Fig. 5
Mutation S652L increases intracellular Ca2+ during simulated action potential firing. a Upper left: Shape of single action potential waveform (APW) mimicked by the following voltage steps: HP: − 80 mV, − 80 to − 60 mV for 2.5 ms, − 60 to + 20 mV in 1 ms, + 20 to − 70 mV in 1.5 ms, − 70 to − 60 mV in 5 ms, − 60 mV for 90 ms. The corresponding ICa of WTL and S652LL are shown below. Right: Representative current responses of WTL and S652LL during 30 s of stimulation with APW-like stimuli at a frequency of 10 Hz. b Peak ICa of S652LL Cav1.3 channels decayed faster than WTL during stimulation. Statistics: unpaired student´s t-test ([mean ± SEM]; WTL, 14.94 ± 2.19, n = 20; S652LL, 30.94 ± 2.85***, n = 21; ***p < 0.001). c Average Ca2+-signal of WTL and S652LL expressing HEK-293 cells upon 30 s of stimulation. Ca2+-signal was normalized to baseline fluorescence (F0 [mean ± SEM]; WTL, 1.65 ± 0.20; S652LL, 1.38 ± 0.18) and current density (pA/pF [mean ± SEM]; WTL, − 11.93 ± 1.46; S652LL, − 8.51 ± 1.04) determined in a ramp protocol before the start of the train. S652LL Cav1.3 channels showed higher levels of [Ca2+] than WTL after 30 s of stimulation. Statistics: unpaired Student’s t test, **p < 0.01. d Ca2+-charge of WTL and S652 LL obtained by integrating the area of the ICa transient normalized to maximum ICa determined in a ramp protocol before the start of the train. Statistics: two-way ANOVA of selected time points (every 5 s), *p < 0.05, **p < 0.01, ***p < 0.001. e Overlay of 1st AP of WTL and S652LL ICa transients normalized to current density (pA/pF) determined in a ramp protocol before the start of the train to demonstrate larger APs induced by mutation S652L. f Normalized representative ICa transients of WTL and S652LL obtained from repolarisations from + 80 mV to − 60 mV (left) or − 40 mV (right); scale bars correspond to the traces of the same color; for parameters and statistics see Table 3. The AP-like command voltage also triggered an outward current component occurring at the peak of the AP spike. We and others (see references in Ortner et al. [29]) have observed this previously. The outward component is likely composed of QON and a passive non-LTCC component (also found in non-transfected cells, [29])