Mammalian TRPC channels are most closely related to the prototypical Drosophila dTRPs both structurally and functionally as they are commonly gated by PLC activation (Hardie, 2007). Although a number of second messengers modulating TRPC4,5 activity have been identified, their prime activator in the PLC pathway remains elusive (Plant and Schaefer, 2005). We hypothesised that, similarly to dTRPs, the PLC substrate phosphatidylinositol 4,5-bisphosphate (PIP₂) can inhibit TRPC4. HEK293 cell lines stably expressing two major splice variants of TRPC4, murine TRPC4α and TRPC4β, were used for patch-clamp recordings performed in symmetrical 124 mM Cs⁺ solutions with internal Ca²⁺ strongly buffered at 100 nM. Infusion of GTPγS (0.2 mM) via pipette activated cation currents which showed characteristic doubly-rectifying I-V relationships with peak current densities at -50 mV of -93.1±34.8 pA/pF (n=7)and -28.9±11.3 pA/pF (n=11) for the TRPC4α and TRPC4β, respectively. These responses were inhibited by the PLC blocker U73122 (2.5 μM) (by 70±6% and 75±9% in the case of the α- and β-isoforms, respectively; n=4, P<0.02 by paired t-test), but not by its inactive analogue U73343. Cell treatment with pertussis toxin (100 ng/ml, 16-18 hrs) inhibited both TRPC4α and –β currents by about 80% (n=8, P<0.01). Thus, distinct G proteins appear to act in synergy in TRPC4 activation. The addition of the stable PIP₂ analogue diC8-PIP₂ to the pipette solution (20 μM) suppressed TRPC4α activation in a voltage-independent manner (by 84%, n=7; P<0.05), but had no significant effect on TRPC4β activation. By in vitro binding assay, we showed that PIP₂ binds directly to the 84 amino acid C-terminal domain which is only present in TRPC4α and known to be an autoinhibitory domain (Schaefer et al., 2002). As TRPC4 is bound to a PDZ domain-containing protein, NHERF (Tang et al., 2000), which in turn interacts with the actin cytoskeleton via the adaptor ERM proteins regulated by PIP₂, we also analysed the consequences of cytoskeleton disruption. Treatment of HEK293 cells with cytochalasin D (5 μM for 2 hrs) completely prevented the inhibitory effect of diC8-PIP₂ on TRPC4α activation. Channel release from the inhibition exerted by PIP₂ and actin cytoskeleton is suggested as a novel mechanism of TRPC4 activation, which is specific to its full-length α-isoform.