Fig 2: CTL1-Mediated Choline Uptake Potentiates IP3-Evoked Ca2+ Signals(A) NG108-15 cells were incubated in HBS alone or with 3 mM choline for the indicated times before adding bradykinin (1 μM) and immediately recording the increase in [Ca2+]i. Results (mean ± SEM; n = 3 with duplicate determinations) show Δ[Ca2+]i evoked by bradykinin.(B) Summary results (mean ± SEM; n = 3) show bradykinin-evoked Δ[Ca2+]i after incubation with the indicated choline concentrations (105 min).(C) WB showing effects of the indicated siRNA (for CTL1) or shRNA (for Sig-1R) and their scrambled counterparts on expression of CTL1 and Sig-1R in NG108-15 cells. Mr markers (kDa) are shown.(D) Summary results (mean ± SD; n = 5) show CTL1 expression in cells treated with the indicated siRNA expressed as a percentage of the matched cells treated with scrambled siRNA.(E) Summary results (mean ± SEM; n = 5 plates with 2 replicates) show the effects of 10 mM choline on bradykinin-evoked Ca2+ signals. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA with Dunnett’s test, relative to control (B and E).(F) Ca2+-mobilizing GPCRs stimulate PLC and PLD, with consequent formation of IP3 and choline. Although we have not resolved how GPCRs stimulate PLD in NG108-15 cells, signals evoked by both PLC and parallel pathways are known to stimulate PLD. IP3 stimulates IP3R, while choline binds to Sig-1Rs, causing them to potentiate IP3R activity. Metabolism of IP3 and choline terminates their signaling. Hence, GPCRs regulate IP3Rs through two parallel, but converging, pathways. Import of extracellular choline by transporters, including the widely expressed CTL1, can also deliver choline to Sig-1Rs.(G) Acetylcholine (ACh) released at cholinergic terminals can activate post- and pre-synaptic receptors, before its rapid hydrolysis to choline by acetylcholinesterase (AChE). Hence, synaptic activity is rapidly followed by a substantial local increase in choline concentration. Transporters (red circles) in the cholinergic terminal (CHT1) and neighboring cells (CTL1-5 and OCT) can import the choline, which will then stimulate Sig-1Rs, providing cells with a paracrine reporter of recent synaptic activity.See also Figure S2.

Fig 3: Choline Is an Agonist of Sig-1Rs(A) Clusters of Sig-1Rs anchored at MAMs are thought to dissociate into monomers when they bind a Sig-1R agonist, freeing Sig-1Rs to interact with their targets, within and beyond MAMs. The targets include IP3Rs.(B) Specific binding of [3H](+)-pentazocine (5 nM) in the presence of choline and related compounds using membranes from Neuro-2A cells stably expressing Sig-1R-GFP (mean ± SEM; n = 5, with 3 replicates for each). Specific binding of 3H-pentazocine was 90% ± 3% of total binding (mean ± SEM; n = 3) for membranes from cells overexpressing Sig-1R, and 13% ± 5% for mock-transfected cells.(C) Choline metabolism (structures from http://www.hmdb.ca).(D) NG108-15 cells were incubated (2 hr, 37°C) with PRE-084 (25 μM) or BD1047 (25 μM), and then, in the continuous presence of the Sig-1R ligands, loaded with Fluo-8 by incubation with Fluo-8 AM in HEPES-buffered saline (HBS) (30 min, 20°C, with a further 30 min to allow de-esterification of Fluo-8). BAPTA (2.5 mM) was then added to chelate extracellular Ca2+ before addition of bradykinin (10 μM). Results show typical responses as means of 3 replicates.(E) Summary results (mean ± SEM; n = 5, each with 3 replicates) show peak increases in [Ca2+]i (Δ[Ca2+]i) evoked by bradykinin. ∗p < 0.05 for maximal responses relative to control, one-way ANOVA with Dunnett’s test.(F) Pooled results (mean ± SEM; n = 20; as percentages of matched control response) for all bradykinin concentrations. The asterisk (∗) denotes 95% confidence intervals that exclude 100%.See also Figure S1A.

Fig 4: Sig-1R and PLD Contribute to Ca2+ Signals Evoked by Agonists of GPCRs(A) Typical pseudocolor images show peak Ca2+ signals (F340/F380) evoked by ATP (50 μM) in Fura-2-loaded NG108-15 cells transfected with control shRNA or shRNA to PLD1 and PLD2, or Sig-1R, each tagged with RFP. Calibration code (F340/F380) and scale bar (20 μm) apply to all panels.(B) Time course of response to ATP (bar; n = 6).(C) Summary (mean ± SD; n = 6) shows Δ[Ca2+]i evoked by ATP. ∗p < 0.05, ANOVA with Bonferroni test, relative to untransfected cells.(D) Δ[Ca2+]i evoked by bradykinin in populations of NG108-15 cells. Histogram (which shares the y axis) compares responses to bradykinin (10 μM) after treatment with scrambled or Sig-1R shRNA. Results are means ± SEM; n = 3 with duplicate determinations. ∗p < 0.05, Student’s t test.(E) WB shows effects of indicated shRNA, each tagged with RFP, on expression of PLD1 and PLD2 in NG108-15 cells. Mr markers (kDa) are shown. Results, typical of 3 WBs, underestimate knockdowns in the cells used for Ca2+ measurements, which used only cells shown to be transfected by expression of RFP (see A).(F and G) Intracellular concentrations of choline (F) and IP3 (G) during stimulation of NG108-15 cells with ATP (50 μM, bar) show the effects of shRNA for PLD1 and PLD2. Results show means ± SD; n = 6.(H) GPCRs that activate PLC and phospholipase D (PLD) initiate two parallel signaling pathways that converge at IP3Rs. IP3 from PLC directly activates IP3R. Choline from PLD activates Sig-1R, which potentiates IP3-evoked Ca2+ release.