Electrostatic interactions between the syntaxin membrane anchor and neurotransmitter passing through the fusion pore

Abstract

Recent experiments have shown that flux through the fusion pore is sensitive to manipulations of the side-chain size of certain residues in the syntaxin (syx) membrane anchor. These residues were proposed to line the wall of the fusion pore of Ca(2+)-triggered exocytosis. Here we continued this line of experimentation to examine possible electrostatic interactions between the pore lining residues and the neurotransmitter norepinephrine (NE). Replacing syx pore-lining residues with aspartate enhanced NE flux above that expected for the size of the aspartate side chain. In contrast, substitution with arginine reduced NE flux below that expected for the size of its side chain. Substituting aspartate and arginine into the nonpore-lining residues did not alter the fusion pore flux. Other amino acids with ionizable side chains had variable effects. These results indicate an electrostatic interaction between the pore-lining residues of syx and NE, and provide additional evidence that the syx membrane anchor is a structural component of the fusion pore.

FIGURE 1

Amperometry spikes recorded from PC12 cells transfected with wild-type syx and two mutants, as indicated. The prespike feet are shaded.

FIGURE 2

Positively charged NE electrostatically interacts with the residues residing in the syx membrane anchor. The mean foot current is plotted against the side-chain volume for residues 276 (A), 283 (B), 275 (C), and 280 (D). Data for nonpolar residues in this plot are from Fig. 3 of reference (2). The line drawn in each plot is the linear fit to the data from nonpolar residues.

FIGURE 3

Current offset (see text) is plotted against the side-chain pK for positions 276 (A) and 283 (B). Linear fits gave p = 0.04 for both plots.