Figure 2

stj but not stol is required for normal MN Ca_v1- and Ca_v2-like current amplitudes. Larval (A) and adult (Ai) Drosophila MN somata show expression of endogenously tagged stol^GFP (magenta) and stj^mCherry (green). (B, Bi) Compared to control (left trace; white box, n = 17) stj^RNAi (middle trace, light gray box, n = 11) but not stol^RNAi (right trace, dark gray box, n = 14) reduces muscle EPSP amplitude in response to MN stimulation (***p < 0.001, Kruskal Wallis ANOVA, Dunn’s post-hoc test). (C, Ci) MN axon terminal boutons on larval muscle 10 contain stj^mCherry puncta (C, top), which are depleted by stj^RNAi (middle), but not by stol^RNAi (bottom). (Ci) stj^mCherry labeling intensity is significantly reduced by stj^RNAi (light gray, ***p > 0.001) but not by stol^RNAi (dark gray, p = 0.21). (D–E) Larval MN Ca_v1 current (D, control, 200 ms command voltage steps in 10 mV increments from − 90 to 0 mV) is reduced by stj^RNAi(Di), but not by stol^RNAi(Dii). Quantification (E) reveals 46% amplitude reduction with stj^RNAi (n = 14; ***p = 1.5*10⁻⁴, one-way ANOVA, LSD post-hoc test) but not by stol^RNAi (n = 7) as compared to control (n = 13). (F–I) Adult MN5 HVA and LVA calcium currents mediated by the Drosophila Ca_v2 homolog cacophony. (F) Control HVA current (200 ms steps from − 90 mV to + 20 mV in 10 mV increments) is reduced by stj^RNAi(Fi), but not by stol^RNAi(Fii). (G) Control LVA current (left, steps from − 90 mV to − 40 mV) is reduced by stj^RNAi (middle) but not by stol^RNAi (right). (H–I) Quantification shows ~ 60% reduction of HVA current amplitude (H) by stj^RNAi (n = 5, **p = 0.006, one-way ANOVA, LSD post-hoc test), but not by stol^RNAi (n = 4) and 36% reduction of LVA current (I) by stj^RNAi (*p = 0.025, one-way ANOVA, LSD post-hoc test) but not by stol^RNAi . (J–K) Pupal MN5 Ca_v2 current (J; K white bar, n = 11) is 53% smaller following stj^RNAi (Ji; K light gray bar, n = 13; ***p = 1.5*10⁻⁵, one-way ANOVA, Tukey post-hoc test) but unaffected by stol^RNAi (Jii; K, dark gray bar, n = 7). Double stj^RNAistol^RNAi did not reduce Ca_v2 current amplitude further than stj^RNAi alone (Jiii, K, black bar, n = 6; p = 0.851, one-way ANOVA, Tukey post-hoc test). Bar diagrams in E–K show means ± SD.