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We have developed a new hypothesis for secretion, particularly at the neuromuscular junction and CNS synapses. Our interpretation of secretion—which is consistent with the structural organization of the neuromuscular junction reported by McMahan and co-workers (1)—is based upon the porocytosis hypothesis (2,3), in which the postsynaptic quantal response results from presynaptic neurotransmitter secretion from many docked vesicles, rather than from a single vesicular exocytotic event (cf. 4,5). In the mechanism we propose, presynaptic vesicles are arrayed at two levels: 1) vesicles are anchored to the active zones of the plasma membrane and juxtaposed to calcium ion-selective channels by proteins such as SNAREs (6) to make a unit; and 2) these vesicle-ion channel-SNARE-membrane-containing units are arranged in spatially periodic arrays. We envision that the organization of the arrayed active zone material at the frog neuromuscular junction described by Harlow and co-workers (1), and the array that we discuss, are one and the same entity. We view this secretory "organelle" (1), which we have called the "synaptomere" (3), to be the unit of secretion, much as the sarcomere is the unit of contraction. The synaptomere contains a scaffold that would prevent vesicular fusion into the terminal membrane and would maintain vesicles in the linear array so that vesicle and terminal unit membranes are in apposition to the receptors on the postsynaptic fold. This arrangement is extendable to synapses, although the fine level of organization of the array structure may vary among secretory systems.
The porocytosis mechanism we propose provides a quantum of neurotransmitter, but without the need to invoke fusion of a single vesicle membrane with the (presynaptic) plasma membrane. The small observed coefficient of variation (<3%) in end plate potentials indicates that there are only about 200 release sites (9), each of which secretes one quantum per action potential (2,7,8,9). The 200 sites found on a small muscle fiber establish a maximum quantitative limit of 1 site per micrometer terminal length for the number of secretory organelles at the neuromuscular junction (2,7,9) and excludes a single vesicle quantum mechanism. Our mathematical modeling efforts have shown that release of neurotransmitter via the quantal vesicular fusion mechanism would result in a coefficient of variation of 14% to 30%. In summary, the notion that neurotransmitter release is mediated through a "single quantum-single vesicle" mechanism would appear to be precluded (2,3).
Strong physiological evidence supports the concept that the repeating components of the synaptomere function as units, each secreting one packet of transmitter (10). Most importantly, the ratio of the large to small class of transmitter packets (MEPPs and sub-MEPPs), and the number of subunits composing the larger class, is readily changed with many treatments and conditions (10), showing that the two classes share the same sub-unit. Decreasing extracellular calcium decreases MEPP frequency and decreases the number of subunits in the MEPP (Fig. 1). In normal calcium, there is a very small percentage of sub-MEPPs, while in reduced calcium concentration most MEPPs are of the sub-MEPP class. A postsynaptic effect is ruled out because the modal size of the sub-MEPP has not changed. Thus, these data indicate that the number of secreting pores in the array is calcium-dependent. The concept that a single vesicle would release only a portion of its contents per flicker is supported by other studies. Neher (11) calculated that a flicker of a pore would secrete about 8% of the contents of a small vesicle. Rahamimoff and Fernandez (12) proposed that a cationic transmitter could exchange with Na ions through a fusion pore to generate the sub-MEPP. In the porocytosis array model, the 200 physiologically described release sites of a neuromuscular junction defined by Katz and Miledi (13) are the synaptomeres, and the attractive "organelles" described by Harlow and co-workers (1).
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The observed constancy of the amount of neurotransmitter secreted with nerve stimulation, attested to by the small value of the coefficient of variance of EPPs, can only be explained by release of small amounts of neurotransmitter molecules from arrays of vesicles at each release site of the neuromuscular junction. Since the coefficient of variation of the quantal packet is a function of 1/square root of the number of contributing vesicles, and there are 30–50 in an array, a standard amount of secretion is guaranteed by the array with each action potential. The array notion is so robust in maintaining a standard packet size, that vesicle contents may vary from full to empty. Since 60% of the acetylcholine in the synapse is present in the cytosol (17,18), transporters on the vesicle membrane would continuously "fill" vesicles. In addition, acetylcholine is readily available for transporters because synthesis, mediated by acetyl-O-transferase, is known to occur on the cytoplasmic surface of the vesicle membrane (19). In addition, it is likely that, concurrently, there are mechanisms for docking and undocking vesicles that are independent of secretion, each process having its own identifiable rate constants. Small variations in amounts of neurotransmitter released are readily accommodated by modulating (e.g., through small changes in calcium dynamics) the amount released from many vesicles whose diameters are observed to vary by 3% to 10% (20,21). Most importantly, the array concept permits quantal size to be frequency-dependent. Thus, to achieve the observed characteristic constancy of "quantal release" (i.e., MEPP size), the synapse must rely on secretion through many vesicles within an array of vesicles. The porocytosis mechanism we have proposed uniquely meets these requirements. We believe that the porocytosis mechanism extends to secretion in other non-synaptic systems.
Footnotes
1 Departments of Radiology, Pharmacology and Physiology, Wayne State University School of Medicine, Detroit, MI; Decision and Information Sciences Division, Argonne National Laboratory, Argonne, IL. 
2 Marine Biological Laboratory, Woods Hole, MA.
3 Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210.
4 Psychiatric Institute, and Department of Anatomy and Cell Biology, College of Medicine, University of Illinois, Chicago, IL 60612.
Literature Cited
This article has been cited by other articles:
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  T. Tsuboi, H. T. McMahon, and G. A. Rutter Mechanisms of Dense Core Vesicle Recapture following "Kiss and Run" ("Cavicapture") Exocytosis in Insulin-secreting Cells J. Biol. Chem., November 5, 2004; 279(45): 47115 - 47124. [Abstract] [Full Text] [PDF]
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