Neuron,
Voi. 4, 643-654,
May, 1990, Copyright
0 1990 by Cell Prep
Properties of the Fusion Pore That Forms during Exocytosis of a Mast ell Secretory A. E. Spruce, L. J. Breckenridge, and W. Almers Department of Physiology and University of Washington Seattle, Washington 98195
A. K. Lee, Biophysics
Summary During exocytosis, secretory vesicles of mast cells generate a current transient that marks the opening of the fusion pore, the first aqueous connection that forms between the vesicle lumen and the cell exterior. By recording and analyzing such current transients, we have tracked the conductance of the fusion pore over the first millisecond of its existence. The first opening of the pore occurs rapidly, generally within 400 us at 23OC. The electric conductance of the pore is a few hundred picosiemens at first, but gradually increases over the subsequent milliseconds. Evidently the pore opens abruptly and then dilates. The initial conductance of the pore suggests a diameter comparable to that of a large ion channel. From an analysis of “capacitance flicker” we infer that a pore can increase its diameter severalfold and still close again completely. This suggests that several early events in membrane fusion are reversible. Introduction During exocytosis, the membrane surrounding a cytosolic vesicle fuses with the plasma membrane, and the contents of the vesicle are released into the extracellular space. Neurons as well as all other eukaryotic cells engage in exocytosis; indeed, exocytosis is but one example of the extensive traffic of vesicles taking place between the various membrane-bounded compartments in every eukaryotic cell. One of the most critical events in this traffic remains a mystery: how do the membranes of the various vesicles and compartments fuse? Current concepts about membrane fusion have been influenced by studies of artificial lipid systems (Verkleij, 1984; Wilschut and Hoechstra, 1984; Finkelstein et al., 1986), by studies of the specific proteins that catalyze the fusion of viral envelopes to host cell membranes (Stegman et al., 1989), and, perhaps most importantly, by quick-freeze electron microscopy. In neurons (Heuser and Reese, 1981) and in secretory cells (e.g., Chandler and Heuser, 1980; Ornberg and Reese, 1981; Schmidt et al., 1983), vesicles frozen in early stages of exocytosis appear connected to the extracellular space by narrow pores. The diameters of such “fusion pores”vary widely, probably because the pores are small at first and then dilate rapidly. Similar pores are observed when influenza virus fusion proteins mediate the fusion of virions to canine kidney cells (Knoll et al., 1988). Throughout, the smallest
Vesic
pores that are readily seen have diameters of IO-20 nm. Do they represent the earliest stage of exocytosis, or do they arise from precursors too small or too short-lived to be detected by present morphologic techniques? Recently it has become possible to detect, in real time, the exocytosis of single secretory vesicles by monitoring the electrical capacitance of the plasma membrane. Stepwise increases in capacitance are seen when the membrane of single vesicles is added to the cell surface (Neher and Marty, 1982; Fernandez et al., 1984). The largest secretory vesicles are found in the mast cells of a mutant mouse, the “beige” mouse. Like normal mast cells of rats and mice, those of beige mice readily undergo exocytosis in response to the mast cell secretagogue, compound 48/80 (Poon et al., 1981), and to internal application of GTP$% In time-resolved recordings of single exocytic events (Breckenridge and Almers, 1987a; Zimmerberg et al., 1987; Almers and Breckenridge, 1988; Alvarez de Toledo and Fernandez, 1988), the giant vesicles of these mast ceils allow one to detect details that are hard to see during exocytosis of the smaller vesicles found in more normal cells. In particular, the exocytosis of each giant vesicle generates a brief electric current that flows through the fusion pore at the instant the pore first opens (Breckenridge and Almers, 198713). Analysis of such transients has shown that the pore has an initial conductance not much larger than that of a single gap junction channel and hence must have molecular dimensions. Here we analyze such transients to gain insight into the first millisecond of the exocytic process. Results Time-Resolved Recording of Exocytosis at the Level of Single Vesicles Figure IA monitors secretion in a degranulating mast cell from a beige mouse. The membrane capacitance, a measure of the cell surface area, is plotted as a function of time. Between IO and 20 s before the trace started, CTPyS (20 KM) had begun to enter the cell through the patch pipette. In mast cells, CTPyS bypasses some of the normal stimulus-secretion coupling reactions and stimulates secretion more directly (Fernandez et al., 1984). As the GTPyS took effect, the cell began to secrete and the capacitance increased in steps. Each step represents the exocytosis of one secretory vesicle (Breckenridge and Almers, 1987a; Zimmerberg et al., 1987; Almers and Breckenridge, 1988). Figure IB plots the membrane conductance (G,,) measured with an 800 Hz alternating current. C,, includes the conductance through ion channels in the plasma membrane, as well as contributions from fusion pores as they form electric connections between the inside of exocytosing vesicles and the outside of
Neuron 644
7 previous results dez, 1986).
Figure 1. Capacitance Degranulating Mast
and Cell
Conductance
CC,,)
Changes
of
a
(A) Capacitance. (B) C,,. The trace starts about IO-20 s after whole-cell contact was established and GTPyS (20 PM) started to diffuse into the cell. A 20 s section without steps was excised from the trace (parallel lines). (C and D) The first capacitance step in (A) (arrow) and the accompanying changes in G,, plotted at higher speed. Each point represents the time average over a 12.5 ms period or ten sinusoidal cycles. Between each 12.5 ms period, the sinusoid was switched off for 112.5 ms to record membrane current without disturbance from the sinusoid. (E) Schematic drawing showing an exocytosing vesicle connected to the plasma membrane by a fusion pore. The resistance of the fusion pore is l/g, and C, is the capacitance of the vesicle membrane. (F) Membrane current during the 112.5 ms interval marked by arrows in (D), i.e., the interval immediately preceding the jumps in capacitance and C,,. Note the outward current transient at the end of the trace that occurs as the fusion pore opens and the vesicle discharges its membrane capacitance through the pore. No such transient was observed in the ten intervals preceding or following the episode shown. The capacitanceof this cell was 8.4 pF at the beginning of the experiment (dashed line in [A]) and 12.4 pF at the end. The conductance connecting the pipette with the cytosol was 0.20 PS at the beginning and 0.19 uS at the end. Hence we consider that our voltage clamp controlled the membrane potential with time constants varying from 41 us at the beginning to 67 TIS at the end of the experiment.
the
cell.
We
used
alternating
current
because
the
con-
of a fusion pore (g in Figure IE) is electrically in series with the vesicle membrane and hence will not show up in conventional direct current conductance measurements with voltage steps. G,, shows several transient increases, each associated with a vesicle fusion event, but only a modest permanent increase. Evidently, secretion occurred without a significant activation of ion channels in the cell membrane, and the membrane of the secretory vesicles contained few or no open ion channels. This confirms
ductance
on
rat mast
cells
(Lindau
and
Fernan-
The Last 200 ms of a Secretory Vesicle Figures IC and ID magnify the first exocytic event in Figures IA and IB (arrow). The capacitance increase reports the opening of this vesicle’s fusion pore. Note that the capacitance step does not rise instantly and that it is accompanied by a transient increase in G,,. Both features are expected if the conductance of the connection (g in Figure IE) is small at first and grows with time. As in previous work (Breckenridge and Almers, 198713; Spruce et al., 1989), the pore conductance can be calculated independently from the capacitance and G,, traces. If we take the capacitance of the vesicle (C,) as equal to the final amplitude of the capacitance step, then the pore conductance is 2.61 nS immediately after the initial jumps in capacitance and G,,, 3.1 nS when capacitance and G,, were measured again 112.5 ms later, %I nS after an additional 112.5 ms, and too large to measure thereafter. Figure IF traces the current collected from the mast cell membrane during the last 112.5 ms before the jump in capacitance and G,,. During the final 5 ms, there is an outward current transient as the exocytosing vesicle discharges its membrane potential through the nascent fusion pore (Breckenridge and Almers, 198713). Similar transients were recorded prior to all of the 26 other exocytic events subsequently observed in this cell. These transients represent the first step in membrane fusion that can be recorded in electrophysiologic experiments and are the subject of this paper. The transient of Figure IF is shown magnified in Figure 2A. It was analyzed to determine the conductance of the fusion pore during the first millisecond of its existence. First, the time integral of the current transient
was
formed
(Figure
28).
Its
final
amplitude
di-
vided by C, equals the difference in potentials across plasma (E,) and vesicle (E,) membrane at the instant the pore first opens (Breckenridge and Almers, 1987b). It was 138 mV in this vesicle. Figure 2C represents the potential that drives the current through the fusion of time. It was obpore, i.e., (E, - E,) as a function tained by subtracting the trace in Figure 2B from its final value and dividing it by C,. Starting at 138 mV, it declined to zero as the vesicle adjusted the charge on its membrane capacitance and as its membrane potential approached that of the plasma membrane. Finally, the trace in Figure 2D was obtained by dividing the trace in Figure 2A by that in 2C. It represents g, the conductance of the fusion pore as a function of time. The conductance increased abruptly as the pore opened and continued to increase more slowly thereafter. The two circles connected to Figure 2D with a dashed line were calculated from the first measurements of capacitance and G,, taken after the transient (see Figures IC and ID). They show that the conductance of the fusion pore continued to increase for the next IO ms.
Fusion 645
Pores
and Exocytosis
-6 -5
u sm P 2 2 m
4: -3
5 -
-2
_I
10
1 01
ms after the conductance jump. Vesicles that exocytose with a large initial conductance discharge their membrane potential rapidly; their transients and conductance traces are shorter, and hence they are underrepresented in this signal average. To illustrate the size of the structure that might give rise to the conductances in Figure 2, the right-hand ordinate of Figure 2D converts conductance into pore diameter (Equation 8-1 in Hille, 1984). We attribute the entire conductance to a single dilating pore (see below). The pore is assumed to be filled with a salt solution of about the same resistivity (100 Qcm) as that of our bathing fluid and to have the length of a gap junction channel (15 nm; Unwin and Zampighi, 1980), the only ion channel known to span two membranes. Under these assumptions, the pore was at first only 2-3 nm wide. After 10 ms, however, when the capacitance and G,, were next measured, the pore diameter had more than doubled. Later G,, measurements in Figures IC and ID suggest that after another 250 ms, the pore had grown to 16 nm, approaching the size of the smallest fusion pores observed in quick-freeze electron micrographs of secreting mast cells (Chandler and Heuser, 1980).
0.5 ms Figure
2. Analysis
of Current
Transients
(A) Current transient in Figure ID at faster sweep speed. (B) Charge carried by the transient, represented by the time integral of (A). (C) Potential across the fusion pore obtained by subtracting trace (B) from its final value and dividing the result by the C, (286 fF). (D) Pore conductance calculated by dividing trace (A) by trace (C). The two circles connected to (D) with a dashed line were calculated from the first measurements of capacitance and G,, taken after the transient (see Figures IC and 1D). They show that the conductance of the fusion pore continues to increase for the next 10 ms. (E) 219 traces similar to that in (D) were selected from our data baseof 904 traces, aligned in time to coincide at the point where the conductance first reached a value of g,/2, and averaged (g, is defined in Figure 3, inset). We included all conductance traces whose points retained a variance of less than 10,000 (pS)* for at least 0.8 ms after the beginning of the current transient. The variance of individual points was estimated as follows. First, the variance of points about the baseline of the conductance trace was calculated from the section of the trace preceding the current transient. The average root-meansquare (rms) noise of the conductance baseline was 23 f 0.3 pS (n = 617). In later portions of the conductance trace, the variance increased as the current trace was divided by a progressively smaller voltage. To allow for this, we assumed that the noise on the current trace (i.e., on trace [A]) remained constant. Once the transient has started, the variance of successive conductance measurements should then increase in inverse proportion to the square of the voltage across the fusion pore. Hence we scaled the voltage trace (i.e., trace [Cl) to have an initial amplitude of 1.0 and estimated the variance of individual conductance measurements to be equal to the baseline variance divided by the square of the scaled voltage.
Figure 2E represents the signal average of 219 traces of the type in Figure 2D. The trace confirms that the conductance increases rapidly as the pore first opens and then contihues to increase more slowly. The initial pore conductance, gi, in Figure 2E, is less than average because we selected traces lasting at least 0.8
The Pore Conductance Is Variable Figure 3 presents analyses of experiments like those in Figure 2 and includes data at lower tern (shaded in Figure 3A; solid symbols in Figure 3C), which are discussed later. To estimate gi, a straight line was fitted to the conductance traces as illustrated in the inset of Figure 3A. A histogram shows that g, varies over an order of magnitude. Its median, 283 pS, is slightly larger than that obtained in a previous, simpler analysis (Breckenridge and Almers, 1987b). The broad histogram may well hide multiple subconductance states. Such subconductance states may have failed to appear as separate peaks because an individual gi measurement is not very accurate (see Figure 3, legend) and because the pore conductance may be influenced by the variable pH inside the vesicles (not all vesicles accumulate the membrane-permeant fluorescent base quinacrine; Breckenridge and Almers, 1987a). Figure 3B shows a histogram of C, values for the 620 vesicles on which Figure 3A is based; vesicles varied widely in size (Breckenridge and Almers, 7987aI. gi grows with vesicle size, increasing 2-to 3-fold as C, increases g-fold (Figure 3C). But gi is independent of E, (range -140 to -50 mV) and of (f, - EJ (range 60-160 mV). Once the pore had opened, its conductance continued to increase at a relatively slower, but highly variable rate, a. In some fusions the conductance increased at rates of thousands of picosiemens per miflisecond, whereas in others it remained constant over the duration of the transient. a showed a weak positive correlation with gi (data not shown; regression line is a = (0.29 gi + 176) (pS/ms); correlation coefficient r = 0.12, significant at the P < 0.01 level, twotailed t-test).
0
0
500
1000
1500
Si @‘I B
8-
es Fa,
0
cunmn
0
0
400 C,
’
I-
3
1
800
4I +
Figure 3. The Initial Vesicle Capacitance
200
C,WI
Pore Conductance
600
400
and
Its Relation
Effects of Temperature Cooling by @‘C-lO°C (shaded in Figure 3A; of vesicle size (Figure effect is probably due of electrolyte conductivity. stance, the conductivity 28% upon cooling by 1972). The small effect
reduced gi by about one-third median 190 pS), independently 3C, filled symbols). Most of this to the temprature dependence In skeletal muscle, for inof cytoplasm diminishes by 10°C (Hodgkin and Nakajima, of temperature fits with the
idea that the fusion pore is an electrolyte-filled channel. In cooled cells, there were more transients of the type shown in Figure 44 (see below). The frequency of transients like the one in Figure 4B was diminished (from 15% to 5%), and that of transients like the one in Figure 4D was increased (from
Voi. 4, 643-654,
May, 1990, Copyright
0 1990 by Cell Prep
Properties of the Fusion Pore That Forms during Exocytosis of a Mast ell Secretory A. E. Spruce, L. J. Breckenridge, and W. Almers Department of Physiology and University of Washington Seattle, Washington 98195
A. K. Lee, Biophysics
Summary During exocytosis, secretory vesicles of mast cells generate a current transient that marks the opening of the fusion pore, the first aqueous connection that forms between the vesicle lumen and the cell exterior. By recording and analyzing such current transients, we have tracked the conductance of the fusion pore over the first millisecond of its existence. The first opening of the pore occurs rapidly, generally within 400 us at 23OC. The electric conductance of the pore is a few hundred picosiemens at first, but gradually increases over the subsequent milliseconds. Evidently the pore opens abruptly and then dilates. The initial conductance of the pore suggests a diameter comparable to that of a large ion channel. From an analysis of “capacitance flicker” we infer that a pore can increase its diameter severalfold and still close again completely. This suggests that several early events in membrane fusion are reversible. Introduction During exocytosis, the membrane surrounding a cytosolic vesicle fuses with the plasma membrane, and the contents of the vesicle are released into the extracellular space. Neurons as well as all other eukaryotic cells engage in exocytosis; indeed, exocytosis is but one example of the extensive traffic of vesicles taking place between the various membrane-bounded compartments in every eukaryotic cell. One of the most critical events in this traffic remains a mystery: how do the membranes of the various vesicles and compartments fuse? Current concepts about membrane fusion have been influenced by studies of artificial lipid systems (Verkleij, 1984; Wilschut and Hoechstra, 1984; Finkelstein et al., 1986), by studies of the specific proteins that catalyze the fusion of viral envelopes to host cell membranes (Stegman et al., 1989), and, perhaps most importantly, by quick-freeze electron microscopy. In neurons (Heuser and Reese, 1981) and in secretory cells (e.g., Chandler and Heuser, 1980; Ornberg and Reese, 1981; Schmidt et al., 1983), vesicles frozen in early stages of exocytosis appear connected to the extracellular space by narrow pores. The diameters of such “fusion pores”vary widely, probably because the pores are small at first and then dilate rapidly. Similar pores are observed when influenza virus fusion proteins mediate the fusion of virions to canine kidney cells (Knoll et al., 1988). Throughout, the smallest
Vesic
pores that are readily seen have diameters of IO-20 nm. Do they represent the earliest stage of exocytosis, or do they arise from precursors too small or too short-lived to be detected by present morphologic techniques? Recently it has become possible to detect, in real time, the exocytosis of single secretory vesicles by monitoring the electrical capacitance of the plasma membrane. Stepwise increases in capacitance are seen when the membrane of single vesicles is added to the cell surface (Neher and Marty, 1982; Fernandez et al., 1984). The largest secretory vesicles are found in the mast cells of a mutant mouse, the “beige” mouse. Like normal mast cells of rats and mice, those of beige mice readily undergo exocytosis in response to the mast cell secretagogue, compound 48/80 (Poon et al., 1981), and to internal application of GTP$% In time-resolved recordings of single exocytic events (Breckenridge and Almers, 1987a; Zimmerberg et al., 1987; Almers and Breckenridge, 1988; Alvarez de Toledo and Fernandez, 1988), the giant vesicles of these mast ceils allow one to detect details that are hard to see during exocytosis of the smaller vesicles found in more normal cells. In particular, the exocytosis of each giant vesicle generates a brief electric current that flows through the fusion pore at the instant the pore first opens (Breckenridge and Almers, 198713). Analysis of such transients has shown that the pore has an initial conductance not much larger than that of a single gap junction channel and hence must have molecular dimensions. Here we analyze such transients to gain insight into the first millisecond of the exocytic process. Results Time-Resolved Recording of Exocytosis at the Level of Single Vesicles Figure IA monitors secretion in a degranulating mast cell from a beige mouse. The membrane capacitance, a measure of the cell surface area, is plotted as a function of time. Between IO and 20 s before the trace started, CTPyS (20 KM) had begun to enter the cell through the patch pipette. In mast cells, CTPyS bypasses some of the normal stimulus-secretion coupling reactions and stimulates secretion more directly (Fernandez et al., 1984). As the GTPyS took effect, the cell began to secrete and the capacitance increased in steps. Each step represents the exocytosis of one secretory vesicle (Breckenridge and Almers, 1987a; Zimmerberg et al., 1987; Almers and Breckenridge, 1988). Figure IB plots the membrane conductance (G,,) measured with an 800 Hz alternating current. C,, includes the conductance through ion channels in the plasma membrane, as well as contributions from fusion pores as they form electric connections between the inside of exocytosing vesicles and the outside of
Neuron 644
7 previous results dez, 1986).
Figure 1. Capacitance Degranulating Mast
and Cell
Conductance
CC,,)
Changes
of
a
(A) Capacitance. (B) C,,. The trace starts about IO-20 s after whole-cell contact was established and GTPyS (20 PM) started to diffuse into the cell. A 20 s section without steps was excised from the trace (parallel lines). (C and D) The first capacitance step in (A) (arrow) and the accompanying changes in G,, plotted at higher speed. Each point represents the time average over a 12.5 ms period or ten sinusoidal cycles. Between each 12.5 ms period, the sinusoid was switched off for 112.5 ms to record membrane current without disturbance from the sinusoid. (E) Schematic drawing showing an exocytosing vesicle connected to the plasma membrane by a fusion pore. The resistance of the fusion pore is l/g, and C, is the capacitance of the vesicle membrane. (F) Membrane current during the 112.5 ms interval marked by arrows in (D), i.e., the interval immediately preceding the jumps in capacitance and C,,. Note the outward current transient at the end of the trace that occurs as the fusion pore opens and the vesicle discharges its membrane capacitance through the pore. No such transient was observed in the ten intervals preceding or following the episode shown. The capacitanceof this cell was 8.4 pF at the beginning of the experiment (dashed line in [A]) and 12.4 pF at the end. The conductance connecting the pipette with the cytosol was 0.20 PS at the beginning and 0.19 uS at the end. Hence we consider that our voltage clamp controlled the membrane potential with time constants varying from 41 us at the beginning to 67 TIS at the end of the experiment.
the
cell.
We
used
alternating
current
because
the
con-
of a fusion pore (g in Figure IE) is electrically in series with the vesicle membrane and hence will not show up in conventional direct current conductance measurements with voltage steps. G,, shows several transient increases, each associated with a vesicle fusion event, but only a modest permanent increase. Evidently, secretion occurred without a significant activation of ion channels in the cell membrane, and the membrane of the secretory vesicles contained few or no open ion channels. This confirms
ductance
on
rat mast
cells
(Lindau
and
Fernan-
The Last 200 ms of a Secretory Vesicle Figures IC and ID magnify the first exocytic event in Figures IA and IB (arrow). The capacitance increase reports the opening of this vesicle’s fusion pore. Note that the capacitance step does not rise instantly and that it is accompanied by a transient increase in G,,. Both features are expected if the conductance of the connection (g in Figure IE) is small at first and grows with time. As in previous work (Breckenridge and Almers, 198713; Spruce et al., 1989), the pore conductance can be calculated independently from the capacitance and G,, traces. If we take the capacitance of the vesicle (C,) as equal to the final amplitude of the capacitance step, then the pore conductance is 2.61 nS immediately after the initial jumps in capacitance and G,,, 3.1 nS when capacitance and G,, were measured again 112.5 ms later, %I nS after an additional 112.5 ms, and too large to measure thereafter. Figure IF traces the current collected from the mast cell membrane during the last 112.5 ms before the jump in capacitance and G,,. During the final 5 ms, there is an outward current transient as the exocytosing vesicle discharges its membrane potential through the nascent fusion pore (Breckenridge and Almers, 198713). Similar transients were recorded prior to all of the 26 other exocytic events subsequently observed in this cell. These transients represent the first step in membrane fusion that can be recorded in electrophysiologic experiments and are the subject of this paper. The transient of Figure IF is shown magnified in Figure 2A. It was analyzed to determine the conductance of the fusion pore during the first millisecond of its existence. First, the time integral of the current transient
was
formed
(Figure
28).
Its
final
amplitude
di-
vided by C, equals the difference in potentials across plasma (E,) and vesicle (E,) membrane at the instant the pore first opens (Breckenridge and Almers, 1987b). It was 138 mV in this vesicle. Figure 2C represents the potential that drives the current through the fusion of time. It was obpore, i.e., (E, - E,) as a function tained by subtracting the trace in Figure 2B from its final value and dividing it by C,. Starting at 138 mV, it declined to zero as the vesicle adjusted the charge on its membrane capacitance and as its membrane potential approached that of the plasma membrane. Finally, the trace in Figure 2D was obtained by dividing the trace in Figure 2A by that in 2C. It represents g, the conductance of the fusion pore as a function of time. The conductance increased abruptly as the pore opened and continued to increase more slowly thereafter. The two circles connected to Figure 2D with a dashed line were calculated from the first measurements of capacitance and G,, taken after the transient (see Figures IC and ID). They show that the conductance of the fusion pore continued to increase for the next IO ms.
Fusion 645
Pores
and Exocytosis
-6 -5
u sm P 2 2 m
4: -3
5 -
-2
_I
10
1 01
ms after the conductance jump. Vesicles that exocytose with a large initial conductance discharge their membrane potential rapidly; their transients and conductance traces are shorter, and hence they are underrepresented in this signal average. To illustrate the size of the structure that might give rise to the conductances in Figure 2, the right-hand ordinate of Figure 2D converts conductance into pore diameter (Equation 8-1 in Hille, 1984). We attribute the entire conductance to a single dilating pore (see below). The pore is assumed to be filled with a salt solution of about the same resistivity (100 Qcm) as that of our bathing fluid and to have the length of a gap junction channel (15 nm; Unwin and Zampighi, 1980), the only ion channel known to span two membranes. Under these assumptions, the pore was at first only 2-3 nm wide. After 10 ms, however, when the capacitance and G,, were next measured, the pore diameter had more than doubled. Later G,, measurements in Figures IC and ID suggest that after another 250 ms, the pore had grown to 16 nm, approaching the size of the smallest fusion pores observed in quick-freeze electron micrographs of secreting mast cells (Chandler and Heuser, 1980).
0.5 ms Figure
2. Analysis
of Current
Transients
(A) Current transient in Figure ID at faster sweep speed. (B) Charge carried by the transient, represented by the time integral of (A). (C) Potential across the fusion pore obtained by subtracting trace (B) from its final value and dividing the result by the C, (286 fF). (D) Pore conductance calculated by dividing trace (A) by trace (C). The two circles connected to (D) with a dashed line were calculated from the first measurements of capacitance and G,, taken after the transient (see Figures IC and 1D). They show that the conductance of the fusion pore continues to increase for the next 10 ms. (E) 219 traces similar to that in (D) were selected from our data baseof 904 traces, aligned in time to coincide at the point where the conductance first reached a value of g,/2, and averaged (g, is defined in Figure 3, inset). We included all conductance traces whose points retained a variance of less than 10,000 (pS)* for at least 0.8 ms after the beginning of the current transient. The variance of individual points was estimated as follows. First, the variance of points about the baseline of the conductance trace was calculated from the section of the trace preceding the current transient. The average root-meansquare (rms) noise of the conductance baseline was 23 f 0.3 pS (n = 617). In later portions of the conductance trace, the variance increased as the current trace was divided by a progressively smaller voltage. To allow for this, we assumed that the noise on the current trace (i.e., on trace [A]) remained constant. Once the transient has started, the variance of successive conductance measurements should then increase in inverse proportion to the square of the voltage across the fusion pore. Hence we scaled the voltage trace (i.e., trace [Cl) to have an initial amplitude of 1.0 and estimated the variance of individual conductance measurements to be equal to the baseline variance divided by the square of the scaled voltage.
Figure 2E represents the signal average of 219 traces of the type in Figure 2D. The trace confirms that the conductance increases rapidly as the pore first opens and then contihues to increase more slowly. The initial pore conductance, gi, in Figure 2E, is less than average because we selected traces lasting at least 0.8
The Pore Conductance Is Variable Figure 3 presents analyses of experiments like those in Figure 2 and includes data at lower tern (shaded in Figure 3A; solid symbols in Figure 3C), which are discussed later. To estimate gi, a straight line was fitted to the conductance traces as illustrated in the inset of Figure 3A. A histogram shows that g, varies over an order of magnitude. Its median, 283 pS, is slightly larger than that obtained in a previous, simpler analysis (Breckenridge and Almers, 1987b). The broad histogram may well hide multiple subconductance states. Such subconductance states may have failed to appear as separate peaks because an individual gi measurement is not very accurate (see Figure 3, legend) and because the pore conductance may be influenced by the variable pH inside the vesicles (not all vesicles accumulate the membrane-permeant fluorescent base quinacrine; Breckenridge and Almers, 1987a). Figure 3B shows a histogram of C, values for the 620 vesicles on which Figure 3A is based; vesicles varied widely in size (Breckenridge and Almers, 7987aI. gi grows with vesicle size, increasing 2-to 3-fold as C, increases g-fold (Figure 3C). But gi is independent of E, (range -140 to -50 mV) and of (f, - EJ (range 60-160 mV). Once the pore had opened, its conductance continued to increase at a relatively slower, but highly variable rate, a. In some fusions the conductance increased at rates of thousands of picosiemens per miflisecond, whereas in others it remained constant over the duration of the transient. a showed a weak positive correlation with gi (data not shown; regression line is a = (0.29 gi + 176) (pS/ms); correlation coefficient r = 0.12, significant at the P < 0.01 level, twotailed t-test).
0
0
500
1000
1500
Si @‘I B
8-
es Fa,
0
cunmn
0
0
400 C,
’
I-
3
1
800
4I +
Figure 3. The Initial Vesicle Capacitance
200
C,WI
Pore Conductance
600
400
and
Its Relation
Effects of Temperature Cooling by @‘C-lO°C (shaded in Figure 3A; of vesicle size (Figure effect is probably due of electrolyte conductivity. stance, the conductivity 28% upon cooling by 1972). The small effect
reduced gi by about one-third median 190 pS), independently 3C, filled symbols). Most of this to the temprature dependence In skeletal muscle, for inof cytoplasm diminishes by 10°C (Hodgkin and Nakajima, of temperature fits with the
idea that the fusion pore is an electrolyte-filled channel. In cooled cells, there were more transients of the type shown in Figure 44 (see below). The frequency of transients like the one in Figure 4B was diminished (from 15% to 5%), and that of transients like the one in Figure 4D was increased (from
