Transient receptor potential (TRP) superfamily of cation channels includes about 30 related proteins which are responsive to a large array of physical and chemical stimuli, typically in a polymodal manner. However, one common emerging feature of TRP regulation appears to be their promiscuous interaction with various phospholipids, which often alters the voltage dependence of the channel gating (Nilius et al. 2007). Several members of the TRPV and TRPM sub-families have been well characterised, especially in terms of their interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂), the ubiquitous anionic lipid. Apart from PIP₂, lysophospholipids (LPLs) such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) could both activate the cold- and menthol receptor TRPM8. We recently showed that TRPM8 activity reduced in inside-out patches due to PIP₂ depletion could be fully restored and even augmented above the cell-attached level following application of LPC or LPI to the intracellular side of the membrane. Ca²⁺ store depletion resulting in Ca²⁺-independent iPLA₂ activation/LPLs production appears to be the main coupling mechanism in this alternative to cold “chemical” mode of TRPM8 activation (Abeele et al. 2006). Regulation of members of the “canonical” TRPC subfamily by PIP₂ is much less characterised. Here we focused on one member, TRPC4, which is typically activated by phospholipase C (PLC)-coupled receptors (Plant & Schaefer, 2003). Its two most abundant transcripts, TRPC4α and TRPC4β (the latter lacks 84 amino acids in the cytosolic C terminus), are widely expressed in various smooth muscles (Walker et al. 2001), and in the gastrointestinal tract TRPC4 is an essential component of the cation channel activated by muscarinic receptor stimulation (Lee et al. 2005). Both for the heterologously expressed channels and the native current the signal transduction downstream of PLC activation is still debated. We thus investigated a possible role of PIP₂ in the regulation of TRPC4. Two mouse isoforms, TRPC4α and TRPC4β, were expressed in HEK293 cells and studied with the patch-clamp recording techniques using Cs⁺-rich (125 mM) external and internal solutions (internal [Ca²⁺]_i was clamped at 100 nM using 10 mM BAPTA to avoid Ca²⁺-dependent channel modulation). Infusion of 200 μM GTPγS via pipette slowly activated cation currents with peak densities at -50 mV of -93.1±34.8 pA/pF and -28.9±11.3 pA/pF for the TRPC4α and TRPC4β, respectively (n=7-11). The currents showed characteristic doubly-rectifying current-voltage relationships. The non-metabolisable PIP₂ analogue diC8-PIP₂ added to the pipette solution (20 μM) strongly suppressed TRPC4α activation (peak current was reduced to -14.7±3.5 pA/pF, n=7; P<0.05 by non-paired t-test), but had no effect on TRPC4β activation (peak current of -25.4±7.6 pA/pF, n=11). Activation of both TRPC4 isoforms was strongly inhibited by the PLC blocker U73122 (2.5 μM, n=4; U73343 used as a negative control had no effect, n=4) as well as by pre-treatment with pertussis toxin (100 ng/ml for 16-18 hrs) indicating involvement of distinct G proteins. Consistent with these findings, in in vitro binding study we showed that PIP₂ binds directly to the 84 amino acid C-terminal domain that is only present in TRPC4α. Since PIP₂ can act as an anchor for various signalling molecules and cytoskeleton we further investigated possible causal relation between PIP₂ effects and cytoskeleton disruption. Treatment of HEK293 cells with cytochalasin D (5 μM for 2 hrs) completely prevented inhibition of TRPC4α by diC8-PIP₂. Finally, we confirmed by Western blotting that both channel variants were expressed in guinea-pig ileal myocytes in which carbachol-induced currents showed essentially similar properties (i.e. diC8-PIP₂ suppressed the current while cytochalasin D treatment accelerated its activation, produced a negative voltage shift of channel gating and abolished the inhibitory effect of diC8-PIP₂). Our results suggest that TRPC4 channel is constitutively inhibited by PIP₂, and its primary mechanism of activation may thus involve release from inhibition by PIP₂ hydrolysis evoked by PLC-coupled receptor stimulation. Cytoskeleton also plays a crucial role in maintaining TRPC4 inactivation.