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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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ERG Recordings Show That Synaptic Transmission Is Not Blocked in the syx3–69 Mutant Fly at Restrictive Temperatures(A) ERGs are obtained from the wild-type fly at the permissive temperature (20 °C), during the restrictive temperature (38 °C), and during recovery at 20 °C following a brief white-light stimulation of the compound eye. The spikes before and following the sustained photoreceptor potential are called “on” and “off” transient potentials (arrows), respectively. They are thought to reflect synaptic transmission from the photoreceptor to downstream interneurons. Note that these transient potentials are not significantly affected at 38 °C. The extracellular recording electrode also detects light-induced high-frequency action potentials (arrowheads), which normally result in startle escape.(B) The “on” and “off” transient potentials are absent in Shits1 flies exposed at 33 °C, consistent with a conditional block of vesicle recycling.(C) Under the same experimental conditions, the “on” and “off” transient potentials in the syx3–69 mutant fly remain essentially similar to those observed in the wild-type fly. Even though the amplitude of photoreceptor potentials is reduced and the duration of recovery is prolonged, synaptic transmission is not blocked at 38 °C in both the wild-type and the mutant fly. Additionally, the mutant fly also displays light-induced high-frequency action potentials (arrowheads), even though it is paralyzed.
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pbio-0050072-g003: ERG Recordings Show That Synaptic Transmission Is Not Blocked in the syx3–69 Mutant Fly at Restrictive Temperatures(A) ERGs are obtained from the wild-type fly at the permissive temperature (20 °C), during the restrictive temperature (38 °C), and during recovery at 20 °C following a brief white-light stimulation of the compound eye. The spikes before and following the sustained photoreceptor potential are called “on” and “off” transient potentials (arrows), respectively. They are thought to reflect synaptic transmission from the photoreceptor to downstream interneurons. Note that these transient potentials are not significantly affected at 38 °C. The extracellular recording electrode also detects light-induced high-frequency action potentials (arrowheads), which normally result in startle escape.(B) The “on” and “off” transient potentials are absent in Shits1 flies exposed at 33 °C, consistent with a conditional block of vesicle recycling.(C) Under the same experimental conditions, the “on” and “off” transient potentials in the syx3–69 mutant fly remain essentially similar to those observed in the wild-type fly. Even though the amplitude of photoreceptor potentials is reduced and the duration of recovery is prolonged, synaptic transmission is not blocked at 38 °C in both the wild-type and the mutant fly. Additionally, the mutant fly also displays light-induced high-frequency action potentials (arrowheads), even though it is paralyzed.

Mentions: Consistent with the observation that synaptic transmission persists along the giant fiber pathway, light-induced “on” and “off” transient potentials of electroretinograms (ERGs) were not blocked by exposure of the syx3–69 fly to the restrictive temperature (Figure 3). These transients are thought to reflect synaptic transmission from photoreceptors to downstream interneurons in the retina [33]. The control fly, Shits1, lost its transient potentials at 33 °C, consistent with a depletion of the vesicle pool [21,29,32] (Figure 3B). However, the findings from the syx3–69 fly differ from those reported earlier [21], which showed that the restrictive temperature reversibly blocked these transients. In our experiments, we carefully monitored the temperature of the syx3–69 fly by placing a temperature probe adjacent to the experimental fly. Additionally, we mounted another syx3–69 fly beside the experimental fly so that we could observe the paralysis during the exposure at 38 °C and the recovery afterward. In a total of eight experiments, we never saw a loss of these transient potentials. In fact, our results showed that the “on” transient potential was slightly increased in amplitude at 38 °C (see Figure 3C). Additionally, we also observed spontaneous and light-induced high-frequency “bursting” activities typically indicative of enhanced neuronal activity in both the wild-type and the syx3–69 flies (see arrowheads in Figure 3A and 3C; see also [34]). Hyperactivity of the thoracic ganglion was also observed independently by Dr. Bruno van Swinderen's laboratory when syx3–69 flies were exposed to the restrictive temperature (B. van Swinderen, personal communication).


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)

ERG Recordings Show That Synaptic Transmission Is Not Blocked in the syx3–69 Mutant Fly at Restrictive Temperatures(A) ERGs are obtained from the wild-type fly at the permissive temperature (20 °C), during the restrictive temperature (38 °C), and during recovery at 20 °C following a brief white-light stimulation of the compound eye. The spikes before and following the sustained photoreceptor potential are called “on” and “off” transient potentials (arrows), respectively. They are thought to reflect synaptic transmission from the photoreceptor to downstream interneurons. Note that these transient potentials are not significantly affected at 38 °C. The extracellular recording electrode also detects light-induced high-frequency action potentials (arrowheads), which normally result in startle escape.(B) The “on” and “off” transient potentials are absent in Shits1 flies exposed at 33 °C, consistent with a conditional block of vesicle recycling.(C) Under the same experimental conditions, the “on” and “off” transient potentials in the syx3–69 mutant fly remain essentially similar to those observed in the wild-type fly. Even though the amplitude of photoreceptor potentials is reduced and the duration of recovery is prolonged, synaptic transmission is not blocked at 38 °C in both the wild-type and the mutant fly. Additionally, the mutant fly also displays light-induced high-frequency action potentials (arrowheads), even though it is paralyzed.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0050072-g003: ERG Recordings Show That Synaptic Transmission Is Not Blocked in the syx3–69 Mutant Fly at Restrictive Temperatures(A) ERGs are obtained from the wild-type fly at the permissive temperature (20 °C), during the restrictive temperature (38 °C), and during recovery at 20 °C following a brief white-light stimulation of the compound eye. The spikes before and following the sustained photoreceptor potential are called “on” and “off” transient potentials (arrows), respectively. They are thought to reflect synaptic transmission from the photoreceptor to downstream interneurons. Note that these transient potentials are not significantly affected at 38 °C. The extracellular recording electrode also detects light-induced high-frequency action potentials (arrowheads), which normally result in startle escape.(B) The “on” and “off” transient potentials are absent in Shits1 flies exposed at 33 °C, consistent with a conditional block of vesicle recycling.(C) Under the same experimental conditions, the “on” and “off” transient potentials in the syx3–69 mutant fly remain essentially similar to those observed in the wild-type fly. Even though the amplitude of photoreceptor potentials is reduced and the duration of recovery is prolonged, synaptic transmission is not blocked at 38 °C in both the wild-type and the mutant fly. Additionally, the mutant fly also displays light-induced high-frequency action potentials (arrowheads), even though it is paralyzed.
Mentions: Consistent with the observation that synaptic transmission persists along the giant fiber pathway, light-induced “on” and “off” transient potentials of electroretinograms (ERGs) were not blocked by exposure of the syx3–69 fly to the restrictive temperature (Figure 3). These transients are thought to reflect synaptic transmission from photoreceptors to downstream interneurons in the retina [33]. The control fly, Shits1, lost its transient potentials at 33 °C, consistent with a depletion of the vesicle pool [21,29,32] (Figure 3B). However, the findings from the syx3–69 fly differ from those reported earlier [21], which showed that the restrictive temperature reversibly blocked these transients. In our experiments, we carefully monitored the temperature of the syx3–69 fly by placing a temperature probe adjacent to the experimental fly. Additionally, we mounted another syx3–69 fly beside the experimental fly so that we could observe the paralysis during the exposure at 38 °C and the recovery afterward. In a total of eight experiments, we never saw a loss of these transient potentials. In fact, our results showed that the “on” transient potential was slightly increased in amplitude at 38 °C (see Figure 3C). Additionally, we also observed spontaneous and light-induced high-frequency “bursting” activities typically indicative of enhanced neuronal activity in both the wild-type and the syx3–69 flies (see arrowheads in Figure 3A and 3C; see also [34]). Hyperactivity of the thoracic ganglion was also observed independently by Dr. Bruno van Swinderen's laboratory when syx3–69 flies were exposed to the restrictive temperature (B. van Swinderen, personal communication).

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