Mutations in layers 4 and 6 severely affect transmitter release. The mutations in layers 4 and 6 are shown in Fig. 1. (Ai and Bi) Mean secretory responses in Ca²⁺-triggered release are dramatically reduced in the L70A (Ai) and the F77A (Bi) mutant. For comparison, traces from double knockout cells (DKO) are included. Double knockout traces are pooled from an independent dataset and were not recorded in parallel to L70A and F77A measurements. Mean ± SEM of Ca²⁺ levels is shown. (Aii and Bii) Detailed view of normalized traces in Ai and Bi. (A: WT rescue, n = 23; L70A, n = 15; double knockout, n = 21; B: WT rescue, n = 8; F77A, n = 13; double knockout, n = 21.)