Fig. 8: Loss of NPC1 function spatially and functionally remodels RyR–Cav1.2–KV2.1 signaling nanodomains. | Nature Communications

Fig. 8: Loss of NPC1 function spatially and functionally remodels RyR–Cav1.2–KV2.1 signaling nanodomains.

From: NPC1-dependent alterations in KV2.1–CaV1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease

Fig. 8

A Schematic diagram of the hypothesis: NPC1 deficient neurons have ER-PM domains enriched in RyR—CaV1.2–Kv2.1—SERCA and increased spontaneous Ca2+ activity at KV2.1-asociated ER–PM MCSs. B Left, representative super-resolution Airyscan images taken at a focal plane near the PM of CTL (black) and U18-teated (red) neurons co-immunolabeled for CaV1.2 and RyR. Right, quantification of % soma occupied by CaV1.2–RyR, CaV1.2–RyR cluster density and CaV1.2–RyR cluster size of CTL (black) and U18-treated (red) neurons in the soma (top, yellow) and dendrite (bottom, orange) region. N = 20–21 (CTL) and n = 26 (U18) neurons and n = 52–53 (CTL) and n = 54–55 (U18) dendrites were analyzed across 2 indepdendent isolations. C Top, schematic diagram of hypothesis: NPC1 deficient neurons (U18, red) have increased Ca2+ activity and KV2.1-associated CaV1.2, while disrupting Kv2.1–Cav1.2 or KV2.1–VAPA/B interactions (CCAD, purple or FFAT, red-brown) abrogates such effects. Middle left, representative super-resolution TIRF images of neurons transfected with GCamP3-Kv2.1P4O4W. Bottom left, intensity time series and kymographs of spontaneous activity taken from the square region of interest. Middle right, quantification of GCamP3-Kv2.1P4O4W peak amplitude of CTL (black), U18 (red), CCAD + U18 (purple) and FFAT + U18 (red-brown) neurons. N = 85 (CTL), n = 141 (U18), n = 23 (CCAD + U18) and n = 10 (FFAT + U18) GCamP3-Kv2.1P4O4W peaks were analyzed across 3 independent isolations. All error bars represent SEM. Statistical significance was calculated using the following tests: Unpaired (two-tail) and Mann–Whitney t tests (two-tail) in (B) and Kruskal–Wallis test in (C). ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. #P < 0.05; ##P < 0.01; ####P < 0.0001. # indicates comparison with the CTL condition. CTL is control, SCRBL is scramble, U18 is U18666A, CCAD is calcium channel association domain, and FFAT is two phenylalanines (FF) in an acidic tract.

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