FIGURE 2.

Amperometry recording from chromaffin cells. A, amperometry traces from chromaffin cells: DKO cells, littermate controls, and DKO cells expressing untagged WT syb2, EGFP-Syb2, Syb2-RST-mVenus, lipid-anchored mutants, and ΔTMD-94. Untransfected DKO cells and DKO cells expressing ΔTMD-94 exhibited low secretion in response to depolarization by a solution containing 105 mm KCl (indicated by bars), but littermate and DKO cells expressing untagged WT syb2 or N-terminal tagged EGFP-Syb2 produced robust trains of spikes. Expression of C-terminal tagged Syb2-RST-mVenus construct in DKO cells produced much less support for exocytosis compared with N-tagged and untagged syb2. DKO cells expressing ΔTMD-CSPα, ΔTMD-CAC, ΔTMD-LC, and ΔTMD-CIIL all secreted poorly. B, spike frequencies of cells expressing the indicated protein during the first depolarization. Syb2-RST-mVenus, the lipid-anchored constructs, and ΔTMD constructs produced frequencies statistically indistinguishable from untransfected DKO cells. n = 26–62 cells from 2 to 7 DKO embryos. C, cumulative spike counts versus time in the first response to depolarization (indicated by the bar) from chromaffin cells expressing the indicated proteins. D, slopes of the rises in cumulative spike count plots from 10 to 70% of the plateau. The slopes of littermate control cells and DKO cells expressing N-tagged and untagged WT syb2 were similar and significantly higher than those from Syb2-RST-mVenus, lipid-anchored mutants, ΔTMD, and untransfected DKO cells. All lipid-anchored syb2 constructs were statistically indistinguishable from DKO except ΔTMD-CSPα. The comparison with EGFP-syb2 or DKO for one-way analysis of variance with post test is indicated by bars. **, p < 0.01; ***, p < 0.001; n.s., not significant. Paired comparisons of slopes with a t test were also performed in GraphPad Prism, and the slopes of Syb2-RST-mVenus and all mutants (except ΔTMD-94) were significantly higher than DKO.