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doi:10.1111/j.1745-7270.2006.00129.x |
Alpha-latrotoxin Triggers
Extracellular Ca2+-dependent Exocytosis and Sensitizes
Fusion Machinery in Endocrine Cells
Zhi-Tao HU1#, Ping ZHAO1#, Jie LIU1#, Zheng-Xing WU1*, and Tao XU1,2*
1 School
of Life Science and Technology, Huazhong University of Science and Technology,
Wuhan 430074, China;
2 National Laboratory of
Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences,
Beijing 100101, China
Received:
August 15, 2005
Accepted:
November 3, 2005
#
These authors contribute equally to this
work
This
work was supported by the grants from the National Science Foundation of China
(30025023, 3000062, 30130230 and 30370647), the Major State Basic Research
Program of China (G1999054000 and 2004CB720000), the Chinese Academy of
Sciences Project (KSCX2-SW-224), and the National High Technology Research and
Development Program of China (2002AA214061)
*
Corresponding authors:
Tao XU: Tel, 86-10-64888469; Fax, 86-10-64867566; E-mail,
[email protected]
Zheng-Xing WU:
Tel, 86-27-87792024; Fax, 86-27-87792024; E-mail, [email protected]
Abstract a-Latrotoxin from the venom of
black widow spider induces and augments neurotransmitter and hormone release by
way of extracellular Ca2+ influx and cellular signal transduction
pathways. By using whole cell current and capacitance recording, the photolysis
of caged Ca2+, and Ca2+ microfluorometry and
amperometry, we investigated the stimulating effect and mechanism of a-latrotoxin on exocytosis in rat
pancreatic b cells, LbT2 cells and latrophilin
plasmid-transfected INS-1 cells. Our data indicated that: (1) a-latrotoxin increased cytosolic Ca2+ concentration through the
formation of cation-permitting pores and subsequent Ca2+ influx with the presence of
extracellular Ca2+; (2) a-latrotoxin
stimulated exocytosis in normal bath solution and its stimulating effect on
secretion was eradicated in Ca2+-free bath solution; and (3) a-latrotoxin sensitized the molecular
machinery of fusion through activation of protein kinase C and increased the
response of cells to Ca2+ photolysed by a flash of ultraviolet
light. In summary, a-latrotoxin induced exocytosis
by way of Ca2+ influx and accelerated vesicle fusion by
the sensitization of fusion machinery.
Key words a-latrotoxin; exocytosis;
calcium; Ca2+-sensitivity of fusion; protein kinase C
(PKC); capacitance measurement; amperometry
Over the last 20 years, a-latrotoxin (a-LTX) from the
venom of black widow spider has been widely used to study the molecular
mechanisms of neurotransmitter and hormone release. a-LTX elicits robust
neurotransmitter release in neurons, and stimulates hormone release in
endocrine cells, including adrenal chromaffin cells, pituitary gonadotropes and
secretory terminals of the posterior pituitary [1–4].
a-LTX can form non-selective cation pores on cell membrane and
subsequently stimulate secretion though Ca2+ influx
[3,5,6]. Evidence shows that the pores are large enough to conduct small
compounds including neurotransmitters [7–9].
Two classes of a-LTX receptors have been
identified: neurexin Ia and calcium-independent receptor for latrotoxin (CIRL)/latrophilin.
Neurexin Ia, first discovered by Petrenko et al. [10], is a member of a
highly polymorphic family of neuronal cell membrane proteins [11]. The binding
of toxin to neurexin Ia is Ca2+-dependent [12]. The ability of a-LTX to act in the absence
of extracellular Ca2+ led to the discovery of another Ca2+-independent
receptor: CIRL/latrophilin. Latrophilin belongs to the G protein-coupled
receptor protein family [13]. Studies have verified that a-LTX binds to
two classes of receptors in tetramers or dimers [14].
Accumulated evidence indicates that a-LTX evokes
secretion in the absence of extracellular Ca2+ by binding to
latrophilin and activating the G protein-phospholipase C (PLC)-inositol
1,4,5-triphosphate (IP3) and diacylglycerol (DAG) signal
transduction pathway [7,15]. Activation of PLC leads to the production of DAG
and IP3, two important intracellular second messengers. Activation of
protein kinase C (PKC) by DAG sensitizes the fusion molecular machinery and
augments secretion [16,17]. IP3 mobilizes the Ca2+
release from intracellular calcium stores to increase the local and global [Ca2+]i,
which triggers and modulates exocytosis of vesicles [18]. However, in this
study, in spite of the preservation of the secretagogue effect, we did not
detect the elevation of cytosolic Ca2+ concentration
by a-LTX
in the absence of extracellular Ca2+, suggesting
that the IP3 signal pathway did not play an important role in the stimulation
effect on exocytosis of a-LTX, and there might be another pathway for a-LTX to regulate
exocytosis, possibly by the activation of PKC.
Challenging cells with a-LTX by
extracellular perfusion in the Ca2+-containing normal and the Ca2+-free
bath solution, we studied the effect of toxins on the intracellular Ca2+
level and exocytosis. Our data indicated that a-LTX directly evoked the
robust secretion by way of Ca2+ influx, and augmented the response of
the toxin challenged cells to the step-like [Ca2+]i
elevation elicited by a short flash of ultraviolet (UV) illumination. The
mechanism underlying the latter effect was that a-LTX sensitized molecular
fusion machinery through PKC activation, which was elicited by the
latrophilin-hetero G protein-PLC-DAG-PKC signal transduction pathway.
Materials and Methods
Construction of latrophilin
expression plasmid
The plasmid pcDNA3.1-latrophilin was
kindly provided by Dr. Y. Ushkaryov
(Department of Biochemistry, Imperial College, London, UK). The challenge of using
latrophilin-enhanced green fluorescent protein (EGFP) fusion protein is that
EGFP may alter the physiological function of latrophilin. We took advantage of
the internal ribosome entry site (IRES)-EGFP cDNA vector, which contains the
IRES of the encephalomyocarditis virus and the EGFP-coding region, to
co-express latrophilin and EGFP for electrophysiological assay. The EcoRI/NotI-digested
IRES-EGFP sequence of pIRES2-EGFP was ligated into EcoRI/NotI-digested
pcDNA3.1-latrophilin vector to generate the pcDNA3.1-latrophilin-IRES-EGFP
plasmid. All DNA cloning was performed using Escherichia coli DH5a competent
cells. Construction integrity was verified by restriction enzyme analysis with HindIII
(data not shown). Restriction enzymes and other standard molecular biology
reagents were obtained from New England Biolabs (Beverly, USA).
Cell preparation
The pancreatic islets of male Wistar rats
(150–200 g) were prepared by collagenase V digestion, and further
digested by dispase II to dissociate single b cells in a Ca2+-free
Krebs-Ringer bicarbonate buffer, as described previously [19]. The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, USA)
supplemented with 25 mM HEPES, 2 mg/ml NaHCO3, 100 IU/ml
penicillin, 100 mg/ml streptomycin and 10% fetal calf serum (Gibco) in 5% CO2 at
37 ºC. The cells of insulin secreting insulinoma cell line INS-1 were grown in
DMEM in the same conditions as used for b cells. Approximately 72 h
before use, latrophilin was introduced into the endogenous receptor-lacking
INS-1 cells by transfecting with pcDNA3.1-latrophilin-IRES-EGFP plasmid using
Lipofectamine 2000 (Invitrogen, Groningen, Switzerland) according to the
manufacturer’s instructions. Cells expressing latrophilin were identified by green
fluorescence (excitation wavelength 488 nm).
[Ca2+]i measurement and Ca2+ uncaging
To measure the [Ca2+]i
response of primary pancreatic b cells, LbT2 and latrophilin-expressing INS-1 cells to a-latrotoxin (Alomone
Labs, Jerusalem, Israel), the cells were loaded with fura-2/AM by incubation at
37 ºC for 20 min in normal bath solution supplemented with 3 mM fura-2/AM. [Ca2+]i was
measured by dual-wavelength excitation (340/380 nm) microfluorometry using
either fura-2 or fura-6F as the Ca2+ indicator.
[Ca2+]i was calculated as follows:
[Ca2+]i=Keff´(R–Rmin)/(Rmax–R)
where Keff, Rmin
and Rmax are constants and obtained from intracellular calibration as
described previously [20]. Fura-2 and fura-6F were purchased from Molecular
Probes (Eugene, USA). All other agents were purchased from Sigma (St. Louis,
USA).
Step-like homogenous global [Ca2+]i
elevation was elicited by a flash of UV light generated by a Rapp flash lamp
(Rapp Optoelektronik, Hamburg, Germany). The flash was followed by a series of
illuminations alternating between 340 nm and 380 nm, which allowed radiometric
determination of the Ca2+ concentration. The duration of these
illuminations was adjusted to maintain relatively constant Ca2+
concentrations, as illumination at 340 nm or 380 nm also leads to the
photolytic release of Ca2+. Trains of light alternating at 340 nm
and 380 nm were generated from a monochromator (Till Photonics, Planegg,
Germany). The fluorescence was acquired by a photodiode (Till Photonics). The
DM-nitrophen-EGTA (DMNP-EGTA; Molecular Probes) containing pipette solution
(110 mM Cs-glutamate, 2 mM MgATP, 0.3 mM GTP, 35 mM HEPES and 5 mM DMNP-EGTA)
was adjusted to pH 7.2 using CsOH or HCl (osmolarity, 300 mOsm). The free Ca2+
concentration was measured to be ~200 nM in vitro by fura-2.
Membrane capacitance
measurement and current recording
Cell capacitance measurement was carried
out during whole cell recordings at 30 ºC –33 ºC using an EPC9
amplifier (Heka Electronics, Lambrecht, Germany). A sine+DC protocol was
applied using the Lockin amplifier of the Pulse program (Heka Electronics). The
cells were voltage-clamped at a holding potential of –70 mV and a sine wave
voltage command with amplitude of 20 mV and frequency of 1024 Hz was applied.
Currents were filtered at 2.9 kHz and sampled at 15.6 kHz. The currents induced
by extracellular application of a-latrotoxin were recorded in the whole cell
configuration using the EPC9 amplifier. Gö6983 (1 mM) was included in the
pipette solution to block PKC activation, in addition, Gö6983 (500 nM in normal
bath solution) was also incubated extracellularly for 10 min. The standard
extracellular bath solution consisted of 138 mm
NaCl, 5.6 mm KCl, 1.2 mm MgCl2, 2.6 mm CaCl2, 5 mm D-glucose and 10 mm HEPES (adjusted to pH 7.4 with NaOH,
osmolarity=310 mOsm). The Ca2+-free external bath solution was similar
to the standard bath solution, except that CaCl2 was
substituted by 1 mM EGTA.
Data analysis
Data analysis was performed using IGOR
Pro 4.02 (WaveMetrics, Lake Oswego, USA) and the results were presented as
mean+/–SEM. Statistical significance (P<0.05) was evaluated by
Student’s t test or the Mann-Whitney rank sum test according to the
normality of datum distribution in SigmaStat 3.11 (Systat Software, Point
Richmond, USA).
Results
a-LTX formed Ca2+ permitting channels on plasma
membrane and induced elevation of global [Ca2+]i
As shown in Fig. 1, the
extracellular application of 6 nM a-LTX by way of local perfusion induced
remarkable [Ca2+]i elevation (D[Ca2+]i) in primary rat pancreatic b cells (n=6, 471.3+/–41 nM),
latrophilin-expressing INS-1 cells (n=5, 681.2+/–56.3 nM) and LbT2 cells (n=6,
850.7+/–78.2 nM) in standard bath solution. However, a-LTX did not
elicit [Ca2+]i increase in these cells immersed in the Ca2+-free
extracellular solution (Fig. 1). These results suggested that a-LTX increased
[Ca2+]i by way of Ca2+ influx. To investigate the mechanism of
Ca2+ influx, we measured the currents induced by a-LTX in LbT2 cells in
whole cell configuration at different holding potentials in the normal (2.6 mM
Ca2+) and Ca2+-free bath solution (Fig. 2). The data showed that a-LTX could evoke
inward currents not only in the normal bath solution, but also in the Ca2+-free
extracellular solution [Fig. 2(A)]. The results suggested the formation
of cation-permitting pores by a-LTX on plasmalemma was Ca2+-independent
and the conductance was not Ca2+ selective. By measuring and analyzing
the current at three different holding potentials (–40, –70 and –100 mV), we
estimated the characteristics of the conductance of pores or the channels
formed by a-LTX. The histogram of current amplitudes versus frequencies, shown
in Fig. 2(B), shows that the whole cell currents in LbT2 cells at a
holding potential of –70 mV had two distinct Gaussian distributions. Of the electrical
events, those distributed around 0 pA (with amplitudes from –1.5 pA to +1.5
pA) were noise. The currents elicited by a-LTX had normal
distribution around –6 pA (–4 pA to –8 pA). The voltage relationship of currents [Fig. 2(C)]
demonstrated that the channel activity of a-LTX was unitary. Our
results agree with previous reports that a-LTX induced inward current
by forming pores or channels which have a unitary conductance [21].
a-LTX induced robust secretion by way of
Ca2+
influx
We examined the effect of a-LTX on
exocytosis using the whole cell capacitance measurement and amperometry with
the EPC9 patch amplifier. In the normal bath solution (2.6 mM Ca2+),
the application of a-LTX by local perfusion with pipettes pointing to the cells elicited
robust secretion in the primary pancreatic b cells (n=5) and LbT2 cells (n=6)
[Fig. 3(A)]. However, the stimulatory effect on the secretion of a-LTX was
eliminated in the Ca2+-free bath solution [Fig. 3(B)].
The results of the capacitance measurement were further confirmed by our
amperometry in primary b cells. The cells were preloaded with serotonin
(5-hydroxytryptamine, 5-HT) for 4–16 h and sensitized by incubation in 10 mM forskolin,
which induces a big increase in the cytosolic cAMP level and sensitizes the
secretory apparatus by way of the activation of protein kinase A, as reported
previously [22]. Extracellular application of 6 nM a-LTX elicited numerous
spikes of 5-HT in normal bath solution [Fig. 4(A)], but very few spikes
in the Ca2+-free solution [Fig. 4(B)]. 5-HT is taken up by
insulin-secreting vesicles and co-released with insulin. The quanta spikes,
recorded with 5 mm carbon fiber electrodes, coincided with that reported previously (Fig.
4) [22]. Our results indicated that a-LTX induced robust
secretion by way of Ca2+ influx through the cation-permitting
pores formed by a-LTX [23].
a-LTX sensitized the molecular machinery
of fusion
To examine and identify the possible
effect and underlying mechanism of a-LTX on secretion in the absence of
extracellular Ca2+, we used weak flash stimuli to evaluate whether a-LTX has any
sensitization effect on fusion machinery. The photolysis of Ca2+-caging
compound by a flash of UV light of about 800 hundred microseconds releases its
caged Ca2+ and leads to homogenous global calcium elevation in the cytosol.
The Ca2+ stimulus triggers vesicles to fuse with plasmalemma. After the
flash photolysis, exocytosis proceeds with an initial, rapid exocytotic burst
followed by a slower, sustained phase. The initial burst component represents
the fusion of the readily releasable vesicles [24,25]. The kinetics of the
burst component may reflect the processes of Ca2+ binding and
unbinding to the so-called Ca2+-sensor and the final fusion. The
maximum rate of release is a reliable indicator for evaluation of Ca2+
sensitivity of fusion at a certain calcium level. Fig. 5(A) shows the
flash response in the a-LTX-treated and control latrophilin-expressing INS-1 cells. a-LTX increased
the amplitude of the exocytotic burst and the rate constant of release (7.3 s–1 for
a-LTX-treated
cells and 3.3 s–1 for control) at similar post-flash
calcium levels. The kinetics of the response in the a-LTX+Gö6983-treated cells
was similar to that in control cells [Fig. 5(B)]. Fig. 5(C)
summarizes the maximum fusion rates of exocytotic bursts of the control, a-LTX and a-LTX+Gö6983
challenged cells. Our data showed that a-LTX markedly increased the
maximum fusion rate of latrophilin-expressing INS-1 cells in response to photolysed Ca2+
stimuli, when compared to the control (175+/–68 fF/s, n=10) and
the a-LTX treated INS-1 cells (590+/–131 fF/s, n=8, P<0.01). The maximum
fusion rates of the a-LTX+Gö6983 challenged cells (189+/–24 fF/s, n=8) were
not significantly different to that of control cells (P=1), but were
notably different to that of a-LTX treated cells (P<0.01), demonstrating
that the exocytosis effect of a-LTX was completely blocked by application of Gö6983.
Discussion
a-LTX is capable of stimulating neurotransmitter and hormone release,
and it has been used widely in the study of exocytosis as a potent toxin tool [26,27].
It is reported that there are two pathways in the mechanism underlying the
effect of a-LTX: (1) by way of extracellular influx; and (2) by way of cellular
signal transduction [2,3,6]. Our data indicate that very low dosage of the
toxin can induce the robust intracellular Ca2+ level
increase in primary pancreatic b cells, latrophilin-expressing INS-1 and LbT2 cells in the presence of
extracellular calcium. The [Ca2+]i elevation
induced by the toxin is attributable to the formation of the Ca2+-permeable
pores or channels and the resultant Ca2+ influx. The
characteristics of ion channels formed by the toxin demonstrated that these
channels are non-selective cation channels with a huge unitary conductance (up
to 200 pS). The channel activity remains in the absence of extracellular Ca2+.
But when other divalent cations such as Mg2+ are omitted,
the currents induced by a-LTX disappear (data not shown). Our results are identical with
former reports [5,23,28]. These indicate that the Ca2+
influx is efficient to evoke robust exocytosis.
a-LTX binds with CIRL/latrophilin and activates the receptor-mediated
pathway [7]. Latrophilin is a G protein-coupled receptor which links with Gaq/11 [6,13]. The
downstream effector of Gaq/11
is PLC. Activation of PLC leads to the generation of IP3 and
DAG, two important intracellular second messengers. IP3
mobilizes intracellular calcium stores to release Ca2+
and induces the exocytosis [29]. However, we failed to observe that a-LTX increases
[Ca2+]i in primary rat b cells, latrophilin-expressing INS-1 cells or LbT2 cells when
the Ca2+ was omitted from the extracellular solution, arguing against the
hypothesis that a-LTX mobilizes intracellular calcium stores.
The Ca2+ sensitization
of fusion machinery by PKC is an important and effective way to increase the
release of neurotransmitters and hormones [29–32]. PKC is able to
increase Ca2+ sensitivity of the molecular machinery of fusion and to accelerate
secretion [16,29]. As endocrine cells share similar secretory apparatus with neurons,
we used INS-1 cells as a model for secretion. Using global homogenous Ca2+ to
stimulate secretion in latrophilin-expressing INS-1, we demonstrated that a-LTX elicits a
much faster secretory response compared with the control, and the effect of toxin
on exocytosis is completely eradicated by the application of Gö6983, a specific
PKC blocker (Fig. 5). The results indicate that a-LTX increases the Ca2+
sensitivity of fusion machinery by way of activation of PKC, and helps to
explain the long-recognized extracellular Ca2+-independent
effect of a-LTX on exocytosis. In addition, our results argue against the
hypothesis that a-LTX directly regulates some pivotal proteins of fusion machinery
after insertion into the membrane [33].
Acknowledgements
We thank Mrs. X. P. XU for the skilled
technical support in cell preparation and plasmid transfection, W. ZHOU for the
support of carbon fiber electrodes (CFE), and the Partner Group Scheme of the
Max Planck Institute for Biophysical Chemistry (Goettingen, Germany).
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