Figure 6.

Mutation of the conserved basic residue in either C₂ A or C₂B decreases Ca²⁺-dependent interactions between anionic phospholipids and synaptotagmin in both Drosophila and mammals. Phospholipid binding for wild-type (black filled circles), C₂A mutant (gray open squares; A-RQ in both Drosophila and mammals), and C₂B mutant (gray open diamonds; B-RQ in Drosophila and B-KQ in mammals) C₂AB domains are graphed versus Ca²⁺ concentration. A, Immobilized WT or A-RQ or B-RQ mutant versions of Drosophila C₂AB were assayed for binding PS/PC liposomes. The A-RQ and the B-RQ mutations each decreased the apparent Ca²⁺ affinity of binding by ~1.5-fold compared with WT without altering the Hill coefficient. The EC₅₀ (mean ± SD in micromolar concentration) was 368 ± 35 for A-RQ, 344 ± 32 for B-RQ, and 238 ± 20 for WT. B, Immobilized WT, A-RQ, or B-KQ mutant versions of mammalian C₂AB were assayed for binding PS/PC liposomes. The A-RQ and the B-KQ mutations each decreased the apparent Ca²⁺ affinity of binding by ~1.5-fold compared with WT without altering the Hill coefficient. The EC₅₀ (mean ± SD in micromolar concentration) was 154 ± 10 for A-RQ, 147 ± 7 for B-KQ, and 98 ± 6 for WT.