FIGURE 5 | Muscarinic inhibition of the M current is accompanied by hydrolysis of PtdIns(4,5)P₂.

a | Hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) was monitored using the green-fluorescent protein (GFP)-tagged pleckstrin homology (PH)-domain of phospholipase-C (PLC–PH). This binds to the inositol phosphate head-groups of PtdIns(4,5)P₂ in the inner leaflet of the membrane. When muscarinic receptors (M1) are stimulated, phospholipase-C (PLC) is activated. This hydrolyses PtdIns(4,5)P₂, forming diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃), which enters the cytosol. The GFP-tagged PLC–PH bound to Ins(1,4,5)P₃ then leaves the membrane and translocates to the cytosol. b | The translocation of the probe (left) following stimulation of the muscarinic receptors with oxotremorine-methiodide (Oxo-M). After washing out the drug, the probe returns to the membrane as the Ins(1,4,5)P₃ is hydrolysed and PtdIns(4,5)P₂ is resynthesized (not illustrated). The right panel shows line scan profiles of the GFP–PLC–PH fluorescence intensity in the presence and absence of Oxo-M. c | Shows simultaneous recording of the decline in membrane current that occurs as the M channels are inhibited and the increase in cytosolic fluorescence after two applications of Oxo-M to a rat sympathetic neuron voltage-clamped with a perforated-patch electrode (J. Winks and S. J. Marsh, unpublished observations). Right panel in b adapted, with permission, from Ref. 48 © (2005) Society for Neuroscience.

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