From the Thomas C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Protonated aminosulfonate compounds directly
inhibit connexin channel activity. This was demonstrated by
pH-dependent connexin channel activity in Good's pH
buffers (MES (4-morpholineethanesulfonic acid), HEPES, and TAPS
(3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence
of pH-dependent channel activity in pH buffers without an
aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity
relation was shifted according to the pKa of each
aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on
connexin channel activity previously attributed to protonation of
connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific
for heteromeric connexin channels containing connexin-26; it does not
occur significantly for homomeric connexin-32 channels. The
identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to
facilitate understanding of cellular modulation of connexin channels
and lead to the development of reagents for use in structure-function studies of connexin protein.
Changes in intracellular pH
(pHi)1
affect gap junction conductance between cells (1). The sensitivity of
junctional conductance to changes in pHi varies with cell
type and connexin isoform (2-5). Decrease of pHi from
physiological levels typically produces a decrease in junctional
conductance (6-8) and in permeability to large tracers (9, 10). The
decrease in junctional conductance is usually reversible with return of
pHi to normal physiological values. The molecular
mechanisms that underlie this modulation of connexin channel activity
are unclear and may differ among connexin isoforms and cell types. It
has been proposed that the modulation is due to direct protonation of
connexin (8), changes in ionized calcium concentration (11), and
activation of calmodulin (12-14). For connexin-43 and for
connexin-32/connexin-38 chimerae, recent work strongly indicates a
pH-dependent interaction between segments of the C-terminal
domain and the single cytoplasmic loop that inhibits channel activity
(3, 15-21).
In this study, we set out to investigate modulation of connexin channel
activity as a function of pH, using in a reconstituted system connexin
channels immunoaffinity-purified from native tissues. Channel activity
was monitored using transport-specific fractionation (TSF) of liposomes
into which connexin channels were reconstituted.
Channel activity in this system was affected by changes in pH. To our
surprise, the changes in channel activity were accounted for by the
direct action of the protonated form of the aminosulfonate compounds
used as pH buffers, rather than of proton concentration itself. There
is no evidence for the direct action of pH alone on the activity of
these connexin channels in this system in the absence of aminosulfonate
compounds. The sensitivity to protonated aminosulfonates was connexin
isoform-specific. Heteromeric channels containing connexin-26 (Cx26) in
addition to connexin-32 (Cx32) were highly sensitive, whereas homomeric
Cx32 channels were not. Taken together with recent work by others on
the structural basis of pH sensitivity of connexin channels in cells,
these data suggest testable hypotheses for cellular and molecular
regulation of connexin channel activity. They also provide an
opportunity for development of pharmacological and affinity reagents
for structure-function studies of connexin channels. This is the first
report of a noncovalent modulatory activity of a biological molecule on
connexin channels (see Ref. 22). Preliminary reports of this work have
appeared in abstract form (23-25).
Materials
Egg phosphatidylcholine, bovine phosphatidylserine, azolectin
(soybean L-phosphatidylcholine), and lissamine rhodamine
B-labeled phosphatidylethanolamine were purchased from Avanti Polar
Lipids. N-Octyl-D-glucopyranoside
(octylglucoside) was from Calbiochem. Bio-Gel (A-0.5 m; exclusion
limit, 500,000 Da) was purchased from Bio-Rad. CNBr-activated Sepharose
beads were obtained from Amersham Pharmacia Biotech. 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. 26
and 27, with the modification that 5 mM EGTA was included
in the homogenization and phosphate buffers. Rat liver yields homomeric
Cx32, and mouse liver yields heteromeric Cx32/Cx26. Homomeric Cx26
channels were not available from a native tissue source; because Cx26
forms heteromeric channels with Cx32, such a source would have to lack Cx32. Homomeric Cx32 can be obtained from rat liver because the Cx26
content of rat liver is very low. There is no wild-type animal for
which the converse is true. It was occasionally possible to obtain
fractions of connexin from mouse liver that varied in Cx32/Cx26 ratio
by taking advantage of the finding that more Cx26 elutes from the
immunobeads (along with Cx32) in the initial fractions than in later
fractions. Except where noted, the heteromeric Cx32/Cx26 channels were
from pooled elution fractions.
Gel Electrophoresis, Protein Blots, and Immunoblots
Gel electrophoresis, blotting, and staining of blots were
carried out as described in Ref. 27.
Antibodies
The monoclonal antibody (M12.13) used in the immunoaffinity
purification and for specific staining of Cx32 on Western blots is
directed against a cytoplasmic domain of Cx32 (28).
pH Buffers and pKa Values
TAPS, HEPES, MES, taurine, and maleate were obtained from Sigma.
Because all TSF experiments were carried out at 37 °C, the pKa values for each compound used in calculations of protonated aminosulfonate concentrations were those either measured at
37 °C (TAPS, 6.0; HEPES, 7.1; MES, 8.1 (29)) or calculated from the
value at 25 °C and the measured enthalpy of ionization (taurine,
8.78 (30)). Unless otherwise noted, the pKa values
given in the text are those at 37 °C.
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. (26). Liposomes were formed by
gel filtration of a 1 mg/ml mixture of PC, PS, and rhodamine-labeled PE
at a molar ratio of 2:1:0.03 in urea buffer (see below) containing 80 mM octylglucoside and immunoaffinity-purified connexin. The size of the liposomes was monodisperse with an approximate mean diameter of 900 Å, shown by HPLC gel filtration (33). The
protein/lipid ratio of the liposomes was typically 1:60 (w/w),
corresponding to an amount of connexin equivalent to <~1 hemichannel
per liposome (see under "Data Analysis," below). Minor variation in
the amount of protein used, the amount damaged in purification, the
reconstitution efficiency, and the amount of lipid retained on the
column produced variations in the percentage of liposomes containing
active channels under standard conditions. The protein/lipid ratios
used yielded functional channels in 30-50% of the liposomes.
Transport-specific Fractionation
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. (26). The
principle of using a density shift to fractionate liposomes was adapted from Goldin and Rhoden (35). Liposomes containing functional channels
are separated from liposomes without functional channels by TSF
achieved by centrifugation through an isoosmotic density gradient
formed by urea and sucrose solutions. Urea buffer contained 10 mM KCl, 10 mM HEPES, 0.1 mM EDTA,
0.1 mM EGTA, 3 mM sodium azide, and 459 mM urea at pH 7.6. Sucrose buffer was identical except that
an osmotically equivalent concentration of sucrose (400 mM)
replaced the urea. Osmolality of urea and sucrose buffers was 500 mosmol/kg, and their specific gravities
(d420) 1.0056 and 1.0511.
An aliquot of liposomes was layered on each 4.4 ml gradient. Gradients
were centrifuged at 300,000 × g for 2-3 h in a
swinging bucket rotor (Sorvall TST 60.4) at 37 °C. Liposome bands
were recovered by aspiration. The distribution of the liposomes in the
gradient was calculated from the specific intensity of rhodamine fluorescence (Perkin-Elmer 650-10S or L550B spectrofluorometer; 560 nm
excitation; 590 nm emission) and the volume of each collected band.
During the centrifugation, liposomes without functional channels move
into the gradient a short distance, being buoyed by the (lighter)
entrapped urea buffer and form a band in the upper part of the
gradient. Liposomes with functional channels continuously equilibrate
their internal solution with the external solution and move to a
position in the lower part of the gradient corresponding to the density
of the liposome phospholipid membrane.
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. Calculations show that the assay is insensitive to
large changes in channel Po down to 0.001.
It is formally possible that a modulatory compound could affect the
proportion of liposomes that shift to the lower position by restricting
the diameter of the pores, rather than moving Po close to 0. However, for this to occur, the channels would have to
become impermeable to urea and to sucrose (liposomes permeable to urea
only move to an intermediate position (36, 37)). Such a change in
diameter would effectively eliminate the ability of connexin channels
to mediate molecular signaling between cells and therefore can be
regarded as a decrease in channel activity.
Data Analysis
Correction for More Than One Channel per Liposome--
Previous
work with the TSF system suggested that the channels distribute among
the liposomes in a manner described by the Poisson distribution (26).
This means that for a given protein-lipid ratio ( Normalization of TSF Data--
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. The
maximum value was almost always at the highest pH value for the series.
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 each buffer for
calculation of means and standard errors.
Curve Fitting--
The activity data was fit with a
four-parameter logistic function of the form f(x) = a/(1 + exp(b*(x Modeling of Heterogeneity--
Channel activity data was modeled
assuming that protonated taurine binds to one or more sites on a
connexin hemichannel to inhibit channel activity. The calculations
assume: 1) the pKa 37 °C of taurine
is 8.78 (30), 2) the Cx32/Cx26 channel population consists of five
equal subpopulations each having a different taurine dissociation
constant (Kd) or cooperativity
(nHill), and 3) channels with
Po < 0.001 are binned as closed by the TSF. At
each protonated taurine concentration the fraction of active channels
is summed over all the subpopulations. Because the number of
subpopulations, the way that the Kd or
nHill values are distributed, and the
Po cutoff value are not known, these calculations only demonstrate the general adequacy of the model rather
than provide estimates of binding parameters.
For each subpopulation of channels, a Po
versus [protonated taurine] relation was calculated from a
standard binding isotherm Kdn/(Kdn + [protonated taurine]n) where Kd is the
dissociation constant and n is nHill as defined above. The binning of the liposomes into active and inactive
populations by the TSF was simulated according to (3) above, giving a
step function in the activityTSF versus
[protonated taurine] relation for each subpopulation. For different
binding parameters the step occurs at different [protonated taurine]. Summing the activityTSF versus [protonated
taurine] step functions for each subpopulation produces an aggregate
activityTSF versus [protonated taurine]
relation that is a series of steps. The binding parameters
Kd or nHill were adjusted so
that the relation approximated the TSF data.
Connexin was immunopurified from octylglucoside-solubilized crude
plasma membranes from rat and mouse liver using a monoclonal antibody
specific for Cx32, as previously described and characterized (26, 27,
38). Earlier 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. The heteromeric channels are functionally heterogeneous
with regard to permeability to large molecules, presumably due to
heterogeneities of isoform stoichiometry and/or arrangement (27).
The activities of channels formed by Cx32 and Cx32/Cx26 were explored
and compared by TSF of liposomes. TSF has been well characterized (32,
34, 36) and effectively used in channel permeability studies (26, 27,
39, 40). In brief, connexin channels are incorporated into the
membranes of unilamellar liposomes. When centrifuged in an appropriate
isoosmolar density gradient, solute exchange through active channels
causes liposomes to become more dense and move to a position deep in
the gradient. Liposomes without active channels remain in the upper
part of the gradient. Any significant channel open probability
(Po) results in sufficient osmolyte exchange to
cause the change in density. Because the change in liposome density can
result from brief channel openings, only when Po
changes above or below a very low value are changes in channel activity
detected by TSF. TSF is therefore an essentially all-or-none assay of
per-liposome channel activity. The technique is more fully described
under "Experimental Procedures."
The effects of test compounds on channel activity were assessed by
exposing connexin-containing liposomes to the compounds during a TSF
spin. The change in distribution of liposomes between the upper and
lower positions, relative to a control gradient without the compound,
is a quantitative measure of the fractional change in activity of the
population of the channels.
Apparent pH Dependence of Activity of Heteromeric Cx32/Cx26
Channels--
To assess the effects of pH on immunopurified,
reconstituted connexin channels, activity was assessed in TSF gradients
in which the pH was adjusted by addition of strong acid or base. In
these initial studies, the reference condition was pH 7.6 in 10 mM HEPES. Activity was assessed at pH 5 and pH 9 and
normalized to that at the reference condition. Experiments were carried
out using homomeric Cx32, and two fractions of a population of
heteromeric Cx32/Cx26 channels with different ratios of Cx32 to Cx26.
The Cx32/Cx26 channels were sensitive to pH changes over this range, whereas the homomeric Cx32 channels were essentially insensitive (Fig.
1). Furthermore, the Cx32/Cx26 channel
population with the greater proportion of Cx26 was more sensitive than
that with less Cx26. Thus, pH sensitivity correlated with Cx26 content.
These results suggest that Cx26 is directly responsible for the
sensitivity to pH or indirectly confers pH sensitivity on the
heteromeric connexin channels. The pH-induced loss of activity with
decreased pH in this system is fully reversible, as it is in cells.
Aminosulfonate pH Buffers Directly Modulate Heteromeric Cx32/Cx26
Channels--
In the experiment shown in Fig. 1, the one-half maximal
effect was near the pKa of HEPES, the pH buffer in
the solutions. To test whether the pH sensitivity was buffer-specific,
other pH buffers were used in place of HEPES in the TSF solutions, all at 10 mM. In MES and TAPS, the Cx32/Cx26 channels showed pH
effects similar to those seen in HEPES. However, the pH range over
which channel activity declined was different for each pH buffer (Fig. 2, filled symbols). A smooth
function was fit to each data set to define half-maximum values for
activity in each buffer. It was found that the half-maximum activity
value in each buffer was displaced toward the pKa of
that buffer (see Fig. 3). Half-maximum
values for MES, HEPES, and TAPS were at pH 6.1, 7.3, and 8.5, respectively, close to the corresponding buffer pKa
values of 6.0, 7.3, and 8.1 (29, 30). (All TSF experiments were carried
out at 37 °C, and all pKa values given are at
that temperature.)
Furthermore, pH buffers that were not in this chemical family, such as
maleate and Tris (Fig. 2, open symbols) and bicarbonate (not
shown), elicited no change in channel activity over the same pH range.
The pH buffers in which activity was pH-dependent were all
Good buffers (41) and are all aminosulfonate compounds (Fig. 3).
Chemical precursors of the Good buffers include the non-aminosulfonates 2-bromoethanesulfonate, isethionate, and 2-propanesulfonate. These compounds, and the amino acid glycine, were tested and were without effect.
The data in Figs. 1 and 2 strongly suggest that the observed changes in
connexin channel activity are not due to protonation of connexin, but
to the action of protonated aminosulfonate compounds on connexin
channels that contain Cx26.
Protonated Aminosulfonates Are Connexin Channel
Inhibitors--
The data in Fig. 2 could be accounted for if either
the deprotonated aminosulfonate acts as a channel agonist or protonated aminosulfonate acts as a channel antagonist.
To help distinguish these possibilities, channel activity was assessed
at pH 7.6 in 50 mM rather than 10 mM HEPES. At
the higher concentration, channel activity was reduced to the level of
activity at 10 mM HEPES at low pH (i.e. when it
is fully protonated) (Fig. 2, asterisk). This indicates that
the protonated (zwitterionic) form of aminosulfonate buffers acts as a
channel antagonist, since in this experiment the concentrations of both
the protonated and deprotonated species were increased equally. This
point is made more rigorously in the following experiment.
Protonated Taurine Directly Modulates Connexin Channels--
The
amino acid taurine (2-aminoethanesulfonic acid), a chemical precursor
of the Good buffers, is an aminosulfonate and also can function as a pH
buffer. Activity of Cx32/Cx26 channels was assessed in 10 mM taurine solutions adjusted to pH values from 7 to 10.5 (Fig. 4A, open circles). A
smooth function fit to the data (curved line) shows that the
half-maximum value for activity modulation by taurine approximates the
pKa value for taurine, consistent with the findings
for the other aminosulfonate buffers (Fig. 2). When taurine
concentration was increased to 50 mM at pH 9.1, the channel
activity was reduced to the same level as at fully protonated 10 mM taurine (Fig. 4A, asterisk), as
was found for the parallel experiment using HEPES.
Channel activity is plotted as a function of protonated taurine
concentration in Fig. 4B. The concentration of protonated taurine can be controlled by taurine concentration as well as by pH.
Therefore, experiments were carried out at constant pH (at pH 8 and at
pH 6), but at taurine levels such that the resulting concentrations of
protonated taurine spanned the range achieved by changing the pH of 10 mM taurine from 7 to 10.5 (i.e. the same range
as in Fig. 4A). From the Henderson-Hasselbalch equation, this concentration range of protonated taurine is 9.84 mM
at pH 7 to 0.19 mM at pH 10.5. pH values of 6 and 8 were
chosen to represent extremes of physiological intracellular pH.
At both pH 6 and pH 8, Cx32/Cx26 channel activity was essentially the
same as that at the same protonated taurine concentrations when taurine
concentration was constant and pH was varied. Protonated taurine
concentration, and not pH, regulates heteromeric Cx32/Cx26 connexin
channel activity.
Cx32/Cx26 Population of Channels Is Heterogeneous in Affinity for
Taurine and/or Cooperativity of Taurine Effect--
Due to the
all-or-none nature of liposome fractionation in the TSF assay, it is
difficult to derive binding parameters from these studies. Classical
binding studies are equilibrium or kinetic measurements, whereas this
assay is neither: liposomes are binned by TSF according to whether the
channel(s) they contain has a Po value greater
than a threshold level. For this reason, under a given condition, a
homogeneous population of channels should either remain entirely at the
upper position or move entirely to the lower position, producing a step
change in the activity-[ligand] relation at the ligand concentration
at which Po becomes detectable by the TSF.
This is clearly not the case; intermediate values (e.g.
partial effects at intermediate protonated taurine concentrations) are
seen. This could arise from heterogeneity in the properties of the
channels. The Cx32/Cx26 channel population is known to be heterogeneous
with regard to molecular selectivity (27). This has been attributed to
channels in this population being composed of several isoform
stoichiometries and/or arrangements. This same structural heterogeneity
could give rise to heterogeneities in pharmacological sensitivity as
well. The summed activity contributions of several populations of
channels with distinct ligand sensitivities would produce the
intermediate channel activity data points seen.
The heterogeneity of the Cx32/Cx26 channels with regard to
aminosulfonate sensitivity was qualitatively verified by experiments utilizing different fractions eluted from the immunobeads during purification. We observed that more Cx26 elutes (along with Cx32) in
the initial fractions from the immunobeads than in later fractions. With care, it was sometimes possible to collect two fractions with
substantially different Cx32:Cx26 ratios (the data in Fig. 1 was
obtained in this manner). Using such fractions, it was established that
Cx26 content correlated with a rightward shift along the pH axis
(indicating greater sensitivity to protonated aminosulfonate), as well
as a greater fraction of the channels being pH-sensitive. Therefore,
the sigmoid relation between pH and TSF channel activity in Figs. 2 and
4A is not a titration curve, but rather the superposition of
responses of several channel forms with different properties. (For this
reason, it is inappropriate to fit the pH data with the Hill equation,
as it suggests erroneous notions of the underlying mechanisms. Instead,
a smooth logistic function was fit to the data solely to determine a
characteristic half-maximal channel activity parameter.)
To show that simple heterogeneities in binding parameters can account
for the findings, the channel activity data was modeled assuming that
the Cx32/Cx26 channel population was composed of several
subpopulations, each of which had a distinct dose-response relation for
inhibition by protonated taurine. The data could be accounted for by
the subpopulations differing in either affinity for taurine
(Kd) or cooperativity of taurine effect
(nHill) (see under "Experimental Procedures"
for details). The calculations are for an arbitrarily simple set of
assumptions and demonstrate only the general adequacy of the model,
suggesting possible ranges of Kd or
nHill for the given assumptions. The
calculations shown are for five equal subpopulations of channels, each
corresponding to a different Cx32/Cx26 stoichiometry (5:1, 4:2, 3:3,
2:4, 1:5; homomeric Cx32 and Cx26 channels are not present (27)).
Fig. 5A illustrates
calculations in which each subpopulation has a constant
Kd but different nHill
constrained to be between 1 and 5. A Kd that gives a
good fit for these assumptions is 3.2 mM. Fig.
5B illustrates calculations in which each subpopulation has
a different Kd and no cooperativity (nHill = 1). Values of Kd for
the illustrated fit to the data range between 1 and 10 µM. The TSF activity data can be accounted for by either
scheme; at present, one cannot distinguish between these two
pharmacological heterogeneities.
When Connexin Channels from Rat Liver Show Aminosulfonate
Sensitivity, It Is because They Contain Cx26--
On rare occasions, a
preparation of immunopurified connexin from rat liver showed a
substantial sensitivity to the aminosulfonate compounds. This
correlated with the presence of detectable amounts of copurified Cx26
on immunostained Western blots (Cx26 is not typically present in such
preparations). The copurification of Cx26 with Cx32 from rat liver
occurred rarely. It is not known what influences the levels of Cx26
that copurify in these preparations; it may result from variations in
the physiological state of the tissue or animal. This result suggests
that heteromeric Cx32/Cx26 channels can exist in rat liver. It also
indicates that the modulation of channel properties by
aminosulfonates that occurs for connexin from mouse liver can also
occur in rat liver when Cx26 levels are increased, as may occur
following hepatic trauma (42, 43).
The data presented here demonstrate a direct modulatory effect of
protonated aminosulfonates on connexin channel activity. The
significance of this finding is 2-fold: 1) it identifies for the first
time a class of cytoplasmic compounds that directly and reversibly
regulates connexin channel activity, and 2) it suggests a mechanism by
which changes in pHi can affect connexin channel activity.
The active compounds all share a common structural motif: a
protonatable amine moiety separated from an ionized sulfonate moiety by
two or three methylene groups. It is possible that compounds containing
other ionized sulfur-containing moieties (i.e., sulfinyl groups) would also be effective. The sensitivity to aminosulfonate depends on isoform composition of the channels, and requires that Cx26
be present.
The data suggest that at least some of the effects on connexin channels
that have been attributed to direct action of low pHi are
mediated instead by protonated aminosulfonates. The data provide no
evidence for an effect on connexin channel activity due to protonation
of connexin. It is possible that pH has direct effects not revealed in
this experimental system and that it can act directly on other
connexins. On the basis of this data one cannot assert that taurine is
a biological regulator of connexin channels (other cytoplasmic
aminosulfonates may have higher affinities and therefore act in
different pH ranges), only that it is present, and protonated, in cells
at levels sufficient to have an effect.
Site(s) of Aminosulfonate Action--
Because on the basis of size
it is likely that taurine can permeate connexin channels, sites
accessible from either the inside or outside of a liposome would be
accessible by external taurine, regardless of the orientation of the
protein in the membrane.
Taurine binds to many proteins, the most prominent of which are the
inhibitory glycine receptor (44-46) and amino acid transporters (47).
Although the structure of the taurine binding site on the glycine
receptor is not known, the most important sequence elements have been
defined (48). The site is thought to be formed by at least two
noncontiguous segments. The amino acid segments are: (a) an
aromatic-small-aromatic sequence (159-161; loop-2) common to the
ligand/gated ion channel family, and (b) a
cationic-X-aromatic-X-small sequence (200-204; loop-3). Inspection of
the amino acid sequences of Cx32 and Cx26 shows that they have four
loop-2-like motifs and three loop-3-like motifs (connexin sequence
alignments and numbering are from Ref. 49). All are in the putative
transmembrane and extracellular domains. However, Cx26 contains an
additional loop-2-like motif (YLF, 212-214) in its small C-terminal
cytoplasmic domain that Cx32 does not. Also, one of the loop-3-like
motifs of Cx26 (K·W·T, 22-26) partially extends into the
cytoplasmic N-terminal (NT) domain, unlike that of Cx32.
These similarities are not strong. However, if they are significant,
they suggest the possibility that both connexins may interact with
taurine in the pore and that the C-terminal (CT) region of Cx26, but
not Cx32, may interact with taurine, perhaps in concert with other
domains. If the latter is the case, a taurine binding site could be
composed of the CT domain (providing a loop-2-like motif) and the
region of the transition between the NT domain and first transmembrane
domain mentioned above (providing a loop-3-like motif). The NT-first
transmembrane domain transition is thought to be at the mouth of the
pore (50). The CT domain of Cx32 does not contain any of the motifs,
which, in this hypothesis, is the reason it is unresponsive to
aminosulfonate. Intriguingly, the CT domain of connexin-43 (which is
required for its pH sensitivity and can confer some pH sensitivity on
Cx32 (17)) contains two loop-2-like motifs, one of which is precisely
aligned with that of Cx26 (YVF, 230-232) and the other of which has
been positively identified as essential for pH-modulation (YAY,
265-267) (18).
It is thus possible that aminosulfonates regulate connexin channels by
occupying a binding site composed of a part of the CT domain and
another domain, perhaps at the NT-first transmembrane domain boundary.
The effect on channel activity could be due to occupancy of the site or
to the conformational changes caused by coordination of disparate parts
of the connexin molecule. These possibilities may be tested directly by
molecular biological approaches.
The amino acid sequences of several high affinity taurine transporters
have been determined, but the residues that interact with taurine have
not yet been identified (51-55). The affinities of these transporters
for taurine range from 4 to 40 µM, and there are several
regions of potentially significant homology with connexins. An
invertebrate odorant receptor has been recently shown to have two high
affinity binding sites for taurine (Kd values of 18 pM and 6 µM), but the amino acid sequence has
not yet been determined (56).
Nature of Aminosulfonate/Connexin Interaction--
The
calculations in Fig. 5 show how connexin channels with (a)
single binding sites of different affinities
(nHill = 1; Kd variable), or
(b) multiple binding sites of identical affinity but
different degrees of cooperativity (nHill
variable; Kd constant), could lead to
pH-dependent connexin channel activities consistent with
the data. A key element is the functional heterogeneity of the
heteromeric channel population, which has been established and seems to
arise from variation in isoform stoichiometry and/or arrangement (27).
The possibility of different subunit isoform stoichiometries or
arrangements suggests a basis for heterogeneity of ligand response. It
also suggests that cellular control of isoform composition could
control the responsiveness to aminosulfonates and changes in
pHi.
The relative potency of the aminosulfonates in affecting connexin
channels is indicated by the relation between the activity curves and
the pKa values. For all the aminosulfonates except
TAPS, the pH at which the effect was half-maximal in 10 mM
buffer was at or below the corresponding buffer pKa. For TAPS, the half-maximal pH was a full 0.4 pH unit basic relative to
the pKa. Thus, a lower concentration of protonated TAPS than the other compounds achieved the same effect. Inspection of
the molecular structures shows that TAPS is the only compound tested
with three methylene groups between the protonatable amine and the
ionized sulfonate moiety. It is possible that this structural difference contributes to the increased efficacy.
Why Are Some Heteromeric Cx32/Cx26 Channels Not
Aminosulfonate-sensitive?--
Not all Cx32/Cx26 channels show
sensitivity to protonated aminosulfonates over the range of
concentrations tested. When protonated aminosulfonate concentration was
increased to ~25 mM (experiments using 50 mM
HEPES or taurine near their pKa values), the
inhibition was no greater than that at 10 mM (experiments using 10 mM pH buffers at low pH). A possible explanation
is that a minimum number of Cx26 monomers must be present in the
hexameric channel for the channel to be sensitive to aminosulfonates.
The apparent aminosulfonate-insensitive component could arise in other
ways as well. If the model based on several Kd values applies, the apparently aminosulfonate-insensitive channels could represent a population of channels with substantially lower affinity for aminosulfonates. If the model based on several
cooperativities applies, the insensitive channels could correspond to a
population of channels with low nHill or
negative cooperativity. In either case, if the protonated
aminosulfonate concentration were to be increased substantially beyond
that used in this study, a greater fraction of the channels would be
expected to be inhibited.
Heterogeneity of binding parameters is most likely to arise from the
structural heterogeneity known to exist in this population of channels
but could additionally arise from differences in phosphorylation states
of Cx32 (Cx26 is not a phosphoprotein), or possibly from proteolytic
degradation of a domain responsible for mediating the action of
aminosulfonate binding.
Relation to Apparent pH Sensitivity of Connexin Channels in
Cells--
Cytoplasmic acidification is known to inhibit cell-cell
coupling (5). The molecular basis for this is not well understood, but
recent work in the paired Xenopus oocyte expression system suggests a molecular mechanism for pH sensitivity of connexin channels.
A proposed model for the pH sensitivity of Cx43 (4) is consistent with
the aminosulfonate interaction with heteromeric Cx32/Cx26 channels
described here. It involves a low pH-facilitated interaction of the
cytoplasmic Cx43 CT domain with a receptor domain elsewhere in the
connexin molecule that causes the channel to close. His95,
which is in the cytoplasmic loop (CL) of nearly all connexins, is
proposed to be part of the receptor (16). There could be a receptor in
each connexin monomer (i.e. six per hemichannel) or a single
receptor could be formed by parts of all six monomers.
In Xenopus, Cx32 channels are only minimally pH-sensitive
(3, 19), consistent with our data. From studies of chimerae of Cx32 and
Cx38 (which is more pH-sensitive than Cx32), a model of pH sensitivity
has been proposed (12) that is mechanistically similar to that proposed
for Cx43, though it differs in the details. It involves a receptor
domain in the N-terminal half of the CL that interacts competitively in
a pH-sensitive manner either with the C-terminal half of the CL to
close the channel or with a proximal region of the CT domain, which
does not close the channel. A Cx43 CT peptide can enhance the degree of
pH sensitivity of Cx32 channels, suggesting conservation of receptor
structure across connexin isoforms (17). However, Cx32 is in a
different subclass of connexins than Cx43 and Cx38 (57), so the
molecular mechanisms of pH sensitivity may differ.
In both models, protonation favors reflexive interactions between
noncontiguous domains of connexin molecules: a region of the CT domain
interacts with a receptor region of the CL in a pH-dependent manner to close the channels. Based on our
data, and the consensus view from the Xenopus system, we
propose two possible mechanisms.
One mechanism is that either protonated aminosulfonate or competent CT
domain (i.e. that of Cx43 or Cx38, but not Cx32) can bind to
the receptor domain to close the channel. The other is that to close
the channel a complex must form between all three elements: protonated
aminosulfonate, competent CT domain, and receptor. Each hypothesis is
consistent with the available data and has distinct consequences and
predictions (Fig. 6).
In cells, the first mechanism involves a competition between the
available CT domains and the available cytoplasmic protonated aminosulfonate. Interaction between the CT domain and receptor would be
pH-sensitive. An ineffective Cx32 CT domain would account for the
relative pH insensitivity of homomeric Cx32 channels, because it would
behave as a competitive blocker in binding to the receptor but not
effecting channel closure. The increased pH sensitivity with Cx26
content that we see would be due to the absence of a significant CT
domain on Cx26; with increased Cx26 in Cx32/Cx26 hexameric
hemichannels, the number of Cx32 CT domains present decreases, and
access to the receptor increases, permitting aminosulfonates in the
solution (or cytoplasm) to interact with the receptor and effect
closure. In this view, Cx26 subunits would either effectively decrease
the local Cx32 CT domain "blocker" concentration, and/or act as
"spacers" to alleviate steric crowding by Cx32 CT domain.
For the second mechanism, pH-dependent channel closure
requires both aminosulfonate and competent CT domain. The
aminosulfonate could interact directly with both other elements, or
could bind to one to enable binding of the resulting two-element
complex to the remaining element. In this case, the pH insensitivity of homomeric Cx32 would arise from an inability of the Cx32 CT domain to
form this complex, rather than from steric crowding or occupancy of the
receptor as above. In cells, pH sensitivity of Cx32 is enhanced by
co-expression of the Cx43 CT domain because it provides a competent CT
domain that can now effectively interact with both the receptor and
cytoplasmic aminosulfonate. For this mechanism to account for the pH
sensitivity of the Cx32/Cx26 channels in our studies, one must
postulate that the small Cx26 CT domain can participate in this complex
formation (perhaps via the loop-2-like motif mentioned above).
Both hypotheses predict that because Cx26 lacks a bulky CT domain,
channels formed by homomeric Cx26 in cells should be pH-sensitive (i.e. allow freer access to the receptor by cytoplasmic
modulators). The published Delmar model, which relies exclusively on a
full CT domain for pH sensitivity, if applied to Cx32 and Cx26,
predicts that homomeric Cx26 would be pH-insensitive. Recent
unpublished work from the Delmar group has established that homomeric
Cx26 channels are in fact highly
pH-sensitive,2 as our models predict.
Functional interaction between aminosulfonate and the CT domain
does not require that they act at the same site: the possibility of
homotropic or heterotropic cooperative linkages allows for interaction
of the effects of the ligands without requiring competition for the
same binding site. For either mechanism, "pH regulation" of
connexin channels between cells would be modified by application of
exogenous aminosulfonates.
In cells, CT domain-truncated Cx32 is not as pH-sensitive as Cx26 (58).
This suggests that for these two isoforms the properties of the
receptor domains differ, or the short CT domain of Cx26 plays a role
that the Cx32 CT domain cannot. If the former, the Cx32 CT domain could
be effective at a Cx43 or Cx26 receptor.
It is possible that the CT domain interacts with the connexin pore in a
manner analogous to that of N-type inactivation of potassium channels,
in which a single bound CT "particle" occludes the pore,
interacting with receptor regions from several subunits (59). For
potassium channels, one particle-receptor complex is sufficient to
block the channel. It is unlikely that a single taurine could serve the
same steric function in the wide connexin pore. Therefore, for the
analogy to hold, taurine molecules would bind to several subunits and
collectively occlude the pore. This would be consistent with
nHill > 1 and inconsistent with multiple affinities. On the other hand, a single taurine molecule could coordinate or permit binding of one or more CT domains to occlude the
pore, consistent with nHill = 1 and multiple affinities.
pH-mediated Sensitivity to Aminosulfonate Compounds in
Cells--
The demonstration that the naturally occurring amino acid
taurine directly modulates connexin channel activity is intriguing. Taurine has diverse biological functions in mammalian tissues. It is
found at relatively high cellular concentrations, up to 50 mM (25, 60). Cellular modulation of taurine concentration could directly modulate the activity of connexin channels (at physiological pH, essentially all taurine is protonated).
Presently, there is no evidence that taurine is an endogenous modulator
of connexin channels in cells. Other cytoplasmic aminosulfonates may be
more effective and therefore act over different ranges of pH. A host of
cytoplasmic compounds have structures that include the motif common to
the active agents. These include taurochloric acid, cysteic acid,
homocysteic acid, tauropine, taurocyamine, N-phosphotaurocyamine, N-phosphohypotaurocyamine,
taurolithocholate, homotaurine, and 5-glutamyl-taurine. Closely related
compounds include cysteinesulfinic acid, homocysteinesulfinic acid,
homohypotaurine, and 3-sulfinoalanine. The efficacies of these
compounds in inhibiting connexin channels are unknown.
There is evidence for changes in pHi under physiological
conditions that could affect the protonation of aminosulfonates with pKa values near resting pHi. For
example, with neuronal activity, glial pHi can undergo
substantial alkalization (up to 0.4 pH unit), followed by a rebound
acidification (up to 0.2 pH unit from resting levels) (61) as the
extracellular pH is regulated. This could produce substantial changes
in the level of a protonated modulator, which would presumably enhance
glial-glial coupling and spatial buffering during the acute phase of
neuronal activity. pH sensitivity of glial and neuronal gap junctions
in the nervous system is well established (10), and it is known that in
astrocytic gap junctions, Cx26 and Cx43, but not the pH-insensitive
Cx32, are present (62).
The direct aminosulfonate effect on connexin channels described here is
likely to both facilitate development of pharmacological tools for
study of connexin channels and lead to greater understanding of the
cellular mechanisms of intercellular communication.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
) in the liposomes,
a Poisson distribution accounts for the fraction of the liposomes that
have functional channels. When
is small, essentially all of the
liposomes in which there are channels contain exactly one channel.
However, as
increases, the fraction of liposomes with two or more
channels increases. When this is the case, a change in the fraction of
liposomes in the lower TSF band does not reflect exactly the change in
channel activity (e.g. liposomes with two channels will move
to the lower band unless both channels are closed by a given
concentration of ligand). This leads to an underestimate of the
inhibitory effect of a test condition, which was corrected in the
following manner:
was estimated from the maximum activity
(percentage of liposomes with active channels) for a given preparation
of liposomes. Using the Poisson distribution, this
was used to
calculate the distribution of channels in the liposome population,
which was used to compensate for the error introduced by some of the
liposomes containing more than one channel. This calculation transforms
the fraction of permeable liposomes in a population into an index
of discrete single channel activity.
c))) + d using the Marquardt-Levenberg algorithm.
A Hill equation was not used because the TSF-pH data does not arise
from a titration curve, but rather from the superimposition of
responses of an unknown number of channel forms with different
properties. For this reason, a smooth function was fit to the data to
determine a characteristic half-maximal channel activity parameter.
RESULTS
View larger version (21K):
[in a new window]
Fig. 1.
Channels containing Cx26 are pH-sensitive in
HEPES. Activity of channels containing various ratios of Cx26 to
Cx32 over a range of pHs were compared using the TSF system. Channel
activities were normalized to that at pH 7.6. Homomeric Cx32 channels
were nearly insensitive to changes in pH over the range 5-9.
Heteromeric Cx32/Cx26 channels were much more sensitive, with the
sensitivity correlating with Cx26 content. Western blots showed the
predominance of Cx32 in the low Cx26:Cx32 sample and the predominance
of Cx26 in the high Cx26:Cx32 sample.
View larger version (23K):
[in a new window]
Fig. 2.
Connexin channel activity as a function of pH
and pH buffer. Cx32/Cx26 channel activity was determined over a
range of pH in five buffer systems. All buffers were at 10 mM. Four parameter logistic fits show that for the
aminosulfonate buffers, the half-maximum values for connexin channel
activity were influenced by the pKa value of each
buffer (MES pKa = 6.0; HEPES pKa = 7.1; TAPS pKa = 8.1). There is no such trend, and
no obvious pH sensitivity, for the pH buffers that are not
aminosulfonates. When the concentration of HEPES was increased to 50 mM but pH kept at 7.6, the channel activity decreased to
the same minimal levels achieved at 10 mM HEPES and lower
pH (asterisk). This indicates that the protonated form of
HEPES acts as an inhibitor of channel activity. Channel activities for
each protein purification (n in inset legend)
were normalized to the maximum values within each data set. Average
activity for data in this figure was 43%, corresponding to ~0.6
functional channels per liposome ( ). Of the functional channels,
~60% were sensitive to aminosulfonates. Bars are
S.E.
View larger version (12K):
[in a new window]
Fig. 3.
Chemical structures of the pH buffers.
MES, HEPES, and TAPS are commonly used Good buffers (41). They share
the common structural motif of a protonatable amine moiety that is
separated from an ionized sulfonate moiety by two or three methylene
groups. This functional motif derives from the naturally occurring
precursor compound, the amino acid taurine.
View larger version (16K):
[in a new window]
Fig. 4.
Protonated taurine modulates connexin channel
activity. Activity of Cx32/Cx26 channels was determined in 10 mM taurine over a range of pH (open circles).
Channel activity was also determined at constant pH (pH 6, squares; pH 8, triangles) but at taurine
concentrations that resulted in levels of protonated taurine that
spanned the same range as when pH of 10 mM taurine was
varied. The channel activity was the same whether the concentration of
protonated taurine was controlled by changes in pH (open
symbols) or by changes in taurine concentration (solid
symbols). Bars are S.E. for six protein preparations.
In A, the data are plotted as function of pH. The
asterisk is the activity at 50 mM taurine at pH
9.1. The data obtained at constant pH (squares and
triangles) are plotted at the pH that corresponds to the
appropriate protonated taurine concentration. In B, the data
are plotted as a function of protonated taurine. Both curves are
four-parameter logistic functions fit to the open symbols. The changes
in channel activity are fully accounted for by changes in protonated
taurine concentration alone.
View larger version (17K):
[in a new window]
Fig. 5.
Modeling of the channel activity. The
relationship between protonated taurine concentration and channel
activity in the Cx32/Cx26 channel population was modeled as described
under "Experimental Procedures." Calculations are for five equal
subpopulations that differ in either Kd or
nHill. The open symbols are the means
of the taurine data presented in Fig. 4. A, calculations for
subpopulations with constant Kd and different
nHills, with 1 nHill
5. Fit to the data (solid
line) is for nHill values of 2.4, 2.8, 3, 4, and 5 and Kd of 3.2 mM. B,
calculations for subpopulations with nHill = 1 and different Kd values. Fit to the data
(solid line) is for Kd values of 1.3, 4.0, 5.0, 6.3, and 10 µM. The calculations only
demonstrate that the TSF activity data can be accounted for by the
model described in the text rather than providing estimates of binding
data. The particular values for nHill and
Kd used to generate these curves are not unique, and
they only suggest possible ranges. The inset in A
shows how the behavior of each subpopulation was modeled. The solid
curve is a binding isotherm
KdnHill/(KdnHill + [protonated taurine]nHill representing the relation
between channel activity and protonated taurine concentration. The
dotted line shows how the smooth relation is transformed by
the TSF into a step function (threshold Po is 0.1 in this
illustration). For the five subpopulations modeled here, the step
functions of the subpopulations are summed.
DISCUSSION
View larger version (38K):
[in a new window]
Fig. 6.
Two mechanisms for aminosulfonate
action. Two mechanisms for aminosulfonate action are diagrammed.
Left, aminosulfonate (e.g. taurine) and
C-terminal region of connexin (CT) independently interact
with a receptor domain in the CL to effect channel closure.
Right, aminosulfonate and the CT domain form a complex that
interacts with other connexin domains (e.g. the CL and/or
the N-terminal region (NT)) to effect channel closure.
![]() |
FOOTNOTES |
---|
* 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: pHi, intracellular pH; Cx, connexin; CT, C-terminal; NT, N-terminal; Po, channel open probability; TSF, transport-specific fractionation (of liposomes); CL, cytoplasmic loop; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydrox-ymethyl)ethyl]amino]-1-propanesulfonic acid.
2 M. Delmar, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|