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The Journal of Neuroscience, August 15, 1998, 18(16):6113-6125
 -Latrotoxin Alters Spontaneous and Depolarization-Evoked
Quantal Release from Rat Adrenal Chromaffin Cells: Evidence for
Multiple Modes of Action
Jun
Liu and
Stanley
Misler
Departments of Medicine and Cell Biology/Physiology and Program in
Neuroscience, Washington University, St. Louis, Missouri 63110
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ABSTRACT |
 -Latrotoxin (-LT) potently enhances both
"spontaneous" and "depolarization-evoked" quantal
secretion from neurons. Here we have used the patch-clamped rat adrenal
chromaffin cell to examine simultaneously the effects of -LT on
membrane current or voltage, cytosolic Ca, and membrane capacitance,
the latter used as an assay for exocytosis. In chromaffin cells exposed
to toxin concentrations of >100 pM, the development of
large conductance, Ca-permeable ion channels, accompanied by a rise in
cytosolic Ca to levels near 1 µM, precedes the initiation
of spontaneous exocytosis. These channels appear to be induced
de novo, because they occur concurrently with
massive reduction or pharmacological block of voltage-dependent Na and
Ca currents. However, enhancement of depolarization-evoked release,
seen in many cells at <50 pM toxin, often occurs in the
absence of a rise in background cytosolic Ca or de novo
channel activity. These results favor Ca entry through toxin-induced
channels underlying initiation of spontaneous release and direct
modulation of the secretory machinery by the toxin-bound receptor
contributing to enhancement of depolarization-evoked secretion as well
as spontaneous release.
Key words:
neurotoxin; exocytosis; membrane capacitance; amperometry; cytosolic Ca; catecholamines
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INTRODUCTION |
-Latrotoxin (a-LT), a 130 kDa
peptide derived from the venom of the black widow spider, is a potent
excitatory neurotoxin that selectively binds to vertebrate presynaptic
nerve terminals to produce skeletal muscle twitching followed by slow
progressive muscle paralysis (Rosenthal and Meldolesi, 1989 ). Within
minutes of its application to an isolated neuromuscular junction, at
picomolar concentrations, the toxin initially increases action
potential-evoked quantal release as well as "spontaneous" quantal
release of transmitter. However, over time, the toxin often blocks
nerve terminal conduction and "depolarization-evoked" release
(Longenecker et al., 1970; Hurlbut and Ceccarelli, 1979). Two proposed
mechanisms of -LT action, each involving the binding of toxin to
membrane receptor(s), might underlie these actions. (1) Binding
of -LT to its receptor(s) might serve to anchor the toxin to the
plasma membrane, thus enabling it to form Ca-permeable, nonselective
cation channels through which Ca2+ and other
divalent cations may enter the nerve terminal (Finkelstein et al.,
1976; Misler and Hurlbut, 1979; Meldolesi et al., 1983). (2) Binding of
-LT to its receptor(s) might alter the interaction of that receptor
with intracellular protein(s) involved with docking or fusion of
secretory granules with the plasma membrane (Petrenko et al., 1991;
Petrenko, 1993). Currently there are two candidate receptors, and each
has been shown to interact, in vitro, with a component of
the secretory apparatus. A Ca-dependent receptor that resembles
neurexin binds synaptotagmin, a putative Ca sensor (Petrenko et al.,
1991; Ushkaryov et al., 1992); a Ca-independent receptor
(CIRL/latrophilin) (Davletov et al., 1996; Krasnoperov et al., 1996)
that resembles members of a family of G-protein-coupled membrane
receptors binds to syntaxin, a putative component of the
vesicle-docking complex (Bennett and Scheller, 1994).
Using amperometry (Wightman et al., 1991; Chow et al., 1992) and
membrane capacitance tracking (Neher and Marty, 1982; Augustine and
Neher, 1992) as single-cell assays of exocytosis, we have demonstrated
previously that crude black widow spider venom and purified -LT
cause massive quantal release from rat adrenal medullary chromaffin
cells (Zhou and Misler, 1995; Barnett et al., 1996; Liu and Misler,
1998). These neural crest-derived, excitable endocrine cells are often
used as a model system to study the mechanisms of exocytosis at nerve
terminals as well as the roles of neurotoxins in modifying transmitter
release (e.g., Bittner et al., 1989). One set of experiments reported
here offers direct evidence strongly supporting the hypothesis that
toxin-induced "channel formation" and the attendant rise in
cytosolic Ca are critical for initiation of spontaneous exocytosis.
Another set of experiments suggests that the enhancement of
depolarization-evoked release, seen at lower doses of toxin, may result
from an "alternative" action of the toxin that is independent of
its ability to enhance cytosolic Ca. It is likely that the latter
action results from the ability of a toxin-bound receptor to modify the
intrinsic machinery of the secretory apparatus. This alternative action
may substantially enhance the potency of channel formation-based,
toxin-related spontaneous exocytosis.
Parts of this paper have been published previously (Liu and Misler,
1998a).
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MATERIALS AND METHODS |
Preparation of cells
Freshly harvested rat adrenals were defatted, decorticated,
minced, and enzymatically digested with a cocktail of collagenase D,
hyaluronidase type 1, and DNase type 1 using a protocol described elsewhere (Neely and Lingle, 1992; Liu and Misler, 1998). The resulting
cells were plated on coverslips coated with Matrigel (Becton Dickinson,
Rutherford, NJ) and maintained for up to 5 d in a 95% air/5%
CO2 incubator at 37°C using a DMEM (Life
Technologies, Gaithersburg, MD) enriched with 10% fetal bovine serum,
100 IU/ml penicillin, 100 mg/ml streptomycin, and 6 mg/ml ascorbic acid and buffered with HEPES and
HCO3 . Fura-2 AM was loaded into
cells; cells were incubated for 20 min in the dark at room
temperature in HEPES-buffered DMEM containing fura-2 AM (5 µM) and then were washed for 20 min with DMEM plus 10%
FBS to permit de-esterification of the dye. This approach provided
uniform loading of the cytoplasm with the Ca-sensitive dye and levels
of photon emission approximately fivefold greater than the
autofluorescence.
Preparation and application of toxin
Purified -LT (2-3 µM stock) was the gift of
Dr. Alexandre Petrenko (New York University). The toxin was diluted to
a 1 µM stock, stored at 4°C, and then further diluted,
as needed, on the day of use. Aliquots were injected into the 1.8 ml
capacity bath from a micropipetter whose tip was ~5-8 mm from the
recording pipette; the tip was then used to gently agitate the bath to
promote convection. The toxin concentrations reported (20 pM to 2 nM) were calculated "equilibrium
concentrations" (i.e., amount injected/bath volume), presumed to be
reached within 3 min after injection.
Recordings
Three to five days after their isolation and plating, single rat
chromaffin cells were monitored at room temperature (20-23°C) in a
HEPES-buffered physiological saline solution (PSS) containing (in
mM): 145 NaCl, 5.5 KCl, 2 CaCl2, 0.1 MgCl2, 10 glucose, and 20 HEPES titrated to pH 7.3 with NaOH. In experiments in which Ca currents were measured, 35 mM NaCl was replaced mole-for-mole with tetraethylammonium
chloride (TEA). A "Ca-free" PSS was made by eliminating
CaCl2 from, and adding 1 mM EGTA to, the
control PSS. An "isotonic-Ca" PSS was made by replacing the NaCl
content of PSS with 98 mM CaCl2. Other
customized solutions are described in the text.
Electrophysiological recordings. Electrophysiological
recordings were made using a perforated-patch variant of the whole-cell recording standard for our lab (Barnett et al., 1996). For
voltage-clamp recording, patch pipettes and salt bridges in contact
with the indifferent Ag/AgCl electrode were filled with a standard high Cs internal solution containing (in mM): 63.7 CsCl, 28.35 Cs2SO4, 47.2 sucrose, 11.8 NaCl, 1 MgCl2, and 20 HEPES titrated to 7.3 with KOH. A
similar solution containing 3-5 µl of nystatin (250 µg/ml) was
added just proximal to the tip of the patch pipette. The membrane
potential was held at  70 mV throughout the experiment, and
sinusoidal, square pulse, or ramp voltage excitation (100 to +60 mV
over 100 msec or 50 to +50 mV over 3 sec) was imposed as needed. In
experiments in which zero current potentials were sought, liquid
junction potentials were calculated using standard ion mobilities
(Barry and Lynch, 1991) and appropriately added algebraically to
interpret individual results (Barry and Lynch, 1991; Neher, 1992). For
current-clamp experiments, a high K internal solution, in which CsCl
and Cs2SO4 were replaced mole-for-mole by KCl
and K2SO4, respectively, was used to
fill the pipette. Current and voltage traces were filtered at 0.5 and
1.5 KHz, respectively, and sampled at 3 KHz.
Our setup for data acquisition and processing has been described in
detail previously (Barnett and Misler, 1997). In experiments in which
capacitance and current were monitored simultaneously, the circuit
parameters of the patch-clamped cell, namely, the parallel combination
of membrane resistance (Rm) and
capacitance (Cm) in series with the
pipette-to-cytoplasm access resistance (Ra), were estimated using an EPC-9
patch-clamp amplifier (Heka Electronic, Lambrecht, Germany) and a newly
described software-based, dual frequency lock-in detector (LID). The
LID was developed as a set of extensions (XOP modules) to the
numerical/graphics package Igor (Wavemetrics). The program was
run on a Macintosh Quadra-650 (Apple). Samples of the analog current
signal were acquired using an ITC-16 analog-to-digital card
(Instrutech, Syosset, NY). Appropriately filtered sinusoidal
stimulation (10 mV peak-to-peak at 400 and 800 Hz) was applied to the
patch-clamp amplifier held at a DC potential of 70 mV. The resultant
membrane current signal at each frequency, which is phase shifted from
the input voltage signal, was fed into the LID that decomposes the
current signal into real (or in-phase) and imaginary (or quadrature)
components of the admittance. Based on these four measurements, a
nonlinear weighted least squares algorithm is used to estimate the
circuit parameters (Rm,
Cm, and Ra),
which are subsequently displayed in real time. The DC membrane current
(Im) at 70 mV, estimated by averaging
the sampled current over the period of the lower frequency sine wave
(i.e., 2.5 msec), is also displayed to track background channel
activity. By interrupting the sinusoidal excitation with square steps
of depolarization, it is possible to examine depolarization-evoked
exocytosis as well. Recently this program has been modified by Dr.
David W. Barnett to trigger a monochrometer and to collect and display
the output of a photomultiplier tube (Till Photonics Photometrics
System; Applied Scientific Instruments, Eugene, OR), thereby permitting
synchronous ratiometric determination of cytosolic Ca in cells loaded
with fura-2.
Amperometric recordings. Amperometric recordings were
performed with polypropylene-insulated carbon fiber electrodes touching the cell surface and held at +780 mV using an EPC-7 amplifier (Heka Electronic) (Zhou and Misler, 1996). Amperometric data, filtered
at 300 Hz using an eight-pole Bessel filter (Frequency Devices,
Haverhill, MA), were acquired simultaneously with membrane current or
membrane voltage data using the software package that runs the digital
LID. Amperometric events were reviewed and tabulated with an
interactive Igor-based program in use in our lab. The criteria for
scoring an amperometric event were (1) an amplitude of at least 1 pA
(approximately three times the peak-to-peak background noise of
acceptable recordings) and (2) a half-height duration of at least 2 msec.
Ratiometric monitoring of cytosolic Ca
Cytosolic Ca was estimated from ratiometric measurements made on
cells loaded with fura-2. Loaded cells were mounted on the stage of an
inverted epifluorescence microscope outfitted for dual wavelength (340 and 380 nm) excitation and appropriate throughput of fluorescence
emission via a bandpass dichroic mirror (500-520 nm). Optical slits
were set to collect emission from a single cell. Average ratiometric
data were converted to cytosolic Ca estimates
([Ca]i) using the equation: [Ca]i = 224 nM (R Rmin)/(Rmax R) × (F380,max/F380,
min), where Rmax and
F380,max and then Rmin
and F380,min were obtained sequentially after
first adding 5 µM ionomycin and then 2.5 mM
EGTA to the bath (Grynkiewicz et al., 1985).
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RESULTS |
Basic phenomenology of -LT action on chromaffin cells
In previous experiments using amperometry (Liu and Misler, 1998b),
we examined key features of toxin action in promoting catecholamine secretion from rat adrenal chromaffin cells. These include (1) its
threshold concentration of ~30 pM and saturating dose of
~1 nM, (2) its requirement of extracellular Ca in the
micromolar range for secretory action but not "functional" binding,
and (3) the association of its secretagogue action with a rise in
cytosolic Ca to ~1 µM but not with the functional state
of endogenous Ca channels. Because these results primarily resembled
those produced by toxin at nerve terminals, they encouraged us to use
the chromaffin cell as a model system to examine features of toxin
action not amenable to investigation at the nerve terminal.
Specifically, we examined the relationship of the channel-forming
action of the toxin to its ability to enhance spontaneous and
depolarization-evoked exocytosis.
Comparison of the time courses of toxin-induced channel activity,
capacitance increases, and quantal releases of catecholamines
From our previous experiments on patch-clamped chromaffin cells,
in which exocytosis was measured by membrane capacitance tracking, it
seemed that the progressive development of toxin-induced channel
activity was followed by the onset of exocytosis measured as an
increase in Cm. Furthermore, the initial
time course of Cm increase was similar to
the initial time course of toxin-induced quantal release, measured
amperometrically from nonpatch-clamped cells (Barnett et al., 1996). To
bridge these two techniques, we performed a set of experiments in which
both assays of exocytosis were monitored simultaneously on the same
patch-clamped cells.
Figure 1 (left) displays
simultaneous traces of membrane capacitance
(Cm), amperometric current
(Iamp), and membrane current (Im), as well as the pipette-to-cytoplasm
access resistance (Ra). Channel activity
began ~30 sec after toxin application. A detectable increase
in Cm, as well as the onset of
amperometric spike events (ASs), was first appreciated 25 sec after
channel activity began, by which time at least 50 pA of inward
current had developed. Thereafter, both the frequency of amperometric
events and the rate of rise in membrane capacitance rapidly increased.
Cm peaked at 2.5 pF above baseline within
a minute of commencement of release, a time when amperometric discharge
was still vigorous and toxin-induced current was still increasing.
However, Cm dropped off over the next 20 sec, in fact undershooting control values by >1 pF.
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Figure 1.
Simultaneous monitoring of secretory response to
-LT by capacitance tracking and amperometry permits estimation of
time courses of exocytosis and endocytosis. Left, Time
courses of membrane capacitance (Cm),
amperometric current events (Iamp),
membrane current (Im), and access
resistance (Ra) recorded from
patch-clamped chromaffin cells held at 70 mV beginning 30 sec after
the addition of 500 pM -LT to the bath. Note that
Ra remained stable throughout the depicted
time interval, thus reducing chances of a "cross-talk" artifact
between changing membrane resistance and
Cm, especially when membrane resistance is
<100 M . Upper Right, Comparison of time course of
 Cm and the running sum of
amperometrically detected quantal release events. Lower
Right, Estimates of time course of total exocytosis, net
exocytosis, and endocytosis after addition of toxin. See Results for
details of computation.
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Figure 1 (right) provides stricter quantitation and an
approach to the interpretation of this data. The upper
panel demonstrates the similar time courses of cumulative
development of Cm
( Cm) and ASs (AS). If we assume
that each amperometric event corresponds to the fusion with the plasma
membrane of a granule that is 300-350 nm in diameter and has a unitary
capacitance equaling 2.4 fF (Chow et al., 1994), the ratio (AS × 2.4 fF/AS)/(observed Cm) is nearly constant at 0.283 (±0.02) over 60 sec. (A similar ratio was
seen in this cell on comparing AS with
Cm during repetitive depolarization.) The lower panel of Figure 1
demonstrates that scaling AS by the factor (2.4 fF/AS × 1/0.283) produces a curve of calculated
Cm versus time; for the initial 60 sec, this calculated curve is virtually superimposable on the curve of
observed Cm versus time. On this
basis we designate the observed Cm
versus time trace as "net exocytosis," the calculated
Cm versus time trace as "total
exocytosis," and the time course of the difference between the two
curves as "endocytosis."
Altogether, the results presented in Figure 1, representative of those
in a series of three similar experiments performed at room temperature
(and one at 30°C), strongly support two ideas. First, amperometry and
the capacitance tracking are monitoring exocytosis from the same pool
of granules, although amperometry is less efficient because it is
monitoring release from a limited area of cell surface. This confirmed
our initial prediction that the dual sinusoidal excitation technique
for capacitance estimation is robust enough to track exocytosis even in
the face of major changes in membrane conductance (Barnett and Misler,
1997). In addition, the intense amperometric discharge was associated
with little or no slow DC displacement of the baseline. The latter might suggest contamination of the record by a cloud of background release from multiple small granules. Second, in the presence of the
toxin, initial vigorous exocytosis can be followed by slow and then
more rapid endocytosis that serves to retrieve the plasma membrane and
restore its surface area to near the resting value. The rapid component
of endocytosis occurs at a rate of ~0.4 pF/sec and actually results
in a 10% undershoot in Cm. This component resembles the "excess retrieval" mode of endocytosis seen with massive depolarization-induced Ca entry sufficient to raise cytosolic Ca to ~10 µM even in cells from which recordings were
made in the perforated-patch mode (Artalejo et al., 1995; Smith and
Neher, 1997).
Relationship of toxin-induced channel activity and
spontaneous exocytosis
In a previous study examining the dependence of toxin action on
external Ca, we found that crude venom gland extract massively increased the resting membrane current and conductance of chromaffin cells held at 70 mV, whether they were bathed in control PSS or a
very low Ca (10-20 µM) PSS, although the latter solution did not support exocytosis. In the very low Ca PSS, the background current developed in a stepwise manner, suggesting the appearance of
long duration, large conductance channels, whereas in control PSS, the
current developed in a more "ragged" manner with very few discrete
steps. We suggested that the toxin might form large conductance,
nonselective cation channels and that Ca interacts with these channels
as a "permeant blocker," permeating yet tightly binding within the
channel pore (Barnett et al., 1996). To characterize toxin-induced
currents more carefully, we applied doses of purified toxin that, based
on our recent dose-response secretion studies using amperometry (Liu
and Misler, 1998a), might allow us to capture initial single-channel
activity as well as subsequent larger increases in membrane current as
the toxin-induced channels dominate membrane conductance. These
experiments were performed using continuous whole-cell voltage-clamp
recording, combined with intermittent voltage-ramp stimulation, on
cells bathed in solutions of varying composition.
Development of a massive toxin-induced current occurs in the
presence of a drastic reduction in endogenous voltage-dependent calcium
and sodium currents
To examine toxin-induced currents in chromaffin cells, we
attempted initially to block endogenous voltage-dependent currents. Figure 2A shows results
from an experiment (typical of a set of three) in which a cell, bathed
in a PSS containing 35 mM TEA, was patched with a
Cs+-nystatin pipette. Under these conditions, in
which the majority of voltage- and Ca-dependent K+
currents are blocked, a 100-msec-long voltage ramp (from 100 to +60
mV) evokes two components of inward current, namely, an initial spike,
peaking at 20 mV, and a later shoulder, most prominent at 0 mV.
Addition of 1 µM tetrodotoxin (TTX), a potent Na channel blocker, and 200 µM Cd, a potent Ca channel blocker,
abolishes both components of inward current and further reduces outward current. Subsequent addition of toxin produces a slowly developing background current that is inward at 70 mV, is mildly outwardly rectifying, and has an average zero current potential
(Erev) of +3 mV (or 5 mV after
correction of liquid junction potential). Figure 2B
shows results (typical of a set of four) from a variant of the latter
experiment in which a cell, bathed in a PSS containing 5 mM
TEA and 4 µM apamin, a more-specific blocker of the small conductance Ca-dependent K+ currents prominently
displayed by these cells, was patched with a
Cs+-nystatin pipette. Under these conditions, in
which the voltage-dependent inward current is initially intact,
addition of toxin results in the apparent disappearance of both the
spike and shoulder components, concurrent with the development of a
background current that here is nearly linear with voltage and has an
Erev of +8 mV (or 1 mV after correction of
liquid junction potential). Continuation of the electrical recording
reveals that the background current slowly wanes, and thereafter both
components of the voltage-dependent current return.
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Figure 2.
Toxin-induced current develops in the face of
previous or concurrent drastic reduction in voltage-dependent sodium
and calcium currents. A, The time course of development
of toxin-induced current after addition of TTX and Cd that
pharmacologically block Na and Ca currents, respectively, is shown.
B, The initial drastic reduction of voltage-dependent
currents, after application of toxin, coincides with the initial
development of toxin-induced current, whereas the slow return of
voltage-dependent current coincides with the subsequent waning of
toxin-induced current. Membrane current was probed by intermittent
imposition of voltage ramp (100 to +60 mV over 100 msec) before and
after development on toxin-induced inward current seen at 70 mV. In
the bottom panels of A and
B, expanded traces recorded at indicated
times, except for the last trace in B
that was recorded 3 min later as part of a different data file, are
presented.
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The ability of the toxin to induce a large membrane conductance under
conditions in which voltage-dependent currents are blocked, as well as
to drastically reduce or abolish the latter currents reversibly,
suggests that toxin-induced current is flowing through "novel"
channels rather than through altered "native" ones. The ability of
the toxin to block voltage-dependent currents necessary for
excitability most likely underlies its capacity, over time, to depress
depolarization-evoked release, a key feature of its effect at
presynaptic terminals.
Toxin-induced channels are generally permeable to cations
including calcium
Figure 3 examines the ionic
selectivity of the toxin-induced membrane conductance. Figure
3A provides evidence that this conductance is overwhelmingly
cation-selective. In three paired experiments, we compared the
current-voltage (I-V) relationships
obtained after applying 200-500 pM toxin in a Ca-free PSS
(with 1 µM TTX and 5 mM TEA) with those
obtained after applying toxin in a modified Ca-free PSS in which
approximately one-half of the NaCl (65 mM) was
isosmotically replaced with sucrose. Partial NaCl replacement shifted
the observed Erev by 15 to 18 mV, consistent
with nearly ideal cation selectivity of the toxin-induced conductance.
Figure 3B provides evidence that large univalent cations are
at least somewhat permeable through this conductance pathway. This
I-V curve was typical of those obtained in three
cells after addition of toxin to a Ca-free PSS in which all of the
total Na + K content but 3 mM was replaced with the bulky
univalent cation N-methyl-D-glucamine (NMDG).
Note that substantial toxin-induced inward current is still seen at
potentials negative to 5 to 10 mV, although outward rectification
is now prominent. Figure 3C provides evidence that Ca is
highly permeable through this conductance as well. Note that after
addition of toxin to isotonic-Ca PSS (residual [Na]o of
<3 mM), impressive inward current is seen at voltages
negative to 0 mV; in fact, the I-V curve is
nearly linear. Altogether these results suggest that toxin-induced
channels are cation-permeable but poorly "selective" among cations,
in part because of their large pore diameter.
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Figure 3.
Survey of ion permeability via -LT-induced
membrane conductance. Currents were recorded in response to voltage
ramps 60-90 sec after the application of toxin to different cells
bathed in Ca-free PSS and in modified Ca-free PSS in which 65 mM NaCl was replaced isosmotically with sucrose
(A), in Ca-free PSS in which Na and K (all but 3 mM) were replaced with NMDG (B), and
in isotonic-Ca PSS (C). See Results for
general overview. Two specific points deserve clarification. (1)
Current-voltage relationships seen before application of toxin are
shown for Ca-free PSS and isotonic-Ca PSS to eliminate nonspecific
effects in these unusual solutions. The lack of voltage-dependent Ca
current in Ca-free PSS is attributed to an external Ca of <10
µM; the lack of Na current is attributed to a partial
block by TTX and partial inactivation by a maintained holding potential
of 50 mV. After toxin application, the "turn-down" in outward
current at very positive membrane potentials may reflect closure of
toxin channels through a flickery subconductance state. Such a
phenomenon was observed after application of the 10 nM
toxin to one side of a lipid bilayer bathed in 150 mM KCl
(W. German and S. Misler, unpublished observations). In the isotonic-Ca
PSS, note that voltage-dependent Ca current peaks at approximately +30
mV, because of the elevated bath Ca and shifts in the activation curve
of the high voltage-activated Ca channels. (2)
Erev denotes the observed reversal potential
of toxin-induced current in each condition. Because in each case, the
liquid junction potentials (LJP) were zeroed before patching the cell,
the shift in Erev can be used to assess the
relative permeability of ions, although in each case we calculate that
the observed Erev is shifted positively by 8 mV from the true Erev because of partial
undoing of the LJP correction after seal formation.
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As illustrated in Figure 4, in several
cells it was possible to record under varying ionic conditions, for up
to 10 sec, clearly recognizable single-channel events before the onset
of massive membrane current. This allowed us to examine our hypothesis
that Ca acts as a permeant blocker of toxin-induced channels. In cells voltage clamped at 70 mV, inward current steps, up to 10 sec long and
averaging 30 pA in amplitude, were seen in Ca-free PSS; using the
observed Erev of +5 to +8 mV, we estimate a
single-channel conductance ( ) of ~400 pS. In contrast, smaller,
more "flickery" openings, averaging 20 pA in amplitude and never
exceeding 0.3 sec in duration, were seen in control PSS (containing 2 mM Ca); from these data we calculated to be ~270 pS.
In two out of six cells examined, clear single-channel openings were
seen in isotonic-Ca PSS; the majority of these averaged 10 pA in
amplitude and appeared to occur as clusters of openings; from these
data we calculated to be ~130 pS.
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Figure 4.
Single-channel currents induced by the toxin.
Left, Sample traces of the first distinct
signs of toxin-induced channel activity recorded after toxin
application at a holding potential of 70 mV in three ionic
conditions: Ca-free PSS, control PSS, and isotonic-Ca PSS.
Right, Histogram of single-channel current amplitudes
recorded under these three conditions.
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Toxin-enhanced spontaneous quantal release is dependent on Ca entry
and cytosolic Ca accumulation
Our early experiments showed that the onset of exocytosis follows
progressive development of toxin-induced channel activity only if
adequate external calcium is present. These results suggested that Ca
entry through the large conductance channels, with a subsequent accumulation in the cytosol, is critical in evoking exocytosis. If this
suggestion is reasonable, then (1) before the onset of release,
cytosolic Ca should rise to levels seen with other conditions, such as
cell dialysis or membrane permeabilization (Dunn and Holz, 1983;
Augustine and Neher, 1992), that evoke sustained release, and (2)
raising the extracellular Ca2+ concentration should
increase the effectiveness of each increment in current to provoke
release. Figure 5 shows that these
predictions are borne out experimentally.
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Figure 5.
Temporal relationship of toxin-induced exocytosis,
measured as an increase in membrane capacitance, to toxin-induced
channel activity and the rise in cytosolic Ca seen at
near-physiological versus near-isotonic extracellular Ca
concentrations. A, Results obtained in control PSS after
addition of 200 nM toxin at 30 sec. B,
Results obtained in isotonic-Ca PSS after addition of 200 nM toxin at 20 sec. Note that in both ionic conditions, the
onset of exocytosis (noted by an arrow) follows a rise
in cytosolic Ca to ~1 µM. However, membrane current
flow and total charge transfer preceding this rise are clearly many
fold greater in control PSS than in isotonic-Ca PSS.
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Figure 5, A and B, compares the time courses of
toxin-induced membrane current, the rise in cytosolic Ca, and the
development of exocytotic release in patch-clamped, fura-2-loaded
cells bathed in control PSS (2 mM Ca) or isotonic-Ca PSS
(98 mM Ca). These records, which are each representative of
a set of four cells, indicate that under both conditions (1)
development of toxin-induced channel activity precedes the rise in
cytosolic Ca and (2) a rise in estimated cytosolic Ca to 0.5-1
µM precedes the onset of a clear rise in membrane
capacitance. However, as predicted above, with isotonic-Ca PSS, the
threshold level of cytosolic Ca is reached after currents as low as 10 pA flow for 5 sec, whereas with standard PSS, the threshold level of
cytosolic Ca is reached only after an average current flow of nearly
100 pA for 15 sec.
Timing of -LT-induced depolarization and release
Since the discovery that -LT increases cation permeability of
membranes, it has been assumed that the ability of -LT to induce
spontaneous quantal release involves, in part, its ability to
depolarize secretory terminals and to set off electrical activity (Longenecker et al., 1970; Nicholls et al., 1982). Figure
6, taken from combined current-clamp and
amperometry experiments, presents direct evidence that -LT indeed
depolarizes chromaffin cells and sets off a barrage of action
potentials before the onset of quantal release, whereas the
slow-to-start quantal release persists long after electrical activity
is blocked. The first signs of toxin activity are small, discrete steps
of depolarization, probably corresponding to the induction of single
channels, beginning 40 sec after application of toxin. (In these cells
with a background membrane resistance of ~0.75 G, an inward
current of 20 pA should produce a 15 mV depolarization.) Subsequent
progressive depolarization drives the membrane potential to the
threshold for evoking action potentials of increasing frequency and
then to a prolonged plateau phase, approaching 0 mV. The latter phase
is consistent with the massive, prolonged opening of nonselective
cation channels and the depolarization-induced inactivation of native
voltage-dependent channels. Note that quantal release is first
detectable near the conclusion of the initial barrage of action
potentials and continues for the duration of plateau depolarization. In
some cells tested, very low doses of toxin (30-50 pM)
evoke infrequent barrages of action potentials that are occasionally
accompanied by brief bursts of amperometric spikes.
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Figure 6.
Time course of membrane excitability and quantal
release of current-clamped chromaffin cells treated with toxin.
Top, Simultaneous recordings of membrane
potential (Vm) and amperometric currents
(Iamp) beginning 10 sec after
application of 150 pM toxin. Bottom,
Expanded Vm trace over the interval in which electrical
activity commences.
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Enhancement by toxin of depolarization-evoked secretion
At nerve terminals, small doses of -LT often enhance
impulse-evoked quantal release by several fold before the onset of
massive increases in spontaneous quantal release. We sought a parallel phenomenon in chromaffin cells to apply patch-clamp recording to
dissect out candidate mechanisms underlying it. We found that low
concentrations of toxin, which do not enhance spontaneous exocytosis,
do enhance depolarization-evoked release.
In experiments presented in Figure 7, we
compared the capacitance increases (Cm)
in response to a brief "test train" of 10 membrane
depolarizations (100- or 200-msec-long pulses from 70 to +10 mV
applied at 1 Hz) applied at intervals to control cells and to test
cells exposed to 20-50 pM toxin. In Figure 7A,
note that in four out of four control cells, identical test trains of
membrane depolarization produced, at most, a 1.15-fold increase in
Cm over 15 min, whereas
depolarization-evoked Ca current changed by <10%. In contrast
in Figure 7B, note that after addition of toxin, 9 out of 14 cells tested displayed substantial enhancement of depolarization-evoked
Cm, despite a <10% change in
depolarization-evoked Ca entry measured as peak Ca currents or the
summed integrals of the current. The enhancement of
Cm averaged 1.85-fold over 10 min and
2.15-fold over 15 min. Data from a sample experiment are shown in
Figure 7C. These experiments established that low concentrations of the toxin can support increases in
depolarization-evoked release out of proportion to any changes in Ca
current.
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Figure 7.
Exposure to small doses of toxin results in
progressive increases in depolarization-evoked quantal release even in
the absence of changes in voltage-dependent Ca current.
A, B, Tabulated results of sets of
experiments in which Cm and membrane
current were measured in response to 10 depolarizations of 200 msec
(from 70 to +10 mV at 1 Hz) at intervals in control cells
(A) and in test cells before and again at
intervals after application of 20-50 pM toxin
(B). C, Sample data
traces from a representative cell before and then
4 min after exposure to 20 pM toxin.
Insets show, with expanded scales, Ca currents recorded
in response to the 1st and 10th depolarizing pulses from 70 to +10
mV. The larger Na+ currents, whose peak values
remained constant at 820 pA, are truncated in these
traces.
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We considered three mechanisms by which toxin might increase
depolarization-evoked exocytosis in the presence of nearly constant depolarization-evoked Ca entry. First, toxin might continuously and
substantially enhance background cytosolic Ca by producing low-frequency channel activity. Given that activation of
depolarization-evoked release (Engisch and Nowycky, 1996) and the
proposed Ca sensor synaptotagmin (Bennett and Scheller, 1994; Littleton
and Bellen, 1995) both appear to require the binding of more than one
Ca ion, background Ca that is subthreshold for evoking exocytosis by
itself might however significantly modulate the cytosolic Ca level
achieved after a constant pulse of Ca entry. Second, intermittent
bursts of toxin-related channel activity or release of Ca from
organelle stores might transiently raise average cytosolic Ca to
300-500 nM for several seconds, a situation known to
potentiate evoked release for up to tens of seconds even after
background cytosolic Ca has returned to baseline (Heinemann et al.,
1993; von Rueden and Neher, 1993). Third, the toxin might enhance
secretion without inducing any rise in cytosolic Ca. Toxin-bound
receptors might interact with a component of the secretory apparatus,
thereby directly enhancing Ca-sensitive exocytosis.
Experiments combining spectrofluorimetry with capacitance tracking in
fura-2-loaded cells allow us to distinguish the relative roles of
these proposed mechanisms. Two basic scenarios were observed. (1) In
six out of eight experiments, during the 7-12 min after addition of
30-50 pM toxin, on average we measured a 2.5-fold (±0.4
SD) increase in depolarization-evoked exocytosis above baseline. In all
cases this occurred with a <5% rise in estimated background cytosolic
Ca and a <7% rise in peak cytosolic Ca estimated to occur during the
depolarization train (for example, compare Fig. 8, left and middle
columns). If we assume a power of 1.5-3 dependence of
quantal release on cytosolic Ca (Augustine and Neher, 1992; Heinemann
et al., 1993; Engisch and Nowycky, 1996), a 7% increase in peak
cytosolic Ca should produce at most a 1.23-fold increase in evoked
release. These considerations favor a mechanism that does not require
sustained enhancement of cytosolic Ca. In addition, during the
recording periods, which included ~30% of the entire interval
between trains, we were unable to detect toxin-induced channel activity
in the background current or an enhanced frequency of brief spontaneous
cytosolic Ca transients that is sometimes seen in these cells. Although
we cannot eliminate the possibility of Ca entry during nonrecorded
periods, in three out of the six cells we observed stable augmentation
of Cm for several determinations in the
absence of interval changes in background cytosolic Ca or Ca handling.
Altogether these data make a mechanism of continuous enhancement of
depolarization-evoked release that is independent of a toxin-induced
rise in cytosolic Ca more plausible than a mechanism of intermittent
enhancement of depolarization-evoked release involving bursts of Ca
entry. (2) In contrast, in three out of the eight cells, brief exposure
(3-8 min in two cells) or more prolonged exposure (12 min in another
cell; see Fig. 8, right column) to toxin resulted in a
rise in background to a level of several hundred nanomolar. Under these
conditions, we observed the largest toxin-induced increments in evoked
release (averaging fourfold). However these increases were accompanied
by intermittent inward current pulses ~20 pA in amplitude. In
addition, in two experiments the half-times for recovery of cytosolic
Ca were prolonged by a factor of nearly 2.5. The latter observations
suggest that steady-state, low-grade channel activity and altered
cytosolic Ca handling can contribute to enhancement of release by
toxin.
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Figure 8.
Simultaneous monitoring of effects of toxin on
baseline and depolarization-evoked changes in membrane capacitance
(Cm) and cytosolic Ca (in this figure
given as the ratio of fluorescence emission after excitation at 340 and
380 nm, i.e.,
F340/F380).
Background membrane current (Ibackground)
and depolarization-evoked currents are also shown. Left,
Pretoxin data; Middle, Right, Post-toxin
data at 7.5 and 12.5 min, respectively. Note that the 2.5-fold increase
in exocytotic response to the depolarizing train seen 7.5 min after
addition of toxin is accompanied by a <10% increase in background
fluorescence ratio (and estimated cytosolic Ca) and only a 5% rise in
the peak level of the fluorescence ratio (and estimated cytosolic Ca)
over those in the control period. However, the further increase in
exocytotic response observed at 12.5 min after addition of toxin occurs
in the presence of a sustained rise in background cytosolic Ca but not
in the peak level of cytosolic Ca. In fact, at 12.5 min, the summed Ca
entry during the train was ~20% less than that at 7.5 min, because
of the initially smaller Ca current and its faster "run-down" with
repeated activity. The discrete steps seen in background current
(Ibackground) at 12.5 min most likely
represent short-lived openings of toxin-induced ion channels. Note at
each time point that the first Cm response
within the train was primarily a step-like change, whereas the
responses to subsequent depolarizations took on a creeping component
that at 7.5 and 12.5 min ultimately dominated the response and
continued for several seconds.
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Comparison of rates of toxin-enhanced spontaneous exocytosis with
rates of asynchronous release after depolarization
The results of the previous section suggested that at low doses LT
enhances depolarization-evoked release at least in part by a mechanism
independent of channel formation and enhancement of cytosolic Ca. At
higher doses of toxin, this alternative mechanism should work in
parallel with channel formation to contribute to the massive
enhancement of spontaneous exocytosis. To estimate the relative
contributions of channel formation and the alternative mechanism to
spontaneous exocytosis, we measured the Ca threshold and the rate of
exocytosis at the higher cytosolic Ca that is seen during this mode of
release and compared these values with those measured for
"asynchronous" exocytosis occurring during and immediately after a
train of depolarizing pulses. As shown in Figure 8, in response to a
train of depolarizing pulses, an increasing fraction of evoked release
occurs as a slow "creep" of capacitance, lasting up to several
seconds after Ca entry has ceased, rather than an abrupt "step" in
capacitance occurring "simultaneously" with (i.e., during or within
a few milliseconds after) Ca entry. Although the step likely represents
exocytosis of granules closely colocalized with Ca channels in response
to a localized rise of Ca to many tens of micromoles per liter, the creep likely represents exocytosis of granules located up to several hundred nanometers from Ca channels in response to much lower levels of
Ca, even approximating the Ca concentration "equilibrated" over the
bulk of the cytoplasm (Klingauf and Neher, 1997). It is likely that
spontaneous exocytosis seen with toxin also represents fusion of
granules poorly colocalized with the Ca entry site, because even when
promoting massive Ca entry through single channels (see Fig.
5B), the toxin only enhances release after the average cytosolic Ca has risen to ~1 µM.
Figure 9 shows our analysis of one of
five experiments in which in the same cell we compared the Ca threshold
and the rates of exocytosis at other cytosolic Ca levels that were seen
with spontaneous toxin-enhanced exocytosis versus asynchronous
exocytosis evoked by trains of depolarizations at two different
frequencies. Note that both modes of exocytosis commence at similar
cytosolic Ca, here expressed as the ratio
F340/F380
(0.45-0.5). However, at similar suprathreshold concentrations
(F340/F380
averaging 0.6) achieved at comparable times after the start of enhanced Ca entry, the rate of spontaneous exocytosis is fivefold faster than
the rate of asynchronous exocytosis. In a series of five similar
experiments, the rate of spontaneous exocytosis in response to the
presence of toxin averaged 2.6 ± 0.5 fold greater than that of
asynchronous depolarization-evoked exocytosis. These results suggest
that although Ca entry is the critical factor in initiating toxin-enhanced spontaneous exocytosis, an alternative mechanism works
to augment, by several fold, the effects of sustained Ca entry.
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Figure 9.
Toxin-enhanced spontaneous exocytosis has a
similar threshold value of [Ca]i but occurs at a faster
rate than does asynchronous exocytosis accompanying trains of
depolarization measured in the same cell. Top, This cell
was subjected to two trains of five 100 msec depolarizations to +10 mV,
first at 1 Hz and later at 0.5 Hz, and changes in
Cm and cytosolic Ca were monitored.
Bottom, Then 1 nM toxin was added,
and these parameters were again monitored. In the case of
depolarization-evoked release, the Ca threshold was determined as the
peak Ca level associated with the first response in the train to show
distinct capacitance creep (see broken arrows downward
and to the right). In the case of toxin-enhanced
"spontaneous exocytosis," the Ca threshold was determined as the
mean value of Ca over the range in which
Cm first averaged 100 fF above a stable
baseline. This criterion was chosen because in most experiments an
increase in background membrane conductance caused by toxin was
reflected as an increase in capacitance noise. The rate of capacitance
increase at the suprathreshold concentration(s) was determined as the
slope of the line tangent to the Cm curve
over the time interval corresponding to the designated cytosolic Ca
range (see broken arrows to the left and
upward). In the case of the asynchronous exocytosis, the
Ca level chosen was the peak level at the end of the train. The average
value obtained for the two runs shown was chosen as the Ca value for
use with toxin-related release.
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DISCUSSION |
Overview
In this study we used the adrenal chromaffin cell as a "model"
system to study the relationship of changes in membrane conductance and
excitability to the well-known secretory effects of -latrotoxin, an
excitatory neurotoxin. Combining membrane capacitance tracking with
spectrofluorometry in the readily patch-clamped chromaffin cell, we
have found important new evidence implicating Ca2+
entry via toxin-induced channels and resultant Ca accumulation in the
cytosol as a critical mechanism by which the toxin initiates the
enhancement of spontaneous exocytosis routinely seen at concentrations of toxin in excess of 100 pM. We present other evidence
indicating the ability of lower doses of toxin (20-50 pM)
to enhance depolarization-evoked exocytosis even under conditions in
which the toxin produces no background rise in cytosolic Ca or in
readily apparent channels. By comparing rates of exocytosis seen during
toxin-enhanced "spontaneous release" with those seen during
asynchronous exocytosis evoked by trains of depolarizations that raise
Ca to similar levels, we obtained evidence suggesting that an
additional action of toxin greatly augments the effects of channel
formation and Ca entry in sustaining toxin-enhanced spontaneous
exocytosis. It is likely that this additional action involves the
binding of toxin to a newly cloned G-protein-related toxin receptor
(CIRL/latrophilin).
Toxin-induced spontaneous exocytosis: dependence on Ca entry
Our experiments examining ion channels induced by the toxin
demonstrated that these channels have interesting and novel features. (1) They have a huge unitary conductance (up to 400 pS). (2) They are
highly selective for cations over anions (Erev
shifts negatively by 15 mV on dilution of PSS by 50%) yet allow
passage of bulky univalent cations such as NMDG. (3) They are highly
permeable to Ca (i.e., in isotonic-Ca PSS, inward currents seen at 70
mV were approximately one-third those in control PSS, whereas
Erev was unchanged). Using a modified form of
the Goldman-Hodgkin-Katz equation (Fatt and Ginsborg, 1958), we
calculate that
PCa/PCs = 0.6. This work confirms and extends an earlier characterization of
toxin-induced channels in neuroblastoma cells (Hurlbut et al., 1994).
Experiments examining cytosolic Ca levels simultaneously with
exocytosis demonstrated that the initiation of toxin-enhanced spontaneous exocytosis is attributable to the ability of membrane-bound -LT to form Ca-permeable channels through which Ca can enter into,
and then accumulate within, the cytoplasm. In control PSS, as well as
in isotonic-Ca PSS, exocytosis begins only after cytosolic free Ca has
risen to 500-1000 nM. This "threshold value" of
cytosolic Ca resembles that seen when patch-clamped chromaffin cells
are dialyzed with patch pipette solutions of known Ca concentration (Augustine and Neher, 1992). In isotonic-Ca PSS, in which the total
toxin-induced inward current is carried by Ca, the rise in Ca and the
onset of secretion follow the sustained (several seconds long) opening
of a single channel. Although there is a consistent delay in the onset
of toxin-induced secretion after the opening of toxin-induced channels,
secretion is continuous after it commences. This feature resembles the
asynchronous exocytosis observed when chromaffin cells are subjected to
prolonged electrical depolarization (Engisch and Nowycky, 1996; Barnett
et al., 1997; Klingauf and Neher, 1997); the latter may reflect
"loose colocalization" between sites of Ca entry and high affinity
release-activating sites (Kd of several
micromolar).
Several lines of evidence make it likely that Ca entry is occurring
through a channel induced de novo by the toxin itself or by
the toxin-liganded receptor. First, the conductance of the toxin-related channel greatly exceeds that of native cation channels. Its estimated single-channel conductance of 130 pS in isotonic external
Ca far exceeds the largest conductance (20-25 pS) measured for an
L-type Ca channel in isotonic Ba or for a stretch-activated nonselective cation channel in isotonic Ca (~10 pS). Its estimated conductance of 400 pS in divalent cation-free PSS far exceeds that of
the Ca channels (60 pS) or even stretch- or transmitter-activated, nonselective cation channels (40-60 pS) measured under similar conditions (see, for example, Hess et al., 1986). Second, toxin-induced channel activity and secretion persists even though voltage-dependent channels are pharmacologically blocked with TTX and Cd. Because the
toxin induces channels with similar conductance and selectivity in
planar bilayers lacking any protein receptors (Finkelstein et al.,
1976), it is likely that the toxin itself is the critical channel-forming agent.
Is there a role for an action of toxin that is independent of
channel forming ability?
Contribution to toxin-enhancement of
depolarization-evoked release?
We have also shown that low concentrations of -LT, incapable of
supporting massive spontaneous quantal release, do enhance release
evoked by repetitive depolarization. Mechanistically, enhancement of
evoked quantal release might occur in a Ca entry-dependent manner,
namely, if the toxin provided a "parallel pathway" for Ca entry,
thereby transiently or persistently raising cytosolic Ca2+. Alternatively it might occur in a manner
independent of toxin-induced Ca entry, especially if the transmembrane
or cytoplasmic domain(s) of a toxin-activated receptor interacted
directly with the secretory apparatus to enhance release. Candidate
substrates for these interactions are now plentiful. In
vitro, synaptotagmin, the putative Ca receptor in Ca-dependent
secretion, binds to a neurexin-like receptor for -LT, whereas
syntaxin, a putative key component of the granule docking and fusion
complex, binds CIRL/latrophilin, the presumed G-protein-coupled
receptor for -LT.
Our observations that enhancement by toxin of depolarization-evoked
release can occur in the absence of demonstrable toxin-induced channel
activity and a sustained increase in cytosolic Ca is consistent with a
mechanism of action independent of channel formation. An interesting
possibility is that toxin increases the size of the readily releasable
pool of granules (RRP). A G-protein-coupled receptor might activate an
effector system such as protein kinase C, whose enhanced activity might
increase the RRP (Gillis et al., 1996). Other possibilities are a
change in Ca sensitivity of release or a change in Ca handling by
sequestration or extrusion machinery, either of which might be caused
by interaction of a liganded toxin receptor with another aspect of the
secretory apparatus. Those possibilities are worth extensive further
investigation. To be sure, an important caveat is that based on our
noncontinuous recording, we cannot rule out the occurrence of transient
bursts of channel activity resulting in Ca-induced enhancement of the
RRP. However, a comparison of the rates of toxin-enhanced spontaneous
exocytosis and asynchronous exocytosis occurring during repetitive
depolarization provides additional evidence in favor of a mechanism
independent of channel formation.
Contribution to spontaneous release?
As discussed above, it is reasonable to assume that the sustained
asynchronous exocytosis evoked by repetitive depolarization and the
toxin-enhanced spontaneous exocytosis, both of which are dependent on
Ca entry but slow-to-start thereafter, result from granule fusions at
sites distant from Ca entry that better reflect global rather than
highly localized cytosolic Ca concentrations. If so, both release
patterns should require a similar threshold concentration of cytosolic
Ca to commence. Our data, although limited, suggest this is so.
However, curiously, even when the rises in cytosolic Ca develop over
similar time courses, the rates of secretion caused by the toxin are
several times higher, suggesting that the toxin exerts some action in
addition to raising cytosolic Ca. Two other recent sets of evidence
support this idea. (1) Toxin can increase release evoked in
detergent-permeabilized cells by a fixed concentration of extracellular
Ca (Bittner et al., 1998), and (2) toxin can enhance release evoked by
application of a fixed concentration of cholinergic agonist without
altering the global cytosolic Ca transient evoked by the agonist
(Michelena et al., 1997).
Conclusions
These results suggest that there may be at least two modes of
toxin action. One, operative at low doses, enhances exocytosis already
stimulated by secretagogue levels of cytosolic Ca; the other,
operative at higher doses, itself insures that those secretagogue levels will be present. The former enhances depolarization-evoked quantal release, the predominant "regulated" mode of secretion in
most excitable cells, and increases the potency of toxin enhancement of
spontaneous exocytosis seen in the absence of ongoing electrical activity. The latter actually initiates the massive enhancement of
spontaneous exocytosis seen at high doses of toxin.
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FOOTNOTES |
Received April 15, 1998; revised May 28, 1998; accepted June 1, 1998.
This work was supported by the Barnes-Jewish Hospital Research
Foundation and the National Institutes of Health Grant DK37380. We
thank Dr. Alexander Petrenko for the gift of purified toxin, Dr. David
Barnett for the design and continuing refinement of our recording
setup, and Dr. Kevin Gillis for invaluable advice.
Correspondence should be addressed to Dr. Stanley Misler, Renal
Division (Yalem 815), Barnes-Jewish Hospital-North, 216 South Kingshighway, St. Louis, MO 63110.
We dedicate this paper as a "Festschrift" to Dr. William P. Hurlbut, a pioneer in latrotoxin research.
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REFERENCES |
-
Artalejo CR,
Henley JR,
McNiven MA,
Palfrey HC
(1995)
Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin.
Proc Natl Acad Sci USA
92:8328-8332[Abstract/Free Full Text].
-
Augustine GA,
Neher E
(1992)
Calcium requirements for secretion in bovine chromaffin cells.
J Physiol (Lond)
450:247-271[Abstract/Free Full Text].
-
Barnett DW,
Misler S
(1997)
An optimized approach to membrane capacitance estimation using dual frequency excitation.
Biophys J
72:1641-1658[Web of Science][Medline].
-
Barnett DW,
Liu J,
Misler S
(1996)
Single-cell measurements of quantal secretion induced by rat adrenal chromaffin cells: dependence on extracellular Ca2+.
Pflügers Arch
432:1039-1046[Web of Science][Medline].
-
Barnett DW,
Liu J,
Misler S
(1997)
Depolarization-induced asynchronous release from rat adrenal chromaffin cells.
Biophys J
72:A157.
-
Barry PH,
Lynch JW
(1991)
Liquid junction potentials and small cell effects in patch-clamp analysis.
J Membr Biol
121:101-117[Web of Science][Medline].
-
Bennett MK,
Scheller RH
(1994)
A molecular description of synaptic vesicle membrane trafficking.
Annu Rev Biochem
63:63-100[Web of Science][Medline].
-
Bittner M,
Krasnoperov VG,
Stuenkel EL,
Petrenko AG,
Holz RW
(1998)
A Ca2+-independent receptor for latrotoxin, CIRL, mediates effects on secretion via multiple mechanisms.
J Neurosci
18:2914-2922[Abstract/Free Full Text].
-
Bittner MA,
Das Gupta BR,
Holz RW
(1989)
Isolated light chains of botulinum toxin inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells.
J Biol Chem
264:10354-10360[Abstract/Free Full Text].
-
Chow RH,
von Rueden L,
Neher E
(1992)
Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.
Nature
356:60-63[Medline].
-
Chow RH,
Klingauf J,
Neher E
(1994)
Time course of Ca2+ concentration triggering exocytosis in neuroendocrine cells.
Proc Natl Acad Sci USA
91:12765-12769[Abstract/Free Full Text].
-
Davletov BA,
Shamotienko OG,
Lelianova VG,
Grishkin EV,
Ushkaryov YA
(1996)
Isolation and biochemical characterization of a Ca2+-independent -latrotoxin-binding protein.
J Biol Chem
271:23239-23245[Abstract/Free Full Text].
-
Dunn LA,
Holz RW
(1983)
Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells.
J Biol Chem
258:4989-4993[Abstract/Free Full Text].
-
Engisch KL,
Nowycky MC
(1996)
Calcium dependence of large dense-cored vesicle exocytosis evoked by calcium influx in bovine adrenal chromaffin cells.
J Neurosci
16:1359-1369[Abstract/Free Full Text].
-
Fatt P,
Ginsborg BL
(1958)
The ionic requirements for the production of action potentials in crustacean muscle fibres.
J Physiol (Lond)
142:516-543.
-
Finkelstein A,
Rubin LL,
Tzeng MC
(1976)
Black widow spider venom: effect of purified toxin on lipid bilayer membranes.
Science
193:1009-1011[Abstract/Free Full Text].
-
Gillis KD,
Moessner R,
Neher E
(1996)
Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules.
Neuron
16:1209-1220[Web of Science][Medline].
-
Grynkiewicz G,
Poenie M,
Tsien RY
(1985)
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:3440-3450[Abstract/Free Full Text].
-
Heinemann C,
Rueden LV,
Chow RH,
Neher E
(1993)
A two-step model of secretion control in neuroendocrine cells.
Pflügers Arch
424:105-112[Web of Science][Medline].
-
Hess P,
Lansman JB,
Tsien RW
(1986)
Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells.
J Gen Physiol
88:293-319[Abstract/Free Full Text].
-
Hurlbut WP,
Ceccarelli B
(1979)
Use of black widow spider venom to study the release of neurotransmitters.
In: Neurotoxins, tools in neurobiology (Ceccarelli B,
Clementi F,
eds), pp 87-115. New York: Raven.
-
Hurlbut WP,
Chieregatti E,
Valtorta F,
Haimann C
(1994)
-Latrotoxin channels in neuroblastoma cells.
J Membr Biol
138:91-102[Web of Science][Medline].
-
Klingauf J,
Neher E
(1997)
Modeling buffered Ca2+ diffusion near the membrane: implications for secretion in neuroendocrine cells.
Biophys J
72:674-690[Web of Science][Medline].
-
Krasnoperov VG,
Beavis R,
Chepurny OG,
Little AR,
Plotnikov AN,
Petrenko AG
(1996)
The calcium-independent receptor of alpha-latrotoxin is not a neurexin.
Biochem Biophys Res Commun
227:868-875[Web of Science][Medline].
-
Littleton JT,
Bellen HJ
(1995)
Synaptotagmin controls and modulates synaptic-vesicle fusion in a Ca2+-dependent manner.
Trends Neurosci
18:177-183[Web of Science][Medline].
-
Liu J,
Misler S
(1998a)
Alpha-latrotoxin alters spontaneous and depolarization evoked quantal release from rat adrenal chromaffin cells.
Biophys J
74:A95.
-
Liu J,
Misler S
(1998b)
-Latrotoxin-induced quantal release of catecholamines from rat adrenal chromaffin cells.
Brain Res
799:56-64.
-
Longenecker HE,
Hurlbut WP,
Mauro A,
Clark AW
(1970)
Effects of black widow spider venom on the frog neuromuscular junction.
Nature
225:701-703[Medline].
-
Meldolesi J,
Madeddu L,
Torda M,
Gatti G,
Niutta E
(1983)
The effect of -latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release.
Neuroscience
10:997-1009[Web of Science][Medline].
-
Michelena P,
de la Fuente MT,
Vega T,
Lara B,
Lopez MG,
Gandia L,
Garcia AG
(1997)
Drastic facilitation by -latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i.
J Physiol (Lond)
502:481-496[Abstract/Free Full Text].
-
Misler S,
Hurlbut WP
(1979)
Action of black widow spider venom on quantized release of acetylcholine at the frog neuromuscular junction: dependence upon external Mg2+.
Proc Natl Acad Sci USA
76:991-995[Abstract/Free Full Text].
-
Neely A,
Lingle CJ
(1992)
Two components of calcium-activated potassium current in rat adrenal chromaffin cells.
J Physiol (Lond)
453:97-131[Abstract/Free Full Text].
-
Neher E
(1992)
Correction for liquid junction potentials in patch clamp experiments.
Methods Enzymol
207:123-131[Web of Science][Medline].
-
Neher E,
Marty A
(1982)
Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc Natl Acad Sci USA
79:6712-6716[Abstract/Free Full Text].
-
Nicholls DG,
Rugolo M,
Scott IG,
Meldolesi J
(1982)
-Latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx and stimulates transmitter release in guinea pig brain synaptosomes.
Proc Natl Acad Sci USA
79:7924-7928[Abstract/Free Full Text].
-
Petrenko AG
(1993)
-Latrotoxin receptor. Implications in nerve terminal function.
FEBS Lett
325:81-85[Web of Science][Medline].
-
Petrenko AG,
Perin MS,
Davletov BA,
Ushkaryov YA,
Geppert M,
Suedhof TC
(1991)
Binding of synaptotagmin to the -latrotoxin receptor implicates both in synaptic vesicle exocytosis.
Nature
353:65-68[Medline].
-
Rosenthal L,
Meldolesi J
(1989)
-Latrotoxin and related toxins.
Pharmacol Ther
42:115-134[Web of Science][Medline].
-
Smith C,
Neher E
(1997)
Multiple forms of endocytosis in bovine adrenal chromaffin cells.
J Cell Biol
139:885-894[Abstract/Free Full Text].
-
Ushkaryov YA,
Petrenko AG,
Geppert M,
Suedhof TC
(1992)
Neurexins: synaptic cell surface proteins related to the -latrotoxin receptor and laminin.
Science
257:50-56[Abstract/Free Full Text].
-
von Rueden L,
Neher E
(1993)
A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells.
Science
262:1061-1065[Abstract/Free Full Text].
-
Wightman RM,
Jankowski JA,
Kennedy RT,
Kawagoe KT,
Schroeder TJ,
Leszczyszyn DJ,
Near JA,
Diliberto Jr EJ,
Viveros OH
(1991)
Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.
Proc Natl Acad Sci USA
88:10754-10758[Abstract/Free Full Text].
-
Zhou Z,
Misler S
(1995)
Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.
Proc Natl Acad Sci USA
92:6938-6942[Abstract/Free Full Text].
-
Zhou Z,
Misler S
(1996)
Amperometric detection of quantal secretion from patch-clamped rat pancreatic
 -cells.
J Biol Chem
271:270-277[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18166113-13$05.00/0
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Top
Abstract
Introduction
