(A) Average ΔCm measured from ribbonless rods filled with 10 mM EGTA and stimulated with a Vstep to −18 mV for durations from 0.5 to 30 ms. Single exponential fits to points from 0.5 to 30 ms. Wild-type results are presented for comparison (see Figure 5A). (B) Comparison of all ΔCm responses that were evoked with 1 and 3 ms steps, with either 0.5 or 10 mM EGTA in the pipette, and for wt and ko rods. All ko responses were significantly smaller than wt values, see text. (C) Plot of peak-ICa over Vstep. Ribbonless rods had significantly smaller peak-ICa at the indicated Vstep values (*, p-values<0.05), with comparisons made between wt vs. ko rods for 0.5 mM EGTA (blue dashed line) and 10 mM EGTA (black dashed line). See Figure 3A for a description of voltage step protocols and analysis. (D) ICa activation kinetics were significantly slower for wt rods filled with 10 mM EGTA (* indicates sig. diff.; see Supplementary file 2). (E, F) Average ICa traces measured from rods filled with 10 mM EGTA illustrate the significant differences in peak-ICa at the onset of depolarization (*; p<0.004 and 0.03 for 1 and 3 ms steps; n: 13 wt and 7 ko cells). See Supplementary file 3 for related results with 10 ms steps. (F) wt rods showed a faster rate of ICa inactivation than ko rods. At the end of the 30 ms steps, ICa amplitudes were no longer significantly different (nd; p>0.3); whereas, at the onset they were different (*; p<0.008; n: 8 wt and 7 ko). Supplementary file 3(G) Summary of average peak-ICa measured from wt and ko rods filled with 10 mM EGTA. The 1 ms steps were essentially tail-currents, and therefore larger in amplitude than peak-ICa measured from step durations ≥3 ms. Only the 9 ms steps did not show a significant difference between wt and ko. (H, I) Comparison of ICa recorded with 0.5 mM EGTA in the pipette. Averaged traces presented in (H). In (I), statistical comparisons show no difference in ICa amplitudes resulting from 1 ms steps (nd; p: 0.13; 9, and 11 cells each), while the currents generated with 3 ms steps were significantly larger for wt rods (*, p: 0.05; 9 and 12 cells each). (J, K) ΔCm in response to 1 and 3 ms steps plotted over peak-ICa in (J), and over QCa in (K). Dashed lines partition data points by step duration. See text for discussion. RRP, readily releasable pool; SV, synaptic vesicle.