Structure/Function Analysis of Ca²⁺Binding to the C₂A Domain of Synaptotagmin 1

Abstract

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.

Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

“…Similar function has been attributed to Ca1 in the case of synaptotagmin I (C2A domain) (29,30). In contrast, Ca1 seems not to be essential for the membrane binding of cytosolic phospholipase A 2 (31).…”

A Gain-of-Function Mutation in Synaptotagmin-1 Reveals a Critical Role of Ca2+-Dependent SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Binding in Synaptic Exocytosis

“…For the hippocampal neuronal cultures, offspring from D232N/D238N-mutant compound heterozygotes were used. All genotyping was performed as described previously (Fernandez-Chacon et al, 2002).…”

“…Thus, a major question is now whether synaptotagmin-1 acts primarily as a Ca 2ϩ -dependent phospholipid binding protein in triggering exocytosis, with its SNARE binding having a separate role (possibly in priming vesicles for subsequent release), or whether the interactions of synaptotagmin-1 with both SNAREs and phospholipids collaborate in Ca 2ϩ triggering of neurotransmitter release. We previously analyzed two aspartate-to-asparagine substitutions in the Ca 2ϩ -binding site of the C 2 A domain (D232N and D238N) that interfere with Ca 2ϩ binding to the C 2 A domain in slightly different ways, but do not alter its atomic structure, and have little effect on Ca 2ϩ -dependent phospholipid binding by synaptotagmin-1 (Fernandez-Chacon et al, 2002). In knock-in mice, the D238N mutation produced no major change in synaptic function, but the D232N mutation caused an unexplained increase of synaptic depression during repetitive stimulation (Fernandez-Chacon et al 2002).…”

“…We previously analyzed two aspartate-to-asparagine substitutions in the Ca 2ϩ -binding site of the C 2 A domain (D232N and D238N) that interfere with Ca 2ϩ binding to the C 2 A domain in slightly different ways, but do not alter its atomic structure, and have little effect on Ca 2ϩ -dependent phospholipid binding by synaptotagmin-1 (Fernandez-Chacon et al, 2002). In knock-in mice, the D238N mutation produced no major change in synaptic function, but the D232N mutation caused an unexplained increase of synaptic depression during repetitive stimulation (Fernandez-Chacon et al 2002). Moreover, a separate study using rescue experiments of synaptotagmin-1 knock-out neurons with D232N-mutant synaptotagmin-1 suggested that the D232N mutation may increase the Ca 2ϩ sensitivity of evoked responses (Stevens and Sullivan, 2003), although again no biochemical basis for the changes was identified.…”

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

“…As expected from structural and functional data, the D405A point mutation should disrupt the most important residue in a putative Ca 2q /phospholipid nucleation site of the 'C2-like' domain III (Strobl et al, 2000;Alexa et al, 2004). A number of site-directed mutagenesis studies with the C2 domains of PKCa (Bolsover et al, 2003), cPLA (Bittova et al, 1999), synaptotagmin (Fernandez-Chacon et al, 2002) and other C2 domain proteins have shown that neutralising mutations of the acidic residues in the Ca 2q -binding loops of C2 domains usually result in an important loss of affinity for membrane lipids. Our data suggest that the contribution of the single acidic-loop residue mutated here is minimal in the context of a heterodimeric calpain.…”