Showing papers by "Thomas Binz published in 2002"

Protein kinase C-dependent phosphorylation of synaptosome-associated protein of 25 kDa at Ser187 potentiates vesicle recruitment.

Abstract: Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser187, in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser187 accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis

Abstract: Synchronous neurotransmission depends on the tight coupling between Ca2+ influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.

Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization.

Abstract: The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn2+ is coordinated by His222 and His226 of the Zn2+ binding motif HisGluXXHis and Glu261, whereas Glu223 coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg362 and Tyr365 of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu350 which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg362 and substitution of Tyr365 with Phe resulted in 79- and 34-fold lower kcat/Km values, respectively. These changes were provoked by decreased catalytic rates (kcat) and not by alterations of ground state substrate binding as evidenced by largely unchanged Kd and Km values. None of these mutations affected the overall secondary structure or zinc content of the ...