Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone

Abstract

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, K_V1.3, and K_Ca3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, K_V1.3, and K_Ca3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC₅₀ values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Keywords: CRAC channel; KCa3.1; KV1.3; Orai; T lymphocyte; nootkatone.

Figure 1

Nootkatone’s inhibitory effect on CRAC channel activity. Membrane currents were recorded via whole-cell voltage clamp. (A) Chemical structure of nootkatone. (B) Nootkatone (1, 3, 10, 100 µM) was applied after confirming the I_CRAC in hORAI overexpressed HEK293T cells. Representative current (I)–voltage (V) relationship curves of normal I_CRAC (black trace) and decreased current in 1, 3, 10, 100 µM concentrations of nootkatone are shown. (C) Relative amplitudes of normalized I_CRAC current are plotted and fitted to a dose-response curve. Data are presented as mean ± S.E.M (n = 4 or 5). Approximate half inhibition is achieved in 10 µM condition (R² = 0.98, IC₅₀ = 12.64).

Figure 2

Nootkatone’s inhibitory effect on Potassium channel activity. (A) Representative chart trace of I_KCa3.1 current inhibition caused by 10 and 100 µM concentrations of nootkatone. After confirming the channel activation, nootkatone solution was changed from lower to higher concentration. TRAM34 (Triarylmethane-34) is used as a potassium channel blocker to show the non-K⁺ current. (B) I-V relationship curve of representative trace. (C) Average value of each I_KCa3.1 with nootkatone treatment was calculated by subtracting TRAM34 inhibited current. All data are expressed as the mean ± SEM (n = 3–5). (D) Representative chart trace of I_KV1.3 current inhibition caused by 3, 10, 100 and 300 µM concentrations of nootkatone. After confirming the channel activation, nootkatone solution was changed from lower to higher concentration. PAP-1((5-(4-Phenoxybutoxy) psoralen) is used as a potassium channel K_V1.3 blocker to show the non-K+ current. (E) I-V relationship curve of representative trace (timestamp of each curve is marked with arrow of same color in Figure 2D). (F) Average value of I_KV1.3 was subtracted by PAP-1 inhibited current and normalized to show dose-response curve. Data are presented as mean ± S.E.M (n = 3–5, IC₅₀ = 10.80, R² = 0.96).

Figure 3

Cell viability was analyzed for 2~3 days after treatment with various concentrations of nootkatone in Jurkat T cells. For (A) 48 h or (B) 72 h after treatment, nootkatone showed no meaningful level of cytotoxicity, as the relative viability was not significantly lower than 80% of the total cell, compared with the control group. (n = 3).

Figure 4

The inhibitory effect of nootkatone on store-operated Ca²⁺ entry (SOCE) was measured via Fura-2 calcium imaging. Activity was calculated with a 340/380 nm ratio, showing intracellular calcium ion concentration. (A) SOCE was induced via CPA (cyclopiazonic acid), with 1.3 mM of extracellular calcium starting calcium entry. Totals of 30 and 100 µM of nootkatone showed a lowering of 340/380 nm ratio; representative traces are shown. BTP2 was used to show the effect of the CRAC channel inhibitor. Representative images of Fura-2 calcium imaging are shown. Red and green signals indicate a higher level of calcium ion entry, while blue indicates lower SOCE activity. (I) Basal ratio, (II) CPA and 1.3 mM Ca²⁺ activated, (III) nootkatone 30 µM added, (IV) 100 µM added, and (V) BTP 10 µM added time points were used for image presentation and statistical comparison. Microscope magnification is 40× (B) Representative 340/380 ratio trace of calcium imaging. (C) Average normalized value of calcium signals in multiple cells in each timepoint, showing significant change after 30 and 100 µM of nootkatone treatment (the experiment was repeated three times, and the average number of cells is 54.3 ± 5.21, one-way ANOVA with bonferroni’s post-hoc test, p < 0.001). (D) Control 340/380 nm ratio trace of calcium imaging. (i) Basal ratio, (ii) CPA and 1.3 mM Ca²⁺ activated, (iii) BTP 10 µM added time points were used for statistical comparison. (E) Average normalized value of calcium signals in multiple cells in each timepoint (i)–(iii). (F) I-V relationship of K_Ca current in human primary CD4+ T cell. (G) Normalized value of K_Ca current. (H) I-V relationship of K_V current in human primary CD4+ T cell. (I) Normalized value of K_V current.

Figure 5

T cell proliferation analysis via flow cytometry (FACS). (A) In the upper panels, anti-CD3 and anti-CD28 untreated (left panel) and treated (middle panel) cell groups show an activation of T cell proliferation, and addition of BTP2 (CRAC channel inhibitor, right panel) showed inhibition of intracellular Ca²⁺ influx could minimalize T cell division. The cells within the red square are proliferating cells. In the lower panels, 10 (left), 30 (middle), and 100 (right panel) μM nootkatone treatment was additionally carried out in anti-CD3/CD28(+) T cells. Activated T cells showed partial inhibition of T cell proliferation, with dose-dependent efficacy. (B) T cell proliferation rate of each group. Statistical comparisons between anti-CD3/CD28(+) T cells and nootkatone treated groups were performed, and significant statistical differences were observed between groups (n = 3, one-way ANOVA with Dunnett’s post-hoc test, ** p < 0.01, *** p < 0.001).