Pathogenic LRRK2 deregulates depolarization-induced Ca ²⁺ entry. (a) Schematic of LRRK2 showing the position of the G2019S mutation (GS) linked to PD. Human SH-SY5Y cell lines stably expressing LRRK2 were generated and then differentiated with RA for analyses. (b) Schematic of the imaging technique used to monitor changes in cytosolic Ca²⁺ through Ca²⁺ channels in the cell membrane in response to K⁺ depolarization using chemical (Fura-2) or genetically encoded (GECO1.2) fluorescent indicators. (c–e) Exemplar Ca²⁺ signals recorded from cells loaded with Fura-2 from the indicated cell line in response to 50 mM K⁺. Gray lines are responses from individual cells. The thick lines are the population average. (f) Ca²⁺ signals from multiple population averages (mean ± SEM) of the indicated line. n = 3 (parental), n = 12 (EV), n = 24 (GS 5), n = 7 (GS 9), n = 18 (WT 3), n = 18 (WT 14), where n refers to the number of independent biological replicates. (g) Summary data (mean ± SEM) quantifying the peak change and the area under the curve for the Ca²⁺ signals in the indicated line. Data were amalgamated for the control lines (parental and EV (n = 15)) and lines expressing wild-type LRRK2 (n = 36) and LRRK2 G2019S (n = 31). Each point represents the mean response from a cell population. ****P < 0.0001 (one-way ANOVA, Tukey’s test). (h) Schematic of the method used to record changes in cytosolic Ca² in cell populations through Ca²⁺ channels in the cell membrane in response to increasing concentrations of K⁺. (i) Summary data (mean ± SEM) quantifying the peak change in the Ca²⁺ signals from the indicated line and K⁺ concentration. n = 10 (EV), n = 13 (GS), n = 13 (WT), where n refers to the number of independent biological replicates. (j) Confocal micrographs of cells expressing GECO1.2. Scale bar: 10 µm. (k) Exemplar Ca²⁺ signals recorded from cells expressing GECO1.2 from the indicated cell line. Cells were stimulated with K⁺ (50 mM) and ionomycin (10 µM) toward the end of the recording. Gray lines are responses from individual cells. The thick line is the population average. (l) Summary data (mean ± SEM) quantifying the peak change in the Ca²⁺ signals from the indicated line. Each point represents the response from an individual transfected cell. n = 44 (WT), n = 66 (GS), where n refers to the number of cells from three independent transfections. *P < 0.05 (Mann–Whitney test). (m) Schematic depicting entry of Ca²⁺ through store-operated Ca²⁺ channels in the cell membrane in response to depletion of ER Ca²⁺ stores with Tg. (n) Ca²⁺ signals (mean ± SEM, n = 3 independent biological replicates) in response to thapsigargin (1 µM). Cells were loaded with Fura-2 and stimulated in the absence of external Ca²⁺, and then, external Ca²⁺ was added back as indicated. (o) Summary data (mean ± SEM, n = 3 independent biological replicates) quantifying the peak change in the Ca²⁺ signals from the indicated line. Each point represents the mean response from a cell population. ns, nonsignificant (unpaired t test). RA, retinoic acid; Tg, thapsigargin; EV, empty vector.