Bioscience Reports, Vol. 15, No. 5, 1995
REVIEW
Transmembrane C a 2+ Gradient and
Function of Membrane Proteins
F. Y. Yang, 1'2 Y. G. Huang, 1 and Y. P. Tu ~
Received September 14, 1995
This review will focus on the recent advance in the study of effect of transmembrane Ca 2+ gradient on
the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca z§ gradient and
sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of u:ansmembrane Ca 2+ gradient on the components
and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper
transmembrane Ca 2+ gradient may play an important role in modulating the conformation and
activity of SR CaZ+-ATPase and the function of membrane proteins involved in the cAMP signal
transduction by mediating the physical state change of the membrane phospholipids.
KEY WORDS: Ca2+-ATPase; cAMP signal transduction; transmembrane Ca 2§ gradient; lipid
fluidity.
Cai, Ca 2+ inside vesicles; Cao Ca z-- outside vesicles; SR, sarcoplasmic reticulum;
PC, phosphatidylcholine; PS, phosphatidylserine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; DPH, 1,6-diphenyl-l,3,5-hexatriene; n-AS, n-(9-anthroyloxy) fatty acids; TMADPH,
1-(4-trimethylammoniumphenyl)-6)-phenyl-l,3,5-hexatriene;
FCCP
carbonylcyanide-ptrifluoromethoxyphenylhydrazone; /3-AR,§
receptors; DHA, dihydroalprenolol; AC,
adenytate cyclase; AC.Lca+ - , higher Ca 2 inside vesicles; AC.Lca- - , lower Ca 2 on both side of
vesicles; AC.Lca+ +, higher Ca 2+ on both side of vesicles; AC.Lca- +, higher Ca 2+ outside vesicles;
cAMP, cyclic adenosine monophosphate; Gs, stimulatory GTP-binding protein; GTP, guanosine
triposphate; GTP~S, guanosine 5'O-(3-thiotriphosphate).
ABBREVIATIONS:
INTRODUCTION
The cytosolic free C a 2+ in most cells is about 10 -6 M, whereas the extracullular
Ca 2+ concentration is around 10-3M and Ca 2+ inside sarcoplasmic reticulum
(SR) is also 1 0 - 3 - 1 0 -2 M. So, it results 1,000-10,000 fold transmembrane Ca 2+
gradient across the plasma membrane [1] or SR membrane [2]. It is well known
that the maintenance of such concentration gradient not only provides the
molecular basis of C a 2+ to act as an intracellular messenger but also initiates cell
migration, exocytosis, lymphocyte kill cell activity, acid secretion, transceullar ion
1National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 100101
Beijing, China.
2 To whom correspondence should be addressed.
351
0144-8463/95/1000-0351$07.50/09 1995PlenumPublishingCorporation
352
Yang, Huangand Tu
transport, neurotransmitter release, gap junction regulation and numerous other
functions [3, 4]. Generally, more attention has been paid to the change in the
activities of cytosolic protein kinases in the consequence of increase in cytosolic
Ca 2+ concentration, while the effect of transmembrane caa+ gradient and its
change on the conformation and activity of transmembrane proteins was more or
less neglected.
In recent years the focus of' our laboratory has been on the effect of
transmembrane Ca 2§ gradient on the function of membrane proteins. By
membrane resolution and reconstitution, we have found that a proper transmembrane Ca 2§ gradient may play an important role in regulating activity and
conformation of membrane proteins, e.g. SR Ca2+-ATPase, erythrocyte Band 3,
glucose transporter and membrane proteins that are related to signal transduction, e.g. adenylate cyclase (AC), stimulatory GTP-binding protein (Gs) and
receptors. This review consists of two parts: 1. Effect of transmembrane Ca 2§
gradient on the enzyme activity and conformation of sarcoplasmic reticulum
Ca2+-ATPase; 2. Effect of transmembrane Ca 2§ gradient on the components and
coupling of cAMP signal transduction pathway.
TRANSMEMBRANE Ca2+ GRADIENT AND SARCOPLASMIC
RETICULUM Ca2+-ATPase (SR Ca2+-ATPase)
It is well known that Ca2§
plays an important role in the control of
contractile activity of muscle cells, and the function of Ca2+-ATPase can be
inhibited by the higher Ca a-- concentration inside SR [5, 6]. During contractionrelaxation cycle of muscle cells, there is a change of transmembrane Ca 2+
gradient in consequence of release Ca 2§ or uptake of Ca 2+ across SR membrane.
In order to explore the role of transmembrane Ca 2+ gradient in the modulation of
SRCa2+-ATPase, the conformation and activity of SRCa2+-ATPase in SR
vesicles or proteoliposomes with or without transmembrane Ca 2+ gradient were
determined and compared.
Alteration of Lipid Fluidity and Ezyme Activity of SR Ca2+-ATPase Containing Proteoliposomes in Consequence of Change of Transmembrane
Ca2+ gradient
By dialysis method, four types of SR Ca2+-ATPase-containing vesicles with
or without transmembrane Ca 2+ gradient were reformed with ATP-dependent
Ca 2+ transport activity. The enzyme activities and lipid fluidity of these
Ca2+-ATPase-containing proteoliposomes were determined and compared [7, 8].
From Table 1, it could be seen that the highest enzyme activity was observed in
the case of proteoliposome A (100/xMCa 2+ on both side, without transmembrane Ca z+ gradient), which is similar to the physiological situation when
Ca 2§ releases from SR and results in the activation of Ca2§
If there
existed transmembrane Ca 2+ gradient, a lower enzyme activity would appear.
And, for the enzyme activities of A, B and C the higher transmembrane Ca z+
Transmembrane Ca 2§ Gradient and Function of Membrane Proteins
353
Table 1. Effect of transmembrane Ca 2§ gradient on enzyme activity and lipid fluidity of
CaZ+-ATPase-containing proteoliposomes
Proteoliposomes
(Ca~:Cao) 1
A
(100:100)
B
(100:1)
C
(100:1)
ATP hydrolysis2
C a 2+ uptake 3
DPH polarization
4.50
0.40
0.157 • 0.002
1.30
0.13
0.166 • 0.001
D
(100:1000)
0.30
0.06
0.175 + 0.002
1 Ca~: Cao =/zM:/zM, 2/*mol/min - mg protein, 3 initial rate (/z mol/min 9
0.20
0.04
0.182 + 0.003
protein).
gradient was, the lower enzyme activity appeared, especially for C (with 1000 fold
transmembrane Ca 2§ gradient), only 7% of enzyme activity of A was observed.
Furthermore, the Ca 2§ uptake of proteoliposomes with different transmembrane Ca 2§ gradient was also compared. The results obtained were similar to
that of ATP hydrolysis activities.
If transmembrane Ca 2+ gradient was dissipated by A23187, enzyme activity
of proteoliposome with Ca 2§ gradient would increase and hence the difference of
enzyme activity in above-mentioned Ca2+-ATPase proteoliposomes would be
diminished [7].
By measuring the polarization of DPH (Table 1) it is interesting to note that
the fluidity of four types of Ca2+-ATPase-containing vesicles decreases in the
same order as the enzyme activities. So, it seems conceivable that the modulation
of SR Ca2+-ATPase by transmembrane Ca 2§ gradient may be correlated with
changes of the physical state of phospholipids.
Transmembrane Ca2+ gradient-mediated Conformation Changes of
Sarcoplasmic Reticulum Ca2+-ATPase
To understand the molecular mechanism of transmembrane C a 2+ gradientmediated modulation of SR Ca2+-ATPase in detail, fluorescence technique and
selective quenching of Trps were applied to the study of transmembrane Ca 2§
gradient-mediated conformational changes of SR Ca2§
The steady-state intrinsic fluorescence spectra of SR vesicles indicated that 1000
fold transmembrane Ca 2§ gradient could result in a reproducible 8% decrease in
intrinsic fluorescence of SR vesicles [9].
Time-resolved fluorescence measurements (Table 2) showed that 13
Table 2. Effect of transmembrane Ca 2§ gradient on life times of
Trp residues in SE Ca2--ATPase
Life times (ns)
SR (Cai:Cao) 1
SR (50:50)
SR (5000:5)
rl (fl)
T2(f2)
~3(f3)
5.50 (0.51)
5.95 (0.53)
2.65 (0.41)
2.75 (0.40)
0.80 (0.08)
0.75 (0.07)
The data present are average of 6 experiments, the standard error is
less than 0.10ns. f is the proportion of each component.
1 Cai:Cao =/zM:/zM.
354
Yang, Huang and Tu
tryptophan residues in SRCa2§
[10, 11] could be divided into three
groups and only the lifetime r~ obviously increased from 5.50 ns to 5.95 ns in
consequence of establishment of 1000 fold transmembrane Ca 2+ gradient. It could
be deduced that this group of Trps, located close to the lipid/protein interface
[12, 13], may be more closely related to the transmembrane Ca z§ gradientmediated conformational changes of SR Ca2§
Hydrophobic hypocrellin B(HB) is peryloquinone derivative which has been
reported to act as a very efficient collisional quencher of Trp residues embedded
in the hydrophobic domain of membrane proteins [14-16]. The results of dynamic
quenching of SR Ca2§
by HB showed that only the quenching of rl by
HB is different in the presence and absence of transmembrane Ca 2§ gradient
while no obvious difference was observed for ~2 and r3 [9]. This result gives us
another indication that the change of environment of the group of Trps with
longest lifetime (Zl) is the characteristics of conformational change of SR Ca 2+ATPase induced by transmembrane Ca 2§ gradient and the establishment of
transmembrane Ca 2§ gradient would lead to these Trps more accessible to HB
[9]. If this group of Trps are thought to be located on the helical segments
spanning the membrane close to the lipid/protein interface [12, 13] and are
closely related to the function of SR Ca2§
the inhibition of its activity by
transmembrane Ca 2§ gradient may be the consequence of change in flexibiltiy of
those transmembrane helixes induced by alteration of lipid physical state. So, in
the following, the role of phospholipids in such a modulation will be further
studied.
PC Plays a Crucial Role in Transmembrane Ca 2+ gradient-mediated
Modulation of SR Ca2+-ATPase
PC is the major phospholipid of SR membrane but its role remains to be
elucidated, especially in the modulation of SRCa2§
by the transmembrane Ca z+ gradient. Thus, the purified Ca2+-ATPase was reconstituted with
different phospholipid mixtures: PC:PE (1 : 1), PS :PE(1 : 1) and PG :PE(1 : 1). The
Ca2§
proteoliposomes prepared were with sufficiently low
Ca 2§ permeability.
Figure 1 showed that reconstitution of Ca2+-ATPase with PC:PE was
associated with a significant inhibition of ATP hydrolysis activity by the
transmembrane Ca :+ gradient (1 mM Ca a§ inside, 50/zM Ca 2§ outside,
Ca1 :Cao = 100:50), while little or no inhibition could be observed in the case of
PS:PE or P G : P E proteoliposomes [7]. And, the highest inhibition effect was
obtained at 50:50 molar ratio of PC:PE. If proteoliposomes contained 25% or
75% of PC, the inhibition effect was very low. This result clearly showed that a
proper percentage of PC (about 50%) is essential for the modulation of
SR Ca:§
by transmembrane Ca 2§ gradient. Similar difference in Ca 2+
uptake of these vesicles was also observed [17].
Furthermore, by comparing the effect of PC differing in acyl chains, higher
inhibition of Ca2§
is observed in vesicles containing DPPC:PE and
DOPC:PE, while no inhibition in DMPC:PE vesicles [17]. It should be noticed
Transmembrane Ca 2§ Gradient and Function of Membrane Proteins
355
4
~
im
v
~
,v-I
1
4.t
a
o
2
2
i
2
1
9 .......
- - ...;
iiii!!iiiiiiiii
PC:PE
PS:PE
PG:PE
Fig. 1. Role of PC in the modulation of ATP hydrolysis activity of SR Ca 2§
ATPase by transmembrane Ca 2-- gradient. 1, Ca~:Cao=501zM:50/zM; 2,
Cai: Ca o = 1000/xM :50/xM.
that most of PC in SR reported by Vrbjar et aL [18] was DPPC (41.5%), followed
by DOPC (21%) and DMPC (only about 1%). So, it could be deduced that under
the physiological condition, DPPC and DOPC are not only a barrier preventing
higher concentration of Ca 2+ from leaking out of SR, but also involved in the
modulation of Ca2+-ATPase by transmembrane Ca 2+ gradient.
T r a n s m e m b r a n e Ca 2+ gradient-mediated Change of Fluidity in the Inner Layer
of Phospholipids Modulates Sarcoplasmic Reticulum Ca2+-ATPase
It is well known that the distibution of the phospholipids in SR membrane is
asymmetrical: PC and acidic phospholipids (PS and PI) are mainly distributed in
the inner layer of the membrane, while 70% of PE is in the outer layer [19].
Based on the above-mentioned results it was suspected that such asymmetrical
distribution might be responsible for the modulation of SR Ca2+-ATPase by the
transmembrane Ca z+ gradient and PC which mainly distributes in the inner layer
of SR membrane, may play a crucial role in such a modulation. So, it seems
interesting to see whether there is any difference in the lipid fluidity of both in
outer and inner layer of Ca2+-ATPase-containing SR vesicles with or without
transmembrane Ca 2+ gradient [20].
D P H is hydrophobic and its degree of fluorescence polarization may reflect
Yang, Huang and Tu
356
Table 3. Fluorescencepolarization (P) of DPH or n-AS (2-AS, 7-AS and 12-AS) in SR
vesicles with or without transmembrane Ca2§ gradient
Fluorescence polarization (P)
SR
(Cai: Cao)1
50:50
5000:5
DPH
2-AS
7-AS
12-AS
0.156 + 0.002
0.163 :~ 0.002
0.167 -4-0.001
0.168 • 0.001
0.217 + 0.001
0.206 • 0.003
0.152 • 0.002
0.150 • 0.001
Fluorescence polarization was measured at 30~ the excitation and emission wavelength
were 360 and 430 nm for DPH, 365 and 440 nm for n-AS. Each number represents an
average of results from five experiments. The t-test for DPH showed p<0.01.
1Cai:Ca~ =/zM:/zM.
the average motion and viscosity of lipid molecules in both layers of SR
membrane. From Table 3 it could be seen that the average fluidity of SR
m e m b r a n e was lower in SR vesicles with a 1000 folds of transmembrane Ca 2§
gradient.
Amphiphatic molecules n-AS (2-AS, 7-AS, 12-AS), intercalated in the outer
phospholipid layer of SR membrane, were used for the measurement of the
fluidity of outer layer of SR membrane. Contrary ot the results obtained using
D P H , there was no obvious difference in the fluidity of SR membrane with or
without 1000 folds of transmembrane Ca z§ gradient (Table 3). These suggested
that the decrease in the average fluidity of whole SR m e m b r a n e resulting from
establishment of 1000 folds of transmembrane Ca 2+ gradient was mainly in
consequence of the change in physical state of phospholipids in the inner layer of
SR membrane.
It was reported that cationic T M A - D P H , could be used to monitor the
asymmetry of plasma membrane fluidity, especially the change of lipid fluidity in
the inner layer of membrane [21]. As acidic phospholipids (PS and PI) of SR
m e m b r a n e were mainly distributed in the inner layer, theoretically cationic
T M A - D P H should become bound preferentially to the inner layer of SR
membrane. Therefore, the difference of the fluorescence lifetime of T M A - D P H in
SR m e m b r a n e with or without transmembrane Ca e+ gradient would mainly reflect
the change of physical state of phospholipids in the inner layer of SR membrane.
Our results showed that the average lifetime of T M A - D P H decreased from
4.5 + 0.1 ns to 3.8 4- 0.2 ns in the case of 1000 folds transmembrane Ca 2§ gradient.
This could be deduced that the establishment of transmembrane Ca 2+ gradient
would result in change in the physical state of phospholipids of SR membrane
especially the decrease of lipid fluidity in the inner layer of SR membrane.
Transmembrane Ca 2+ gradient-mediated Modulation of SR Ca 2+ ATPase is not
Closely Related to the Change of Membrane Potential
Zimniak and Navarro have reported that m e m b r a n e potential had an
important influence on the enzymatic activity of reconstituted SR CaZ+-ATPase
[22,23]. As ionophore A23187 and FCCP could dissipate Ca a§ gradient and
m e m b r a n e potential respectively [24], these two probes were used to determine
Transmembrane Ca 2+ Gradient and Function of Membrane Proteins
357
Table 4. Change of enzyme activity of SR Ca2+-ATPase-incorporated
proteoliposomes in consequence of the dissipation of Ca2§ gradient or
membrane potential
Enzyme activity (/xmol/min 9mg protein)
Proteoliposomes
(Ca~:Cao=/zM:/xM)
1000:50
Control
0.072
+A23187
+FCCP
1.25
0.65
whether transmembrane Ca 2§ gradient-mediated modulation of SR Ca2+-ATPase
could be ascribed to the consequence of membrane potential resulting from Ca 2+
gradient [25].
From Table 4, it could be seen that the dissipation of transmembrane Ca 2+
gradient by A23187 would result in 70% increase of enzymatic activity of
Ca2§
vesicles while FCCP exhibited almost no effect. This
result clearly showed that the change of membrane potential is not closely related
with transmembrane Ca 2§ gradient-mediated modulation of SR Ca2+-ATPase.
Furthermore, our results also indicated that instead of Ca 2§ transmembrane
Sr 2§ gradient exhibited no effect on the enzyme activity of SR Ca2§
[25].
This might provide another evidence that transmembrane Ca 2+ gradient-mediated
modulation of SR Ca2+-ATPase could not be ascribed to the change of membrane
potential.
Summing up, it may be deduced that during the contraction-relaxation cycle
of muscle cells, in consequence of release of stored Ca 2§ through the channel, a
decrease in transmembrane Ca 2§ gradient will be occurred, resulting in an
increase of membrane fluidity, followed by activating of Ca2+-ATPase, which will
uptake Ca 2§ back into SR and reestablish a Ca z§ gradient. This leads to a
decrease in lipid fluidity, and hence a conformation change of Ca2+-ATPase with
concommitant inhibition of its activity. It seems that in addition to the direct
effect of Ca 2§ on SR Ca2§
the transmembrane Ca 2§ gradient-mediated
change in fluidity of phospholipids (mainly the inner layer of membrane) may also
be involved in such modulation process. And, PC, mainly located in the inner
layer of SR membrane, plays an important role in the modulation of A R Ca 2§
ATPase by the transmembrane Ca 2§ gradient.
E F F E C T O F T R A N S M E M B R A N E Ca 2§ G R A D I E N T O N T H E c A M P
SIGNAL TRANSDUCTION PATHWAY
cAMP signal transduction system consists of three distinct types of
membrane-bound proteins: adenylate cyclase (AC), stimulatory GTP-binding
proteins (Gs) and the receptor itself [26]. In order to examine the influence of
transmembrane Ca 2§ gradient, firstly, each of the purified components, /3
adrenergic receptor (/3-AR) [27], adenylate cyclase (AC) [28] and Gs [29], was
reconstituted on asolectin liposomes separately. Then, AC, Gs and /3-AR were
co-reconstituted on asolectin vesicles with (1-1000fold) or without transmembrane Ca 2+ gradient [30]. Activity and conformational change of /3-AR,
358
Yang, Huang and Tu
AC, Gs + AC or AC + Gs +/3 + AR-incorporating proteoliposomes were monitored by biochemical and biophysical approaches.
Effect of Transmembrane Ca 2§ gradient on Ligand Binding of the fl -ARcontaining Proteoliposomes
The initial step in any receptor-mediated signal transduction is the recognition and binding of ligand, which can be defined by the dissociation constant (KD)
and the number of binding sites available (R). The ligand binding to /3-AR
reconstituted on the asolectin liposomes with different Ca 2§ gradient was studied
using the radiolabeled antagonist, [3H]DHA. The results indicated that the
affinity and efficacy of /3-ARs for ligands of the proteoliposomes with a
transmembrane Ca 2§ gradient (lower Ca 2§ inside, similar to the physiological
situation) is higher as compared with that of proteoliposomes without transmembrane Ca 2§ gradient. Following the decrease of transmembrane Ca 2§
gradient, the affinity and efficacy of/3-ARs for the ligands would be reduced [27].
It may suggest that a proper transmembrane Ca 2§ gradient similar to physiological situation is essential for ligands to firmly bind to/3-AR, which favors/3-AR to
trigger the signal transduction of cAMP pathway.
In view of the fact that membrane fluidity change could influence the
activities of membrane-bound enzymes [31-35]. The relationship between the
physical state change of the membrane lipid and the ligand binding of /3-ARincorporating proteoliposomes with different transmembrane Ca 2§ gradients was
further studied by using a series of anthroyloxy fatty acid (n-AF, 2-AS, 7-AS and
16-AP). The results showed that the fluidity near the lipid bilayer center (C16) in
proteoliposomes with transmembrane Ca 2+ gradient similar to physiological
condition was higher than that of proteoliposomes without transmembrane Ca 2§
gradient [27]. This may indicate that a proper transmembrane Ca 2§ gradient
modulates ligands binding to/3-AR by virtue of affecting membrane fluidity near
the hydrophobic core where ligands binding to the receptor occurs, which would
favor ligands binding to the reconstituted/3-AR.
Effect of Transmembrane Ca 2§ gradient on Fluidity and Function of Adenylate
Cyclase (AC)-containing Proteoliposomes
Firstly, proteoliposomes with AC inserting in a highly oriented manner with
most of active sites facing outside, similar to inside-out cell preparations were
prepared. Results showed that these AC-incorporating vesicles could be reconstituted with sufficiently low permeability to Ca 2+ [28]. The enzyme activity,
conformation and fluidity of proteoliposomes with or without transmembrane
Ca 2§ gradient were determined and compared [28, 36].
From Table 5, it could be seen that the highest activity was observed in the
case of A C . L c a + - (lower Ca 2§ outside) which is similar to the physiological
situation. If transmembrane Ca 2§ gradient was in the inverse direction ( A C . L C a - +,
higher Ca 2+ outside), a lowest enzyme activity would appear, proteoliposomes
without transmembrane Ca 2§ gradient exhibited intermediate activities.
Transmembrane Ca 2§ Gradient and Function of Membrane Proteins
Table 5.
359
Effect of transmembrane Ca 2§ gradient (1000 fold) on enzyme activity of AC-containing
proteoliposomes
Proteoliposomes
(Ca~:Cao) 1
Enzyme control activity
(pmol cAMP/mg 9min)
+A23187
AC.Lca+ (1000:1)
AC.Lca- (1:1)
AC.Lca+ +
(1000:1000)
AC.LCa- +
(1:1000)
880
350
370
410
270
250
180
280
Cai:Cao =/xM:tzM.
It is interesting to note that following the dissipation of Ca 2§ gradient by
A23187, the difference in activity between A C . L c a + - and A C . L c a - + would
obviously be diminished. These results provided another indication that a proper
transmembrane Ca 2§ gradient is essential for the higher enzyme activity of AC.
In order to compare the conformation of AC in these proteoliposomes,
fluorescence spectroscopy and CD have been used.
The results showed [36] that the intrinsic protein fluorescence of four
types of proteoliposomes decreased in the order: A C . L c a + - > A C . L c a - - >
AC.Lca++>AC.Lca-+.
It may indicate that the microenvironment of
trypophanyl residues of AC in these types of asolectin vesicles was different from
each other.
The CD spectra of AC-containing vesicles with (1000fold) or without
transmembrane Ca 2§ gradient were also measured and compared. It is interesting
to note that the order of decreasing in the a-helix contents of AC in four types
vesicles coincides exactly with that of increasing in the enzyme activities of AC
[37].
Friendlander [33] reported that lipid fluidity could influence adenylate
cyclase activity. So, the lipid fluidity of four types of AC-incorporating proteoliposomes was measured using D P H as fluorescent probe [38]. The results showed
that the fluidity of four types of proteoliposomes followed the same order
(AC.Lca+ - > A C - L c a - - > AC.Lca+ + > A C - L c a - +) as the enzyme activity.
It was known that higher Ca 2§ concentration (>1/.~M) would inhibit adenylate
cyclase [39]. However, from the results obtained, it seems that in addition to
direct effect, Ca2§
change in lipid fluidity is also involved in the
modulation of adenylate cyclase activity.
From the above-mentioned result, it may deduce that a proper transmembrane Ca 2§ gradient may offer both in the outer and inner layer a suitable
fluidity of phospholipid, favoring the formation of an optimal conformation of the
reconsituted adenylate cyclase with higher enzymatic activity.
Effect of Transmembrane C a 2+ gradient on Gs Function
As stimulatory GTP-binding protein (Gs), a member of the G protein superfamily, mediates the stimulation of adenylate cyclase by a variety of hormones
[40], the transmembrane Ca 2§ gradient modulates the Gs function and its
relationship to the physical state of membrane phospholipids was investigated~
Yang, Huang and Tu
360
Table 6. Effect of transmembrane Caz+ gradient on Gs activities of binding
[35S]GTPz,S and stimulating adenylate cylase
Gs activity of
binding [3sS]GTPrS
(nmol/mg)
Samples
-A23187
Lca+ Lea+ +
Lea- Lea- +
3.318 -4-0.433
1.638 4- 0.374
1.896 4- 0.323
0.882 4-0.197
+A23187"
2.058 4-0.307
1.7224-0.378
1.806 4-0.332
1.6804-0.189
Gs-stimulated adenytate
cyclase activity
(nmol cAMP/min/mg)
-A23187
+A23187"
3.740 4- 0.356
1.6364- 0.170
1.8244- 0.158
0.8644- 0.117
1.808 4-0.190
1.743 4-0.201
1.689 4-0.183
1.584 4-0.159
Each value in Table 1 represents X 4-SD of experiments.
* Proteoliposomes were pretreated with A23187 (10/xg/ml) for 10min at 0~ before
assay.
(1) A Proper Transmembrane Ca 2+ gradient is Essential for Gs Function
As is indicated above that transmembrane Ca 2+ plays an important role in
regulating the activity of the reconstituted adenylate cyclase, which is a
component of the signal transduction system and is activated by Gs protein, it
would be interesting to see whether a transmembrane Ca 2+ gradient also plays a
role in modulating the Gs function of stimulating adenynate cyclase activity and
binding GTP. The data in Table 6 clearly show that Gs activities of both binding
G T P y S and stimulating adenylate cyclase activity were the highest in proteoliposomes type a (Lca+ - , lower Ca 2+ outside), which is similar to the physiological
condition, and the lowest in proteoliposomes type d (Ca z + - +) with an inverse
transmembrane Ca 2+ gradient (higher Ca 2§ outside) [29]. Proteoliposomes
without transmembrane Ca z+ gradient ( L c a + + or L c a - - ) exhibited intermediate activities. It may be suggested from these results that a proper transmembrane
Ca 2+ gradient is important for Gs to exert its physiological function. This idea is
further supported by the finding that dissipation of the transmembrane Ca 2+
gradient by the Ca 2+ ionophore A23 187 leads to a decrease in Gs function in the
proteoliposomes type a and an increase in the proteoliposomes type d (Table 6)
[29].
(2) Transmembrane Ca z+ gradient Regulates Gs Function by Altering Lipid
Fluidity
Figure 2 shows that the three-dimensional depiction of anisotropy decays of
the fluorescent probe, D P H in four types of the proteoliposomes (a, b, c, d) with
different transmembrane Ca 2+ gradient as mentioned above. The results from
measurement of the anisotropy at long times (r=) indicating that a lower order of
lipids in the proteoliposomes with a physiological transmembrane Ca 2+ gradient
(type a, Ca 2+ - , lower Ca 2+ outside) than that of the proteolipisomes with an
inverse Ca 2+ gradient (type d, Ca 2+- +, higher Ca 2+ outside). The order
parameter (S) of D P H in these proteoliposomes calculated from Fig. 2 increased
in the order: a ( 0 . 3 2 8 •
(0.341•
(0.338•
(0.352 • 0.0050). The difference between proteoliposomes type a and d was
Transmembrane Ca 2+ Gradient and Function of Membrane Proteins
361
/I,
"o
0
5
lO
15
Time/tO'gs
Fig. 2. The three-dimensional depiction of anisotropy decays of D P H in
the proteoliposomes with or without transmembrane Ca 2§ gradient. Curve
a, Lca+ - ; curve b, Lca+ +; curve c, L e a - - ; curve d, L c a - +.
significant (P < 0.001). It may indicate that lipid fluidity was higher in proteoliposome type a (Lca+ - ) with a transmembrane Ca 2§ gradient similar to physiological situation than that in proteoliposome type d ( L c a - +) with a transmembrane
Ca 2§ gradient in the inverse direction [29].
(3) Conformational Change of Gs in the Proteoliposomes with Different
Transmembrane Ca 2+ gradients
In order to study the mechanism of effect of transmembrane Ca 2+ gradient
on Gs-stimulation of adenylate cyclase, the conformational difference in Gscontaining proteoliposomes with different Ca 2+ gradient was also monitored using
a fluorescent probe, acrylodan. This probe reacts efficiently and selectively with
thiol group ( - S H ) in protein to give covalently-linked fluorescent derivatives
[41]. Figure 3 shows that the difference in the emission spectra of the
Gs-containing proteoliposomes labeled by acrylodan. It can be seen that the
emission wavelength maximum in proteoliposomes with a transmembrane Ca z+
gradient similar to the physiological situation ( G s . A C . L c a + - , curve a) was
significantly red-shifted 7.8nm (from 461 nm to 468.8nm) and the relative
fluorescence intensity was markedly lower (71 percent) as compared with those in
proteoliposomes with a transmembrane Ca 2+ gradient in the inverse direction
( G s . A C . L c a - +, curve d). The fluorescence spectra in proteoliposomes without
transmembrane Ca z+ gradient (Gs.AC.Lca+ +, curve b or Gs.AC-Lca- - , curve
c) exhibited intermediate changes with emission wavelength maximum for both
being 463 nm and fluorescence intensity being 99.2 and 93.8 respectively. The
results indicate that the thiol of Gs in proteoliposomes with a transmembrane
Ca 2+ gradients similar to physiological situation were exposed in more hydrophobic environment in the membrane compared with those in the inverse direction
362
Yang, Huang and Tu
150
d
12,
/
I
100
O
\,
e
"\ "', \,
i.l ,,~
O
/ //
x
\
&
',
\
\
~ \
////
/ ii'
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\.
/'.~
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[I
0400
I
4,50
I
500
Wavelength (nm)
550
Fig. 3. Fluorescenceemission spectra of acylodan adduct of Gs in proteoliposomesof different
transmembrane Ca2- gradient, a, GsAC.Lca+ -;
b, GsAC.Lca++; c, GsAV.Lca--; d,
GsAC.Lca-+. Samples were excited at 370 nm,
and emissionspectra monitored at 400-550 nm.
of Ca 2+ gradient, which favours GTP-activated Gs to stimulate adenylate cyclase.
In accordance with the changes of the stimulation of adenylate cyclase by Gs, our
results may suggest that a proper transmembrane Ca 2§ gradient is essential for
the maintenance of a suitable conformation of Gs in the proteoliposomes, which
may facilitate its stimulation of adenylate cyclase activity [42].
Effect of Transmembrane Ca2+ gradient on the Coupling of cAMP-signal
Transduction Pathway
As the results described above indicated that a proper transmembrane Ca 2+
gradient is essential for regulating the activity and conformation of the individual
/3-AR, A C and Gs in cAMP signal transduction system, it would be interesting to
explore whether a transmembrane Ca 2§ gradient also plays an important role in
activating the coupling of cAMP signal transduction pathway, cAMP signal
transduction system consists of three distinct types of plasma-membrane associated proteins: adenylate cyclase, stimulatory G protein (Gs) and the receptor
itself. Therefore, the modulation of Gs mediated /3-AR-adenylate cyclase
coupling by transmembrane Ca 2+ gradient and their relation to the physical state
of membrane phospholipids were further investigated. The purpose was reached
Transmembrane Ca 2§ Gradient and Function of Membrane Proteins
363
by an approach of co-reconstitution of the pure/3-AR from duck erythrocytes, Gs
and AC from bovine brain cortices on asolectin liposomes to attempt to form a
complete cAMP signal transduction pathway. And the coupling in the four types
of proteoliposomes with different transmembrane Ca 2§ gradient as above was
compared [30].
The data obtained showed that both the basal and hormone-stimulated
activities of adenylate cyclase in type d proteoliposomes without transmembrane
Ca 2§ gradient across membranes were significantly lower than that in proteoliposomes with a transmembrane Ca 2+ gradient similar to physiological situation
(1000-fold, type a) and this can be further enhanced by the decrease (100-fold) of
Ca 2+ gradient following Ca 2§ influx into cells during signal transduction [43].
DPH was used to measure the effect of transmembrane Ca 2§ gradient on
lipid fluidity of four types of proteoliposomes with different transmembrane Ca 2§
gradient. The results showed that in these proteoliposomes the membrane fluidity
changed in the order, which is well correlated with the changes in the basal and
hormone-stimualted activities of adenylate cyclase [43]. It may indicate that
transmembrane Ca 2+ gradient regulates the function of/3-AR-adenylate cyclase
signal pathway by inducing changes in membrane fluidity proteoliposomes, which
would favour the formation of a suitable conformation of /3-AR with higher
coupling to adenylate cyclase via Gs.
PERSPECTIVES
There are many proteins located in the cell membrane. In addition to the
membrane proteins mentioned in the present paper, the study on the effect of a
transmembrane Ca 2§ gradient on the function of other proteins of plasma
membrane (e.g. human erythrocyte glucose transporter and Band 3 etc.) is still
being carried out in our lab. Preliminary results obtained also showed that they
could also be affected and modulated by transmembrane Ca 2§ gradient [44]. We
hope that more and more attention will be paid on this aspect.
ACKNOWLEDGMENTS
This work was supported by National Natural Science Foundation of China
and the Chinese Academy of Sciences.
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