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Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Lagow RD, Bao H, Cohen EN, Daniels RW, Zuzek A, Williams WH, Macleod GT, Sutton RB, Zhang B - PLoS Biol. (2007)

Bottom Line: At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure.Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain.Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

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Both Constitutive Secretion and Ca2+-Triggered Vesicle Fusion Are Dramatically Enhanced in syx3–69 Mutant Flies(A–C) The rate of spontaneous fusion of synaptic vesicles detected as mEPSPs (or minis) is significantly increased in syx3–69 mutants compared to the wild-type control (+/+). Representative recordings of minis and histograms of mini frequency from the wild-type and the mutant larvae are shown in (A) and (B). The average frequency of minis is increased by 7-fold in the syx3–69 mutant (B), whereas the average amplitude of these minis is similar (C). Note that the increase in the rate of constitutive secretion persists in saline containing 0 [Ca2+] (B). These and all other electrophysiological recordings were conducted at 19–20 °C.(D–F) The amplitude of EPSPs triggered by action potential–evoked Ca2+ entry is significantly increased in the syx3–69 mutant. Representative traces of EPSPs, histograms of average EPSP amplitude, and quantal content from the wild type and the syx3–69 mutant are shown in (D), (E), and (F), respectively.**, p < 0.01; ***, p < 0.001.
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pbio-0050072-g007: Both Constitutive Secretion and Ca2+-Triggered Vesicle Fusion Are Dramatically Enhanced in syx3–69 Mutant Flies(A–C) The rate of spontaneous fusion of synaptic vesicles detected as mEPSPs (or minis) is significantly increased in syx3–69 mutants compared to the wild-type control (+/+). Representative recordings of minis and histograms of mini frequency from the wild-type and the mutant larvae are shown in (A) and (B). The average frequency of minis is increased by 7-fold in the syx3–69 mutant (B), whereas the average amplitude of these minis is similar (C). Note that the increase in the rate of constitutive secretion persists in saline containing 0 [Ca2+] (B). These and all other electrophysiological recordings were conducted at 19–20 °C.(D–F) The amplitude of EPSPs triggered by action potential–evoked Ca2+ entry is significantly increased in the syx3–69 mutant. Representative traces of EPSPs, histograms of average EPSP amplitude, and quantal content from the wild type and the syx3–69 mutant are shown in (D), (E), and (F), respectively.**, p < 0.01; ***, p < 0.001.

Mentions: The level of the SDS-resistant SNARE complex has been shown to correlate well with the level of exocytosis [39,41,42]. We next tested whether this increase in the rate of SNARE complex assembly had any physiological effects on synaptic vesicle fusion. We recorded action potential–independent and constitutive (or spontaneous) miniature excitatory postsynaptic potentials (mEPSPs or minis) from third instar larval body-wall muscles innervated by motoneurons [43,44]. These mEPSPs are caused by constitutive secretion of glutamate from the nerve terminal [43]. Surprisingly, we found that the frequency of constitutive release was dramatically increased some 7-fold in the mutant (n = 9) compared to the wild type (n = 8; p < 0.001) (Figure 7A and 7B). The average mini amplitude was similar in both the syx3–69 mutant (n = 11) and the wild-type larvae (n = 8; p > 0.1) (Figure 7C), suggesting that quanta and postsynaptic receptors likely remain normal. Immunocytochemical studies of glutamate receptors failed to show detectable differences between the mutant and the wild type (unpublished data). This mini recording was conducted in saline containing 0.8 mM Ca2+ and 1 μm TTX, which was also used for evoked synaptic potentials (below). The resting potential was not different between these two genotypes (−69.7 ± 1.2 mV, n = 8, for the wild type, and −69.4 ± 0.9 mV, n = 9, for the mutant; p > 0.5). In these and all other larval recordings shown in this study, the muscle input resistance (between 5–9 MΩ) did not differ between the wild-type and the mutant larvae.


Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Lagow RD, Bao H, Cohen EN, Daniels RW, Zuzek A, Williams WH, Macleod GT, Sutton RB, Zhang B - PLoS Biol. (2007)

Both Constitutive Secretion and Ca2+-Triggered Vesicle Fusion Are Dramatically Enhanced in syx3–69 Mutant Flies(A–C) The rate of spontaneous fusion of synaptic vesicles detected as mEPSPs (or minis) is significantly increased in syx3–69 mutants compared to the wild-type control (+/+). Representative recordings of minis and histograms of mini frequency from the wild-type and the mutant larvae are shown in (A) and (B). The average frequency of minis is increased by 7-fold in the syx3–69 mutant (B), whereas the average amplitude of these minis is similar (C). Note that the increase in the rate of constitutive secretion persists in saline containing 0 [Ca2+] (B). These and all other electrophysiological recordings were conducted at 19–20 °C.(D–F) The amplitude of EPSPs triggered by action potential–evoked Ca2+ entry is significantly increased in the syx3–69 mutant. Representative traces of EPSPs, histograms of average EPSP amplitude, and quantal content from the wild type and the syx3–69 mutant are shown in (D), (E), and (F), respectively.**, p < 0.01; ***, p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1808484&req=5

pbio-0050072-g007: Both Constitutive Secretion and Ca2+-Triggered Vesicle Fusion Are Dramatically Enhanced in syx3–69 Mutant Flies(A–C) The rate of spontaneous fusion of synaptic vesicles detected as mEPSPs (or minis) is significantly increased in syx3–69 mutants compared to the wild-type control (+/+). Representative recordings of minis and histograms of mini frequency from the wild-type and the mutant larvae are shown in (A) and (B). The average frequency of minis is increased by 7-fold in the syx3–69 mutant (B), whereas the average amplitude of these minis is similar (C). Note that the increase in the rate of constitutive secretion persists in saline containing 0 [Ca2+] (B). These and all other electrophysiological recordings were conducted at 19–20 °C.(D–F) The amplitude of EPSPs triggered by action potential–evoked Ca2+ entry is significantly increased in the syx3–69 mutant. Representative traces of EPSPs, histograms of average EPSP amplitude, and quantal content from the wild type and the syx3–69 mutant are shown in (D), (E), and (F), respectively.**, p < 0.01; ***, p < 0.001.
Mentions: The level of the SDS-resistant SNARE complex has been shown to correlate well with the level of exocytosis [39,41,42]. We next tested whether this increase in the rate of SNARE complex assembly had any physiological effects on synaptic vesicle fusion. We recorded action potential–independent and constitutive (or spontaneous) miniature excitatory postsynaptic potentials (mEPSPs or minis) from third instar larval body-wall muscles innervated by motoneurons [43,44]. These mEPSPs are caused by constitutive secretion of glutamate from the nerve terminal [43]. Surprisingly, we found that the frequency of constitutive release was dramatically increased some 7-fold in the mutant (n = 9) compared to the wild type (n = 8; p < 0.001) (Figure 7A and 7B). The average mini amplitude was similar in both the syx3–69 mutant (n = 11) and the wild-type larvae (n = 8; p > 0.1) (Figure 7C), suggesting that quanta and postsynaptic receptors likely remain normal. Immunocytochemical studies of glutamate receptors failed to show detectable differences between the mutant and the wild type (unpublished data). This mini recording was conducted in saline containing 0.8 mM Ca2+ and 1 μm TTX, which was also used for evoked synaptic potentials (below). The resting potential was not different between these two genotypes (−69.7 ± 1.2 mV, n = 8, for the wild type, and −69.4 ± 0.9 mV, n = 9, for the mutant; p > 0.5). In these and all other larval recordings shown in this study, the muscle input resistance (between 5–9 MΩ) did not differ between the wild-type and the mutant larvae.

Bottom Line: At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure.Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain.Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Show MeSH
Related in: MedlinePlus