The fusion pore, 60 years after the first cartoon

Abstract

Neurotransmitter release occurs in the form of quantal events by fusion of secretory vesicles with the plasma membrane, and begins with the formation of a fusion pore that has a conductance similar to that of a large ion channel or gap junction. In this review, we propose mechanisms of fusion pore formation and discuss their implications for fusion pore structure and function. Accumulating evidence indicates a direct role of soluble N-ethylmaleimide-sensitive-factor attachment receptor proteins in the opening of fusion pores. Fusion pores are likely neither protein channels nor purely lipid, but are of proteolipidic composition. Future perspectives to gain better insight into the molecular structure of fusion pores are discussed.

Keywords: exocytosis; fusion pore; membrane fusion.

Fig. 1

Diagram explaining the quantal discharge character of Ach at a motor nerve terminal (N). M: muscle fiber with junctional folds (see Robertson, 1956 [168]). The transmitter is believed to be preformed in intracellular microsomes, which after critical collision with the nerve membrane, release their Ach contents into the intersynaptic space. This phenomenon is illustrated in b and c and it can be assumed that it occurs when certain reactive molecules (represented by dots) of the two surfaces meet. From [3].

Fig. 2

Initial fusion pore conductance from analysis of current transients. (A) Upon fusion the capacitance Cv of the vesicle is added to the plasma membrane (C_M) and any intravesicular potential V_V is rapidly discharged by the current I_Pore that flows across the fusion pore conductance Gp. The initial V_V is obtained from the integrated charge of I_Pore and the C_V. C_V is the difference of C_M before the fusion event and after fusion event. C_M is measured periodically by applying a sine wave voltage to determine the equivalent circuit parameters. R_A is the access resistance from the pipette tip. (B) A typical current transient measured at −80 mV holding potential. (C) Two representative fusion events showing different initial G_P. Modified after reference [8].

Fig. 3

Whole cell capacitance measurements of fusion pore dynamics in cells with large vesicles. (A) Whole cell configuration using the lock-in amplifier technique and equations used for analysis. (B) Minimal equivalent circuit for analysis of fusion pore conductance with access (pipette) resistance R_A, membrane capacitance C_M, vesicle capacitance C_V, and fusion pore conductance G_P. (C) Recording of fusion pore expansion in a horse eosinophil. When the fusion pore conductance increases, the phase shifted current component (Im/ω, red) increases gradually to the full vesicle capacitance C_V while the in-phase component (Re, blue) shows a transient increase. G_P can be calculated from Im and Re or from Im and C_V as indicated. G_P shows a fluctuating increase with an average slope of ~15 nS/s. Data from reference [8].

Fig. 4

Cell attached capacitance measurements of fusion pore dynamics in cells with small vesicles. (A) Cell attached configuration using the lock-in amplifier technique. (B) Minimal equivalent circuit for analysis of fusion pore conductance with components R_A, C_M, C_V, and G_P. In this configuration the measured capacitance is that of the patch (C_Patch). C_M is negligible because it is much larger than C_Patch and enters with its reciprocal value. (C) Recording of fusion pore opening from a human neutrophil [11]. G_P rises from an initial value as small as 35 pS. C_V was calculated from Re and Im as indicated.

Fig. 5

Amperometric measurement of fusion pore dynamics. (A) A CFM is placed close to the cell and a voltage of typically 700 mV is applied. (B) Amperometric current from a single fusion event in a chromaffin cell [54]. The amperometric current reports the flux of catecholamine molecules from the vesicle. Amperometric spikes are typically quantified by quantal size, half width, mean foot current and foot duration as indicated. The foot current reports fusion pore properties as shown in Fig. 6.

Fig. 6

(A) Patch amperometry records simultaneously fusion pore conductance by cell attached capacitance measurement and catecholamine release by amperometry with a CFM inserted into the patch pipette together with the reference electrode. The sine wave for capacitance measurement is applied to the bath electrode. (B) Measurement of a single fusion event with extremely long amperometric foot signal. (C) Foot current fluctuations (green trace) are synchronous with G_P fluctuations (red trace).

Fig. 7

Hypothetical steps in exocytosis. A–E illustrate the hypothesis where a proteinaceous fusion pore is formed, expands and eventually proceeds to full fusion. F–H show a section through the hypothetical fusion pore complex and the position of that complex in the two lipid bilayers of the plasma and vesicle membranes. Once the pore has opened, lipid molecules can diffuse along the amphipathic surfaces exposed between the fusion pore subunits. The entry of lipid molecules between the subunits causes the pore to dilate. Redrawn after [22]

Fig. 8

Geometry of a lipidic pore that is generated by revolving the semicircles (red curves) about the axis of revolution, Y. a = radius of the narrowest part of the pore; b = radius of the generating semicircles; X = shortest distance between the axis of revolution and a point on the semicircle. Inset shows the same geometry with the angle and radii of curvatures. J_p⁻¹ and J_m⁻¹ are the parallel and meridional radii of curvature, respectively. ψ(x) is the angle between the tangent to the semicircle at the point (x, y) and the horizontal axis. (B) A pore spanning two bilayers and the space between them. h=monolayer thickness, d=interbilayer distance, L = pore length = 4h + d. (C) A pore spanning the single bilayer formed on hemifusion of the fusing bilayers (L = 2h). Modified after [27].

Fig. 9

Schematic illustration of a proteolipidic fusion pore formed by SNAREs in cooperation with accessory proteins as indicated. The function of the various components is discussed in the text. Ca²⁺ ions are depicted as black dots.