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Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
Correspondence: Address reprint requests and inquiries to Meyer Jackson, Email: mjackson{at}physiology.wisc.edu.
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ABSTRACT |
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Ca2+-triggered exocytosis proceeds through an intermediate structure known as the fusion pore. One hypothetical model for the fusion pore is a proteinaceous channel-like complex (1
), and recent experiments indicated that the syntaxin (syx) membrane anchor could be part of this complex (2). Altering the side-chain size of residues in the syx transmembrane segment influenced the flux of the neurotransmitter norepinephrine (NE), in the same way that altering the side chains along the ion permeation pathway of channel-forming proteins alters their ionic current (3). The ion channel flux is uniquely sensitive to manipulations of the pore-lining residues of a channel forming protein (3, 4).
NE carries a positive charge at the neutral pH of the extracellular fluid or the acidic pH inside a vesicle. When passing through a fusion pore, an electrostatic interaction is possible if the pore contains a charge. We therefore introduced amino acids with ionizable side chains into the syx membrane anchor. NE release from single vesicles was detected as a spike in amperometry recordings. The "foot" signal preceding a spike corresponds to the leak of NE through an open fusion pore (Fig. 1) (2, 7, 8, 9). The average amplitude of the prespike foot (PSF) was used to evaluate the NE flux through fusion pores.
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), and two of these residues, 276 and 283, were selected for charge substitutions. PSF amplitudes were first evaluated in plots versus amino acid side-chain volume. Linear fits for the nonpolar substitutions provide an assessment of the effect of side-chain size in the steric occlusion of fusion pores. The value for aspartate (D) and glutamate (E), which ionize to form a negative charge, fell above the linear fit for both pore-lining positions (Fig. 2, A and B), indicating enhanced NE efflux. Substituting positively charged arginine (R) at these sites reduced the fusion pore flux below this line, indicating reduced fusion pore flux. Lysine (K) and histidine (H) substitutions failed to alter the fusion pore flux, and this may reflect their weaker tendency to ionize.
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Subtracting the experimentally recorded foot current from that based on the linear fits in Fig. 2, A and B, yields the "current offset", which represents the electrostatic contribution. The current offset varied inversely with the side-chain pK (Fig. 3). (The pK values were for ionization in an aqueous solution (5), and could be altered by the protein/membrane environment.)
These results demonstrate an electrostatic interaction between the syx membrane anchor and NE escaping from a vesicle through the fusion pore. It has been suggested that substitutions that increase the fusion pore flux would provide stronger support for a direct role of the syx membrane anchor (10). Since aspartate substitutions at positions 276 and 283 produce such an increase, this strengthens the case for a direct involvement of syx. The demonstration of two separate physical interactions, steric and electrostatic, between the syx membrane anchor and neurotransmitter indicates that this region of the protein is a structural component of the fusion pore.
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METHODS |
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TOPABSTRACTMETHODSACKNOWLEDGEMENTSREFERENCES |
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). Wild-type syx and mutants were subcloned into the pIRES2-EGFP vector (Clonetech, Palo Alto, CA). All mutants were confirmed by DNA sequencing.
Cell culture and amperometry
PC12 cells were cultured and transfected as previously described (2). Cells were transferred to dishes coated with collagen-I and poly-D-lysine (BD Bioscience, Bedford, MA) and incubated with 1.5 mM NE and 0.5 mM ascorbate (Sigma, St. Louis, MO) overnight. Amperometry recording was performed with 5 µm carbon fiber electrodes connecting to a VA-10 amplifier at a polarization of 650 mV (2, 7, 8). The bathing solution contained (in mM): 150 NaCl, 4.2 KCl, 1 NaH2PO4, 0.7 MgCl2, 2 CaCl2, and 10 Hepes (pH 7.4). Secretion was induced by depolarization using the same bathing solution but with elevated KCl (105 mM) and reduced NaCl (5 mM). Data were analyzed with a computer program to extract foot information (8). Feet were analyzed for spikes larger than 20 pA. The foot current was calculated as the PSF area divided by the foot duration. Feet with durations <0.75 ms (three times the sampling rate) were rejected as too brief for reliable detection.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMETHODSACKNOWLEDGEMENTSREFERENCES |
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Submitted on November 22, 2004; accepted for publication December 28, 2004.
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REFERENCES |
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2 Han, X., C. T. Wang, J. Bai, E. R. Chapman, and M. B. Jackson. 2004. Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis. Science. 304:289–292.
3 Imoto, K., C. Busch, B. Sakmann, M. Mishina, T. Konno, J. Nakai, H. Bujo, Y. Mori, K. Fukuda, and S. Numa. 1988. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 335:645–648.[CrossRef][Medline]
4 Lester, H. A. 1992. The permeation pathway of neurotransmitter-gated ion channels. Annu. Rev. Biophys. Biomol. Struct. 21:267–292.[CrossRef][Medline]
5 Weast, R. C. 1977. Properties of the amino acids. In Handbook of Chemistry and Physics, 58th ed. CRC Press, Cleveland, OH.
6 Cormack, B. 1994. Introduction of a point mutation by sequential PCR steps. In Current Protocols in Molecular Biology. F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, editors. John Wiley & Sons, New York. 397–482.
7 Wang, C.-T., R. Grishanin, C. A. Earles, P. Y. Chang, T. F. J. Martin, E. R. Chapman, and M. B. Jackson. 2001. Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles. Science. 294:1111–1115.
8 Chow, R. H., and L. von Rüden. 1995. Electrochemical detection of secretion from single cells. In Single-Channel Recording. B. Sakmann and E. Neher, editors. Plenum Press, New York. 245–275.
9 Chow, R. H., L. von Rüden, and E. Neher. 1992. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature. 356:60–63.[CrossRef][Medline]
10 Szule, J. A., and J. R. Coorssen. 2004. Comment of "Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis". Science. 306:813.[CrossRef]
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