Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

Abstract

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead, we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation, and suggest a prefusion rather than postfusion role for vesicular cation channels.

Figure 1

Flux of catecholamines during the foot signals is correlated with fusion pore conductance. Patch amperometry recordings with small patch-CFE distance. (a-f) Analysis of an event with exceptionally long foot signal. Amperometric current (a) indicates flux of catecholamine. Real part (b) and imaginary part (c) of patch admittance were used to calculate the time course of fusion pore conductance (d, continuous line). On an expanded scale the amperometric current during the foot signal (d, dotted line) is seen to fluctuate in parallel with the fusion pore conductance. The ratio of catecholamine flux/fusion pore conductance (e) is relatively constant for the first 800 ms of the fusion pore opening and then decreases, suggesting depletion of free catecholamine in the vesicle. The dashed lines indicate the baseline of the respective signals. The 500 ms scale bar at the top is for panels a-e. For the part indicated by the solid line in (e) the flux is plotted vs. fusion pore conductance (f) showing a proportional relationship with a slope (solid line) of ~3×10⁷ molecules s⁻¹ nS⁻¹ in agreement with the mean ratio indicated by the solid line in (e). A second example with shorter foot signal also shows parallel changes in fusion pore conductance and amperometric foot current (g). The flux/conductance (h) is rather constant with a mean value of 3.1×10⁷ molecules s⁻¹ nS⁻¹ (horizontal line). A third example with unusually low catecholamine concentration (i) shows initially lower flux/conductance of ~1.4×10⁷ molecules s⁻¹ nS⁻¹ (j, solid line) and decreasing foot current while fusion pore conductance remains rather constant.

Figure 2

Amperometric foot signals are not accompanied by net outward current. (A) If the positive charge carried by catecholamines (blue) is replaced by cations entering the vesicle through channels in its membrane (red), a net outward current will be generated. (b) Simultaneous detection of catecholamine release and patch current with cell-attached patch amperometry for an individual event shows no net outward patch current associated with the foot signal. The traces from top to bottom show the time course of: Real part of patch admittance change (Re, blue), imaginary part of patch admittance change (Im/ω, red), fusion pore conductance (G_P, turquoise), granule capacitance (C_V, brown), amperometric current (I_amp, green), and patch current (I_patch, black). The horizontal dashed lines indicate the baselines of the respective signals. (c) Similar analysis of an event with an expanding fusion pore conductance (G_P). (d) Averaged amperometric current (green) and patch current (black) from 11 events with foot amplitude > 1 pA and foot duration >20 ms. The events were aligned at the time of rapid fusion pore expansion (end of foot = onset of the spike). The black and grey dotted lines indicate respectively the lower limits for expected patch currents if charge compensation occurs through channels in the vesicle membrane for total release or foot only (arrow). The vertical dashed lines indicate the plateau of the averaged foot current during which the patch current was averaged (see text).

Figure 3

Analysis of fusion pore properties in different extracellular solutions determined by patch amperometry as in Fig. 2. See Methods for compositions of solutions. (a,b) Fusion pore openings were characterized by quantifying initial G_P, average G_P and fusion pore duration as described in Patch Amperometry section of Methods. Panels c-e show statistical analysis of mean fusion pore conductance (c), initial fusion pore conductance (d), and fusion pore lifetime (e) measured as time from fusion pore formation to rapid expansion (onset of amperometric spike). Error bars are s.e.m, n=43 for solution A, n=39 for solution B, n=38 for solution C (* p<0.05, ** p<0.01).

Figure 4

Extracellular ion concentration modulates amperometric foot signals. (a) Representative amperometric events measured in 3 different solutions as indicated. Each signal is shown with full amplitude (left, scale bars 50 pA, 50 ms) and on expanded scale to reveal the foot signal more clearly (right, scale bars 20 pA, 50 ms). The 0 NaCl signal has no detectable foot. (b-f) Statistical analysis of amperometric foot signals measured in solutions with the indicated [NaCl] quantifying frequency of amperometric spike with detectable foot signals (b), foot charge indicating amount of catecholamine release during foot (c), foot duration (d) and mean of detectable foot currents (e), and mean foot currents assuming an average of 0.5 pA for undetected foot currents (f). Error bars are s.e.m., n=121 for 140 NaCl, n=102 for 90 NaCl, n=98 for 40 NaCl, n=69 for 0 NaCl (* p<0.05, ** p<0.01). In panels e and f the symbols indicate foot currents expected from constant field theory (see text and supplementary fig. 1). The intravesicular free catecholamine concentrations [CA]_free were set to reproduce the amperometric current measured in solution containing 140 mM NaCl. (e) Model 1, monovalent cation selective fusion pore (filled squares, [CA]_free=205 mM); Model 2, cation selective fusion pore (filled circles, [CA]_free=200 mM); Model 3, nonselective fusion pore [Cl⁻]_V=0 (open squares, [CA]_free=370 mM); Model 4, nonselective fusion pore [Cl⁻]_V=50 mM (open circles, [CA]_free=280 mM). (f) Model 1, monovalent cation selective fusion pore (filled squares, [CA]_free=135 mM); Model 2, cation selective fusion pore (filled circles, [CA]_free=130 mM); Model 3, nonselective fusion pore [Cl⁻]_V=0 (open squares, [CA]_free=260 mM); Model 4, nonselective fusion pore [Cl⁻]_V=50 mM (open circles, [CA]_free=200 mM). (g) Release of catecholamines through a narrow fusion pore is associated with charge compensation by cation entry through the fusion pore.