KCNQ(Kv7)/M potassium channels are ‘gated’ by membrane phosphatidylinositol-4,5-bisphosphate (PIP₂) (Zhang et al. 2003). Hydrolysis and depletion of PIP₂ probably explains the inhibition of these channels produced by stimulating muscarinic acetylcholine receptors (mAChRs) (Suh et al. 2004). We previously reported that, in rat sympathetic neurons, over-expression of the PIP₂-synthesizing enzyme PI5-kinase reduced inhibition of M-channels by the mAChR-agonist oxotremorine-M (Oxo-M) (Winks et al. 2003). We have now assessed quantitatively how far this effect was due to increased membrane PIP₂ levels. Sympathetic neurons were dissociated from rat superior cervical ganglia (isolated from rats killed according to Home Office regulations), cultured in vitro, and transformed to express the PIP₂-binding fluorophore GFP-PLCδ-PH (Stauffer et al. 1998). Since this construct also binds IP₃ (e.g. Hirose et al. 1999) we used an ‘IP₃-displacement’ assay to estimate membrane [PIP₂]. For this, we patched neurons with pipettes containing different concentrations of IP₃ and measured the fractional translocation of GFP-PLCδ-PH from the membrane to the cytosol. Using the binding constants of Hirose et al. (1999), mean resting [PIP₂] (95% confidence limits in brackets) as ‘seen’ by the probe was calculated to be 261 (192-381) μM under control conditions and 693 (457-1153) μM after over-expressing PI5-kinase (Winks et al. 2003). Oxo-M also induces translocation of GFP-PLCδ-PH (Winks et al. 2003). From this, we calculated the changes in membrane [PIP₂] accompanying mAChR stimulation by incorporating measurements of the effective cytosolic volume available to IP₃ (determined from cell size and comparison of intracellular and aqueous vesicle BODIPY fluorescence) and the membrane:cytosol volume ratio. Expected changes in M-current amplitude were then calculated using the data of Zhang et al. (2003) for KCNQ2/3 channel activation by DiC8-PIP₂ (EC₅₀ 87 μM). The calculations accorded with the experimentally-observed concentration-M-current inhibition curve for Oxo-M (mean IC₅₀ 1.6 μM; maximal inhibition 74%; ~83% fall in membrane [PIP₂]). They also predicted a much smaller fall in [PIP₂] (23%) in PI5-kinase over-expressing neurons, and much less maximal M-current inhibition (2.3%), in accord with previous measurements (4.6%: Winks et al. 2003). This provides quantitative support for the hypothesis that, in these neurons, mAChR-induced M-current inhibition results directly from membrane PIP₂ depletion.