I bungarotoxin competes with ACh an endogenous activator
[125I] α-bungarotoxin competes with ACh, an endogenous activator of α7-nACh receptors by binding to the ACh binding site on the receptor (Albuquerque et al., 2009). For this reason, the effect of curcumin was investigated on the specific binding of [125I] α-bungarotoxin. Saturation curves for the binding of [125I] α-bungarotoxin, in the presence and absence (controls) of curcumin are presented in Fig. 4A. At a concentration of 10 μM, curcumin did not cause a significant alteration in the specific binding of [125I] α-bungarotoxin. The apparent affinity (KD) of the receptor for [125I] α-bungarotoxin was 0.71 ± 0.16 and 0.76 ± 0.21 pM for controls and curcumin, respectively (n = 9 measurement from 3 experiments; ANOVA, P = .089). Further analysis of saturation binding data by Scatchard analysis (Fig. 4B) indicated that, in the absence and presence of curcumin (10 μM), there is no significant alteration in a Bmax values. In the absence and presence of curcumin, Bmax values were 0.746 ± 0.065 pmol/mg and 0.781 ± 0.087 pmol/mg, respectively (n = 9 measurement from 3 experiments, ANOVA, P = .073).
Discussion In the present study, we provide novel evidence that curcumin potentiates the function of human α7-nACh receptors expressed in SH-EP1 cells. Potentiation by curcumin occurs in a time and concentration dependent manner, but appears independent of channel MAZ51 by protein A, C, and CaM type kinases. The findings underscore a role for curcumin in the regulation of α7-nACh receptor signaling in various cells. Our findings suggest that the application of curcumin alone is not sufficient to promote a detectable change in intracellular Ca2+ within cells. However, 10 min. pre-incubation with curcumin can significantly potentiate the Ca2+ transients response of the ligand activated α7-nACh receptors suggesting that curcumin functions as a positive modulator of α7-nACh receptors. Since, Ca2+ transients induced by high K+ containing solutions and activation of α4β2 nACh receptors were not altered by pre-incubation with 10 μM curcumin, the effects of curcumin appear specific to α7-nACh receptors. Interestingly, these functional effects of curcumin appeared dependent on its application prior to the receptor ligand as evidenced by our finding that co-application of curcumin and choline does not alter the choline-induced Ca2+ transients. Although the kinetics of curcumin effect were not studied in detail in this report, our comparison of the effect of curcumin application (before and during) choline stimulation of the receptor indicates that the potentiating effects of curcumin are achieved after several seconds of ligand preexposure. This time dependent effect of curcumin on the choline response of the nACh receptor suggests that additional cellular pathways upstream of the receptor are engaged by curcumin in these cells. However, inhibition of key nACh receptor modulating kinases such as protein kinase A, C, and CaM by specific inhibitors did not reverse the effect of curcumin suggesting that phosphorylation by the kinases is not required for curcumin\'s effects on the nACh receptor. In the future studies, it will be important to test the effects of curcumin on other pathways including those pathways that have been demonstrated to be activated by curcumin such as several integral membrane proteins including enzymes, transporters, and ion channels (Zhang et al., 2014). Notably, the results of radioligand binding experiments indicate that curcumin does alter specific binding of [125I] α-bungarotoxin suggesting that curcumin may act as allosteric modulator of α7-nACh receptor. In this study, curcumin was applied in the concentration range of 10 nM to 10 μM and was found that it can enhance the function of α7-nACh receptors in a concentration-dependent manner with EC50 value of 231 nM. Interestingly, even the major metabolites of curcumin tetrahydrocurcumin, demethylcurcumin, and didemethylcurcumin (Anand et al., 2007) can also potentiate the choline-induced Ca2+ transients, suggesting bioactivity of these curcumin metabolites at the α7-nACh receptor site. The concentration of curcumin in plasma and its ability to pass the blood brain barrier following oral and intravenous administration has been studied previously (Shoba et al., 1998, Anand et al., 2007). When curcumin is given orally at a dose of 2 g/kg to rats, maximum serum concentration of 1.35 μg/ml (3.5 μM) was attained (Shoba et al., 1998). Since curcumin is a highly lipophilic compound with LogP (octanol–water partition coefficient) value of 3.3, its membrane concentration is expected to be considerably higher than blood levels. Therefore, the functional modulation of α7-nACh receptors demonstrated in this study can be pharmacologically relevant.