Comment on "Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis".

Ca2þ-triggered membrane fusion is the defining stage of exocytosis, proceeding by an as-yet-unknown molecular mechanism. Han et al. (1) described a model in which multiple syntaxin transmembrane domains (TMDs) act as subunits of a proteinaceous fusion pore. Although intriguing, the interpretations do not address sequence conservation or other data in the field, and the results are equally consistent with the wellestablished stalk-pore model for membrane fusion (2). As disruptions induced by the mutations used in (1) will include profound effects on local membrane curvature stress, the results of these studies should also be considered in terms of a lipidic fusion-pore model. The model of syntaxin TMDs forming the fusion pore (1) is consistent with the demonstrated lack of critical SNARE (soluble Nethylmaleimide–sensitive factor attachment protein receptor) cytoplasmic domain interactions during the Ca2þ-triggered fusion steps (3, 4). The fusion pore was characterized as a gap junction–like channel (5), and small molecule fluxes might thus be affected by changes in pore-lining constituents, as in ion channels (6, 7). Within the parameters of this model, Han et al. mutated amino acids in the TMD of syntaxin, a protein essential for exocytosis. Tryptophan replacement of isoleucine (I269W), glycine (G276W), and isoleucine (I283W) in the TMD decreased transmitter flux, leading the authors to model a fusion pore composed of five to eight syntaxin monomers, with these residues lining the pore interior (1). However, using the same PC12 cell model, Hua and Scheller (8) previously deduced that three syntaxin-containing complexes are required for Ca2þ-triggered exocytosis; three such TMDs could not form a sufficient pore for the passage of transmitters. In addition, the Han et al. model (1) infers that syntaxin TMDs form the plasma membrane portion of the fusion pore, which must then link with a vesicle-membrane counterpart to complete the pore. However, as with viral fusion, native triggered fusion systems minimally require proteins in only one of the interacting membranes (9–12). Considering the evidence for a conserved membrane merger mechanism in the pathway of Ca2þ-triggered release (13), the proposed model (1) would suggest highly conserved syntaxin TMDs, particularly of the putative pore-lining residues. Based on the rationale that introduction of large tryptophan moieties Bblocked[ the fusion pore, the molecular volume of pore-lining amino acids should be highly conserved. To test this, we aligned the TMD of syntaxin homologs (Fig. 1) and compared the amino acid residues corresponding to positions 269, 276, and 283 of rat syntaxin 1A Eas used in (1); see Table 1^. There is no clear conservation of molecular volumes (14). This lack of correlation appears regardless of the source or type of vesicle (Fig. 1). Interestingly, these seven residues that interchangeably occur at positions 269, 276, and 283 (listed in Table 1) are considered to be among the most hydrophobic (15). Tryptophan, used to test the model (1), is much more hydrophilic than any of the naturally occurring amino acids at these positions. Langosch et al. (16) noted that the TMDs of syntaxin homologs contain an overrepresentation of isoleucine and valine (the two most hydrophobic residues) as compared with other tail-anchored membrane proteins. These residues would thus contribute substantially to the hydrophobic volume of the bilayer added by syntaxin TMD, which would strongly affect membrane curvature stress (17, 18). Local spontaneous curvature is a crucial parameter for membrane merger TECHNICAL COMMENT