assay will not detect the activity of channels with
Po less than 0.001, so this is effectively an
all-or-none assay for channel function based on urea/sucrose permeability.
Data Analysis--
The studies of cyclic nucleotide binding
involve exposure of solubilized connexin channels to several ligand
concentrations. The ligand is essentially locked in place by
incorporation of the connexin into liposome membrane as detergent
diffuses away during reconstitution, and its effect is assessed as a
change in channel activity relative to controls without ligand.
Therefore the channel activity gives a quantitative assessment of the
fraction of channels to which ligand is bound at the moment of
reconstitution in membranes
Rather than looking at the aggregate response of a population of
receptors while association-dissociation reactions take place, this
assay captures those associations at a given instant. Thus, an
irreversible reaction (incorporation into a bilayer) is used to freeze
a reversible reaction (ligand binding) under steady-state conditions.
For each preparation of connexin, the percentage of liposomes in the
lower band of TSF data was normalized to the maximum value obtained for
that preparation. This enabled comparison of modulatory effects across
reconstitutions that produced different amounts of channel activity
(fractions of liposomes with functional channels). Where several
preparations were used, normalized data sets were combined for
calculation of means and standard errors.
Previous work with the TSF system suggested that the channels
distribute among the liposomes in a manner described by the Poisson
distribution (28). For a given protein-lipid ratio ( Connexin was immunopurified from rodent liver using a monoclonal
antibody that recognizes connexin-32. Previous biochemical and
functional studies have characterized connexin purified in this way
from rat liver as homomeric Cx32 hemichannels, and that from mouse
liver as heteromeric Cx32/Cx26 hemichannels (28, 29, 35).
The sensitivities of channels formed by Cx32 and Cx32/Cx26 to several
agents were explored and compared by transport-specific fractionation
(TSF) of liposomes. TSF has been well characterized (32, 34, 36) and
effectively used in studies of channel permeability (28, 29, 37).
Purified connexin is incorporated into unilamellar liposomes by gel
filtration of a mixture of octylglucoside-solubilized connexin and
lipid. TSF fractionates the liposomes on the basis of permeation of
osmolytes (urea and sucrose) through the reconstituted channels.
Specifically, it employs buoyant density sedimentation to separate into
distinct bands liposomes with open channels permeable to urea and
sucrose from liposomes that are without such channels. Liposomes that
do not contain open connexin channels (i.e., are not
permeable to urea and sucrose) migrate to an equilibrium position in
the upper part of the gradient. For liposomes that contain open
connexin channels, the osmolytes exchange through the channels and the
liposomes migrate to a lower position determined only by lipid density.
Any significant channel open probability (Po) results in sufficient osmolyte exchange to cause the required change in
density. The TSF is therefore an all-or-none assay of per-liposome
channel activity.
Effects of test compounds on channel activity were assessed by exposing
connexin or connexin-containing liposomes to the compounds prior to or
during a TSF spin. Effects on channel activity were quantified as
changes in the fraction of liposomes in the lower band relative to that
for connexin or liposomes not exposed to the test compound.
The fractional change in distribution of liposomes between the two
bands is a quantitative measure of the fractional change in activity of
the population of the channels. The change in liposome density can
result from brief channel openings, so only when
Po changes above or below a low threshold value
are changes in channel activity detected by TSF.
Many Compounds That Affect Junctional Communication in Cells Do Not
Have Effects on Connexin Channels in the TSF System--
In initial
studies, the effects of test compounds were assessed by including them
in the TSF solutions. The results are for both homomeric Cx32 channels
and heteromeric Cx32/Cx26 channels, except where otherwise indicated.
Table I lists compounds that can affect
junctional ionic current or dye transfer when applied to cells but that
were without effect in this system. Tables
II, III, and IV list additional
compounds that were also without
detectable effect. Absence of effect indicates that the actions of a
compound on connexin channels either (a) are not direct,
(b) are not dramatic enough to be detected by TSF, or
(c) require the intercellular channel structure (two
end-to-end hexameric hemichannels).
However, positive findings in this system indicate dramatic effects on
channel activity. When applied at 10 mM, but not 1 mM, to reconstituted hemichannels, cAMP inhibited channel
activity (Fig. 1). Activity was not
affected by 10 mM adenine or adenosine. By cytoplasmic
standards, 10 mM is a high concentration of cAMP, so a
sensitivity in this range is not likely to be biological relevant. We
speculated that an apparent low affinity for cAMP could arise from
restricted accessibility to a high affinity site. This could occur if
the binding site were partially obscured by membrane lipids or if the
reconstituted channel was only rarely in a conformation that enabled
access to the site.
High Affinity Inhibition of Solubilized Connexin Channels by cAMP
or cGMP--
A high affinity interaction between cyclic nucleotides
and connexin was established by studies in which connexin in
lipid-detergent micelles was exposed to the nucleotides. In pilot
studies of connexin channel permeation, connexin channels were exposed
to low amounts of cAMP or cGMP as permeability tracers in the presence
of detergent during liposome formation. Even though the nucleotides
were present at only micromolar levels, channel activity after
reconstitution was completely inhibited. This surprising effect was
investigated further: immunopurified connexin was exposed to
nucleotides while in detergent, then incorporated into liposomes, and
channel activity was assessed.
Exposure of purified connexin to as little as 1.5 nM cAMP
or cGMP completely eliminated the channel activity of subsequently reconstituted Cx32 or Cx32/Cx26 channels. This inhibition was not
reversible by increase of the salt in the TSF gradients to 100 mM, change of the TSF gradient pH to 5 or 9, or by omitting the nucleotides from the TSF gradient. Western blots established that
the presence of cyclic nucleotides during reconstitution had no effect
on incorporation of connexin into liposomes (Fig. 2). The inhibition of channel activity is
not due to the action of cyclic nucleotide-dependent
protein kinase as there was none present, nor was there ATP or divalent
cations.
No effect was seen with 1.5 nM AMP, ADP, ATP, cTMP, or
cCMP. Therefore this high affinity effect was specific for cyclic
purine monophosphates (cPMPs). This is the first evidence of a
modulatory high affinity binding site for a second messenger on
connexin channels.
Dose-response data of the cPMP effect were obtained by performing
reconstitutions using aliquots from a detergent-solubilized lipid and
connexin mixture containing different concentrations of cyclic
nucleotide (either cAMP or cGMP). The Hill equation was fitted to the
dose/response data (Fig. 3). Homomeric
Cx32 showed essentially identical high affinity inhibition by both cAMP
(Kd = 0.106 nM;
nHill = 2.1) and cGMP (Kd = 0.108 nM; nHill = 1.5). For
heteromeric Cx32/Cx26 channels, cAMP effects were higher affinity and
somewhat less cooperative (Kd = 0.054 nM; nHill = 1.1) than cGMP effects
(Kd = 0.15 nM;
nHill = 1.6). The homomeric Cx32 channels have
affinities for the two cyclic nucleotides intermediate to those of the
Cx32/Cx26 channels.
Exposure to 1.5 nM cyclic nucleotide fluorescent analogs
1,N6-etheno-cAMP and
2'-(N-methylanthraniloyl)-cGMP or to the photoaffinity reagent 8-azido-cAMP during detergent solubilization also completely inhibited both homomeric Cx32 and heteromeric Cx32/Cx26 channel activity.
Permeabilization of the liposome membranes by exposure to 5%
Me2SO or to octylglucoside levels below the critical
micelle concentration did not reverse the inhibition. Additionally,
attempts to induce the inhibition in reconstituted channels not
previously exposed to cyclic nucleotide by exposure of the liposomes to
10 µM cAMP or 8-(4-chlorophenylthio)-cAMP (a hydrophobic
analog of cAMP) in 5% Me2SO or octylglucoside levels below
the critical micelle concentration were not successful.
These experiments demonstrate the existence of a selective, high
affinity modulatory site for cyclic purine monophosphates on connexin
channels. The site is accessible while the protein is in micelles
and/or during its transition from micellar to bilayer environments, and
it becomes less accessible following incorporation of the channels into membranes.
The inhibition of channel activity by cPMPs could be achieved by
allosteric effects or physical occlusion of the pore. The latter is not
likely because cPMPs can permeate reconstituted Cx32 channels (29). The
much-reduced sensitivity of reconstituted channels may indicate that
after reconstitution the site is accessible only when the protein is in
a restricted set of conditions or conformational states. Alternatively,
the effects at millimolar levels could be due to a distinct, low
affinity site unrelated to the effects at nanomolar levels.
That activity inhibition persists in the absence of free nucleotide
after reconstitution suggests that once ligand is bound and the channel
is incorporated into a membrane, the ligand is precluded from
dissociation. This may occur by conformational changes secondary to
binding or to the transition to a membrane environment. Examples of the
former include the binding-dependent conformational change
that occurs in peptide binding by calmodulin (38, 39) and by the
molecular chaperone DnaK (40). Binding at an active site can involve
conformational changes that stabilize the bound complex (41, 42) or
render it inaccessible to bulk solvent (43). Receptor-ligand complexes
can have half-lives of days (44-47). Alternatively, dissociation of
ligand may be blocked by membrane lipids, where the bound ligand is
shielded from the aqueous phase by the phospholipid bilayer following reconstitution.
Cyclic nucleotides can permeate some junctional channels (48-52), as
well as hemichannels (29), so the inhibition at subnanomolar levels may
seem paradoxical. However, cytoplasmic concentrations of cyclic
nucleotides range between nanomolar and micromolar, well below the
millimolar levels required for effects on connexin channels
already in membranes.
Homomeric Cx32 and heteromeric Cx32/Cx26 have distinguishable but
similar affinities for the nucleotides, suggesting that the binding
sites for the nucleotides on the two connexins are different, but not
dramatically so. The activity of the cyclic nucleotide analogs may be
informative regarding the nature of the binding site. Because
1,N6-etheno-cAMP and 8-azido-cAMP are
derivatized on the purine ring, 2'-(N-methylanthraniloyl)-cGMP is derivatized on the ribose
sugar, and all three are effective at nanomolar levels, it appears that additions to the 2, 8, N6 and 2'-hydroxyl groups
do not preclude steric fit into the putative binding site.
Cyclic nucleotides directly modulate protein function in several
systems. The most well known class is the catabolite activator protein
(CAP) family of proteins, which includes the Escherichia coli CAP, cyclic-nucleotide dependent protein kinases, and
cyclic-nucleotide-gated ion channels (53, 54). However, Cx32 and Cx26
do not contain the amino acid sequences that are strictly conserved at
the cyclic nucleotide binding domains of the CAP family. Neither is
there obvious sequence similarity with the postulated cGMP binding
domain of cGMP-specific phosphodiesterases (55).
Unlike the CAP and phosphodiesterase families of cyclic nucleotide
receptors, which have micromolar affinities, the cAR1 and cAR3
chemoattractant receptors of Dictyostelium have nanomolar affinities for cyclic nucleotides (56), in the same range that we
determined for connexins, and mediate physiological responses at
subnanomolar levels (57). Further analogy with the cAR family is
provided by our finding that 2'-(N-methylanthraniloyl)-cGMP is just as effective as cGMP, suggesting that substitutions at the
ribose 2'-OH position do not interfere with binding to connexin. An
unsubstituted ribose 2'-OH group is required for binding of cGMP by the
CAP family of receptors (58) but not for cAR1 (59)
The amino acids involved in the cyclic nucleotide binding to cAR1 have
not yet been identified. However, a region of 26 amino acids
(148-173), thought to control access to the binding pocket (60, 61),
shows ~25% identity and ~60% homology with a region of Cx32 and
Cx26 that spans the cytoplasmic loop and the third transmembrane
domain. The same level of identity and homology extends through
the eight positions N-terminal to this sequence before dropping off.
This sequence similarity is only suggestive; further analogy with the
cAR family must await positive identification of the relevant amino
acids.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
it is a "snapshot" of binding. In this
way, it is formally analogous to a standard binding assay in which one
assesses the average fraction of receptors occupied for a given
concentration of ligand. The process is irreversible after
reconstitution up to the lifetime of functional channels in the
liposomes (~4 weeks).
) in the
liposomes, a Poisson distribution accounts for the fraction of the
liposomes that have functional channels. A statistical method was used
to correct for the fraction of liposomes that contained more than one
channel (described in the companion paper (33)). This calculation
compensates for the underestimate of the effect of a test compound
introduced by some of liposomes containing more than one channel,
transforming the fraction of permeable liposomes in a population to
accurately reflect discrete single channel activities.
RESULTS
Compounds affecting junctional coupling in cells that did not have
effects on connexin channel activity in the TSF system
Compounds that inhibit other ion channels that were without effect on
connexin channels in the TSF system
Divalent ions without effect on connexin channels in the TSF system
Miscellaneous compounds without effect on connexin channels in the TSF
system
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Fig. 1.
TSF gradients showing inhibition of channel
activity by cAMP. The left tube shows separation of
connexin-containing liposomes by TSF into two populations: one composed
of liposomes without functional channels (upper bands, upper
arrows) and one composed of liposomes with functional channels
(lower band, lower arrow). The liposomes without open
channels come to a position in the upper part of the tube because the
more dense lipid is buoyed up by the entrapped urea solution of lighter
density. Liposomes with open channels equilibrate the intraliposomal
solution with the external solution and move to a lower position
determined solely by lipid density. Liposome membranes contain a trace
amount of rhodamine-PE. The right tube shows the effect on
the same population of liposomes of 10 mM cAMP in the
gradient. Channel activity was completely inhibited, resulting in only
a single band, at the upper position.
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Fig. 2.
Western blots showing no effect of exposure
to cAMP during reconstitution on subsequent incorporation of connexin
into liposomes. A, protein incorporated into liposomes
without exposure to cAMP. B, protein incorporated into
liposomes in the presence of 100 µM cAMP. Both lanes were
immunostained with a monoclonal antibody against Cx32 (30) and
developed with alkaline phosphatase. Exposure of the connexin to a cAMP
concentration 5 orders of magnitude greater than that required to
inhibit channel activity (1 nM) did not affect
incorporation of the connexin into liposome membrane.
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Fig. 3.
Dose-response relations for cAMP and
cGMP. Connexin was reconstituted in a range of concentrations of
cAMP (triangles) or cGMP (circles). Nucleotide
was not present in the TSF gradients. Data were fit using the Hill
equation. Bars are S.E. A, homomeric Cx32
channels had essentially identical responses to cAMP and to cGMP.
B, heteromeric Cx32/Cx26 channels were more sensitive to
cAMP than to cGMP. For both types of connexin channels, binding of
nucleotide inhibited subsequent channel activity and appears to involve
more than one binding site per channel.
DISCUSSION
Hill coefficients of between 1 and 2 for the cPMP effects suggest that there could be two independent binding sites or more than two cooperative sites per channel. Because all six Cx32 subunits comprising a homomeric hemichannel have identical sequences, there are potentially six binding sites per hemichannel. On the other hand, it is conceivable that the sites of action are within the pore and altered by the micelle-membrane transition.
The fact that connexin channels have subnanomolar specific affinities for cAMP and cGMP, and that binding of these cyclic nucleotides closes connexin channels, is likely to be of physiological importance. One possible function is to keep hemichannels closed while they are in Golgi or endoplasmic reticulum membranes. The binding site may be accessible during initial membrane insertion and/or channel assembly, when cytoplasmic cPMPs could bind and thus ensure that connexin hemichannels remain closed during trafficking to the plasma membrane. Docking of apposed hemichannels to form the full intercellular channel could cause dissociation of these nucleotides from connexin channels.
Connexin-32 is co-translationally inserted into endoplasmic reticulum membrane (62, 63). Intriguingly, it has been shown that during co-translational insertion of polytopic membrane proteins, the transmembrane segments inserted through the endoplasmic reticulum membrane are stabilized in a salt-accessible compartment, apparently not interacting directly with lipid (64, 65). Thus, regions of a folded membrane protein that will be later blocked from aqueous access by membrane lipid appear to be transiently accessible to cytoplasmic components.
In view of this, we favor the view that both the micellar environment in our experiments and the partially unfolded state of the protein as it is inserted into a bilayer renders the cPMP site(s) accessible. NMR studies show that nondenaturing detergent such that used in these studies increase molecular motion of and accessibility to protein domains that are exposed to lipid when in bilayers (66, 67). Specifically, there is particularly enhanced accessibility to residues at the membrane-water transition (68, 69). There is evidence that nondenaturing detergents do not fully coat the hydrophobic surfaces of proteins (unlike SDS), leaving a somewhat open structure (70, 71) accessible to small, hydrophilic ligands.
Furthermore, the predicted number of residues for connexin transmembrane helices suggests that the hydrophobic core of the protein is relatively short, composed of five helical turns (28 Å) (72) rather than the six or more typical of channel proteins (73). There may be special lipid requirements for connexin channels in cell membranes to accommodate this. The lipid environment in the present studies is different from that in cell membranes, and may occlude sites that are accessible in them. Differences in the lipid environment may affect accessibility to binding sites, as well as other aspects of channel function (74-78).
This work address two long-standing fundamental issues of
connexin channel function. One is the identification and mechanisms of
biological ligands that control connexin channel activity. The other is
the identification of specific affinity reagents with which to probe
the structure-function of connexin channels. These new findings,
showing that connexin channels can be directly regulated by a
cytoplasmic ligand, are likely to lead to structure-function studies of
connexin channels and better understanding of the molecular basis for
regulation of intercellular signaling.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM36044 and Johns Hopkins University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Structural Biology, Max-Planck-Institut
für Biophysik, Kennedyallee 70, D-60596 Frankfurt am Main, Federal Republic of Germany.
§ To whom correspondence should be addressed: Dept. of Pharmacology & Physiology, New Jersey Medical School, UMDNJ, 185 S. Orange Ave., University Heights, Newark, NJ 07103.
The abbreviations used are: Cx, connexin; CAP, catabolite activator protein; cPMPs, cyclic purine monophosphates cAMP and cGMP; Po, channel open probability; TSF, transport-specific fractionation (of liposomes).
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