A wide variety of ion channels are subject to regulation by membrane PIP2 (phosphatidylinositol-4,5-bisphosphate) (Suh & Hille, 2005). We have concentrated our attention on a species of K+ channel, the M-channel. This is a low-threshold, voltage-gated K+ channel that is widely distributed in central and peripheral neurons, where is serves to regulate neuronal excitability. Native M-channels are composed (primarily) of a heteromeric assembly of Kv7.2 and Kv7.3 subunits. The channels are closed by activating many different receptors that couple to Gq/11 G proteins, most prominently M1 muscarinic acetylcholine receptors (mAChRs), leading to an increased neuronal excitability. Channel opening requires the presence of PIP2 and available evidence suggests that closure following M1-mAChR stimulation results from Gq / phospholipase-C (PLC) mediated PIP2 hydrolysis and consequent PIP2 depletion (Delmas & Brown, 2005; Suh & Hille, 2005). We have tried to follow the dynamics of mAChR-induced PIP2 hydrolysis and depletion in single living sympathetic neurons using (initially) the fluorescently-tagged PH domain of PLCδ, GFP-PLCδPH, in combination with voltage-clamp membrane current recording (Winks et al., 2005). This probe binds to membrane PIP2 then translocates to the cytosol following PIP2 hydrolysis. Since the probe also binds to the cytosolic hydrolysis product inositol-4,5-bisphosphate (IP3), we used an intracellular IP3 displacement assay to calculate changes in membrane PIP2 from the fluorescence signals. Fluorescence changes showed a close temporal and concentration-dependent correlation with mAChR-induced M-current inhibition, and inhibition could be satisfactorily accounted for from the calculated changes in membrane PIP2, with a maximal depletion of ~83%. However, these calculations break down if PIP2 synthesis is accelerated during receptor activation. This has been suggested to occur following activation of another Gq/11-coupled receptor, the B2-bradykinin (BK) receptor, for which M-current inhibition seems to result from the IP3-induced release of Ca2+ rather than PIP2 depletion (see Delmas & Brown, 2005). Hence, we have now used another PIP2-binding probe that does not bind to products of PIP2 hydrolysis, the C-terminus of the transcription factor tubby (Santanaga et al., 2001; Quinn & Tinker, 2004), mutated to reduce affinity for PIP2 (YFP-tubby-R332H). This co-localized with the PLCδ-PH probe and the two translocated with a similar time course following mAChR stimulation. In contrast, BK induced much less translocation of tubby-R332H than PLCδ-PH. BK, but not mAChR-induced translocation was then increased when PIP2 synthesis was inhibited by wortmannin. Thus, BK produced less PIP2 depletion than mAChR stimulation because it accelerated PIP2 synthesis but could produce equivalent depletion when synthesis was inhibited. We further observed that, when the Ca2+-mediated component of M-current inhibition by BK was reduced by depleting Ca2+ stores with thapsigargin, inhibition was restored by wortmannin. In this way we could switch the mechanism of BK-induced inhibition from that mediated by the action of a product of PIP2 hydrolysis (IP3 / Ca2+) to one mediated by PIP2 depletion. Thus, these K+ channels can be regulated by both PIP2 and by products of PIP2 hydrolysis. While the contributions of these two may vary with different agonists (and probably in different cell types), they are not mutually exclusive, and indeed may interact (see Delmas & Brown, 2005).