The IS4B exon is required for Cav2 localization to active zone and for evoked synaptic transmission.
(A-C) Representative confocal projection views of triple labels for GFP tagged Cav2 channels (green), the active zone marker brp (magenta), and HRP to label axonal membrane (blue) in control animals with all Cav2 exons (cacsfGFP, top row, A), in animals with selective excision of either the alternative exon IS4A ( IS4AsfGFP, middle row, B), or the alternative exon IS4B ( ISABsfGFP, bottom row, C). Excision of IS4B is embryonic lethal, so that localization analysis was conducted in heterozygous animals ( ISABsfGFP/+). The gross morphology of the neuromuscular junctions (muscle fibers, bouton numbers and sizes, active zone numbers) was similar in all three genotypes. GFP tagged Cav2 channels localize to active zones (A) as previously reported (Gratz et al. 2019; Krick et al. 2021). Excision of the IS4A exon does neither impact Cav2 channel active zone localization nor labeling intensity (B). By contrast, upon excision of the IS4B exon, Cav2 channel label (white arrowheads) is faint and not strictly co-localized with active zones (C). (D, E) Representative traces of evoked synaptic transmission as recorded in TEVC from muscle fiber 6 upon extracellular stimulation of the motor nerve from a wildtype control animal (CS, blue), an animal with GFP tagged Cav2 channels (cacsfGFP, green), and animals with GFP tagged Cav2 channels and either IS4A exon excision (ΔISA4, orange) or IS4B excision (ΔISAB, transheterozygous over ΔISA4, black trace). (D) Postsynaptic currents (PSCs) are similarly shaped between CS control (blue) and animals with GFP-tagged Cav2 channels (cacsfGFP, green), and (E) PSC amplitudes are not statistically different (p=0.34, two sided Tukey’s multiple comparison test). In animals with homozygous IS4A exon excision (orange), PSC amplitude is slightly but not significantly increased (p=0.18, two sided Tukey’s multiple comparison test). In transheterozygous animals with IS4A excision on one chromosome and IS4B excision on the other one, PSC amplitude is significantly decreased (p=0.0008), two sided Tukey’s multiple comparison test). (F) Quantal size (mPSC amplitude) and spontaneous release frequency (G) show no significant difference between genotypes. (H, I) Since animals homozygous for IS4B exon excision are lethal, we created mosaic animals that were heterozygous for Cav2 in most neurons but hemizygous for either cacsfGFP or ΔISAB in motoneurons innervating muscle M12 (see methods, cacFlpStop). In control with all Cav2 exons (cacsfGFP, green, top row) cacsfGFP colocalizes with brp (magenta) in presynaptic active zones on M12 (H) and evoked synaptic transmission induces PSCs of about 100 nA amplitude (I). By contrast, upon deletion of IS4B (H, bottom row) in motoneurons to M12 fuzzy Cav2 label is found throughout the motor terminals, but Cav2 (green) does not strictly colocalize with brp (magenta) in active zones (H) and evoked synaptic transmission is reduced by more than 90%, Student’s T-test, p < 0.0001 (I). (J) HVA Cav2 currents as recorded from the somata of adult flight motoneurons in mosaic animals with only one copy of the Cav2 locus in flight motoneurons (see methods). HVA currents are measured by starting from a holding potential of -50 mV (LVA inactivation) followed by step command voltages from -90 mV to +20 mV in 10 mV increments (left). In GFP-tagged controls (cacsfGFP / cacFlpStop) this reveals transient and sustained HVA current components. By contrast, following excision of the IS4B exon ( ISABsfGFP / cacFlpStop), the sustained HVA current is nearly absent. Current-voltage (IV) relation of sustained HVA for controls with all Cav2 exons (cacsfGFP, green circles, n = 8) and following excision of IS4B ( IS4BsfGFP, dark green squares, n = 4).