C 60 fullerenes disrupt cellular signalling leading to TRPC4 and TRPC6 channels opening by the activation of muscarinic receptors and G-proteins in small intestinal smooth muscles

Abstract The effect of water-soluble pristine C 60 fullerene nanoparticles (C 60 NPs) on receptor-operated cation channels formed by TRPC4/C6 proteins in ileal smooth muscle cells was investigated for the first time. Activation of these channels subsequent to acetylcholine binding to the expressed in these cells M 2 and M 3 muscarinic receptors represents the key event in the parasympathetic control of gastrointestinal smooth muscle motility and cholinergic excitation-contraction coupling. Experiments were performed on single collagenase-dispersed mouse ileal myocytes using patch-clamp techniques with symmetrical 125 mM Cs + solutions and [Ca 2 + ] i ‘clamped’ at 100 nM in order to isolate the muscarinic cation current (mI CAT ). The current was induced by intracellular infusion of 200 μM GTPγS, which activates G-proteins directly, i.e. bypassing the muscarinic receptors. C 60 NPs applied at 10 − 6  M at peak response to activation of G-proteins caused mI CAT inhibition by 47.0 ± 3.5% (n = 9). The inhibition developed rather slowly, with the time constant of 119 ± 16 s, was voltage-independent and irreversible. Thus, C 60 NPs are unlikely to cause any direct block of TRPC4/C6 channels; rather, they may accumulate in the membrane and disrupt G-protein signalling leading to mI CAT generation. C 60 NPs may represent a novel class of biocompatible molecules for the treatment of disorders associated with enhanced gastrointestinal motility.