It has been suggested that IK(M) (KCNQ/K_V7) channels are regulated by phosphatidylinositol-4,5-bisphosphate (PIP₂:Suh & Hille, 2002; Zhang et al., 2003). We have used a novel approach employing N-terminal palmitoylated decapeptides (palpeptide) to study this transduction pathway (Robbins & Brown, 2003).Rat superior cervical ganglion cells (isolated from rats killed by approved Schedule 1 methods) in primary culture were superfused at 23⁰C with (mM) NaCl 144, KCl 2.5, MgCl₂ 0.5, CaCl₂ 2, Hepes 5, glucose 10, pH 7.4. The amphotericin perforated variant of the patch clamp technique was used with electrodes filled with (mM) K acetate 80, KCl 30, Hepes 40, MgCl₂ EGTA 3, CaCl₂ 1, pH 7.2. Cells were clamped at -20mV and stepped to -50mV for 1 second to generate IK(M) deactivating tail currents. All peptides were synthesised by ABC, Imperial College, London, purified to >90% with RPHPLC and dissolved in DMSO at 10mM. The original peptide (H-palmitoyl-HRQKHFEKRR-CONH₂) is based on the putative PIP₂ binding site on KCNQ2-5 (Zhang et al, 2003) and a range of variants produced: K4M, H5C, K8M, K4MK8M, non palmitoylated and a fluorescein labelled version.The PIP₂ palpeptide inhibited IK(M) with an IC₅₀ of 1.5 ± 0.2µM (n=7); the non- palmitoylated version was ineffective up to 10µM (n=6). The muscarinic receptor antagonists, atropine (1µM) and pirenzepine (0.3µM), did not reduce the effectiveness of the PIP₂ palpeptide. PIP₂ palpeptide had no effect on other potassium currents such as IK(A) and IK(DR). The fluorescein labelled version confirmed a plasma membrane localisation of the PIP₂ palpeptide. To investigate the residues likely to be involved in PIP₂ binding we used a number of mutants and assessed their potency in inhibiting IK(M). The K4M and H5C mutants showed no significant change in potency, IC₅₀s 0.7 ± 0.1µM (n=5) and 0.9 ± 0.3µM (n=5) respectively. Conversely the K8M and the double mutant K4MK8M were significantly (p<0.05 ANOVA) less potent on the current (IC₅₀s 3.2 ± 0.9µM (n=5) and 3.4 ± 0.5µM (n=5) respectively). All slope values were not significantly different from each other or unity. Further, low concentrations of PIP₂ palpeptide increased the sensitivity (IC₅₀) of IK(M) to oxotremorine-M evoked inhibition from 0.8 ± 0.1µM (n=6) to 0.5 ± 0.1µM (n=6) 0.2 ± 0.04µM (n=6) at 0.1 and 0.3µM PIPpalpeptide 2 respectively.We suggest that the PIP₂ palpeptide is effectively buffering PIP₂ levels in the membrane so reducing IK(M) amplitude and increasing its sensitivity to muscarinic receptor mediated inhibition. Furthermore the lysine residue near the C-terminal end of the palpeptide seems to be important for PIP₂ binding.