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. We tested this hypothesis in chromaffin cells using cell-attached patch amperometry measuring simultaneously catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% (if any) of cations 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.Neurotransmitter and hormone release occurs by exocytosis, which begins with the formation of a narrow fusion pore 1,2 . Fusion pores in mast cells and chromaffin cells have typically an initial conductance of ~330 pS 1,3 through which vesicular serotonin and catecholamines are released, respectively 4,5 . The initial fusion pore of a synaptic vesicle is typically >280 pS and release of neurotransmitter should occur with a time constant <500 μs due to the small vesicle volume 6 . Many neurotransmitters and hormones are organic ions that carry charge with them. Among these acetylcholine, serotonin and catecholamines are mostly monovalent cations at physiological or more acidic vesicular pH. Efflux of charged molecules through a narrow fusion pore would rapidly charge the small capacitance of the vesicle and a mechanism of charge compensation is required.Release of catecholamines from a single vesicle can be detected electrochemically 7 . The initial flux of catecholamines through the early narrow fusion pore can be measured as an amperometric foot signal preceding the amperometric spike 3,5,8 . The amplitude of amperometric foot currents is typically ~5 pA. Since most catecholamine molecules will be in monovalent cationic form but two electrons are transferred per molecule in the oxidation giving rise to the amperometric current 9 , the catecholamines released during the foot signal carry an ionic current of ~2.5 pA with them. This would charge a typical bovine chromaffin granule 4correspondence should be addressed to M.L. (ml95@cornell.edu).

The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

“…It has been proposed that the outer part of the fusion pore may be a ring of Ϸ5 or 7 syntaxin transmembrane domains (5, 6). However, the transmembrane domains of syntaxin as well as synaptobrevin are hydrophobic, and it is not immediately evident how they could form a pore that allows for permeation of charged transmitter molecules as well as other ions in a way similar to an ion channel (16). An alternative hypothesis is that fusion pore formation occurs via a hemifusion intermediate (7,8).…”

“…Fusion pore openings in cells overexpressing SNAP-25⌬9 show variable properties (Fig. 2), as do nontransfected bovine chromaffin cells (16). Typically (Fig.…”

“…The amperometric foot current amplitude indicates the flux of molecules through the pore, and the duration of the foot signal indicates the time interval from fusion pore opening to its rapid expansion (14,16). In cells overexpressing SNAP-25⌬9, the mean foot duration (Fig.…”

“…The opening of single fusion pores can be detected by carbon fiber amperometry as a ''foot signal'' (13) preceding an amperometric current spike that ref lects the f lux of catecholamines released from individual vesicles. The amperometric foot signal reflects flux of transmitter through narrow fusion pores with conductance Ͻ2 nS by electrodiffusion (14)(15)(16).…”

Role of the synaptobrevin C terminus in fusion pore formation

“…In an alternative proteinaceous fusion pore model, the fusion pore is lined by the TM domain of syntaxin (9) and possibly synaptobrevin (10). However, it is not immediately evident how the hydrophobic transmembrane domains can line an aqueous fusion pore that allows for ion permeation by electrodiffusion (11). When the C-terminal SNARE domain interactions are reduced by mutating or deleting the C terminus of SNAP-25, or when flexible linkers are introduced between the sybII TM domain and its SNARE domain, the rate of exocytosis is reduced (12)(13)(14)(15)(16) and the flux of transmitter through the early fusion pore is decreased (16,17), consistent with a structural change in the fusion pore.…”