Anionic phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PI4,5P₂), are involved in a variety of cellular processes. One example is the regulation of various potassium channels e.g. depletion of PI4,5P₂ causes inhibition of the G-protein regulated inwardly rectifying potassium channel, Kir3.1/3.4. Using electrophysiological techniques with wild type and mutant proteins the C-terminal domains of Kir6.2 and Kir3.1/3.4 were identified to play a part in the binding of PI4,5P₂ (Schulze et al., 2003, Huang et al., 1998.). We have developed an assay to demonstrate the direct binding of anionic phospholipids to C-terminal cytoplasmic domains of members of the inwardly rectifying potassium channels, including the G-protein regulated Kir3.x family, the ATP-sensitive Kir6.x family, and a member of the voltage gated potassium channels, KCNQ1.The N- and C-terminal cytoplasmic domains of the respective channels were cloned into the pMALc2x vector (NEB) to give a N-terminal fusion protein with maltose binding protein (MBP). The fusion proteins were subsequently expressed and purified. Lipid-protein overlay assays were carried out using PIP Strips (Echelon) that are spotted with 100 pmol of a variety of phospholipids. Statistical significance was determined using one-way ANOVA with Dunnett correction (n>3).Direct and qualitative binding was observed for the C-terminals of Kir3.1, 3.2 and 3.4, Kir 6.1 and 6.2 and the unrelated KCNQ1. Different potassium channel domains showed varying affinity to phospholipids. Differences in affinity to phospholipids were observed within the Kir3.x family in that only the Kir3.1 C-terminus was shown to significantly bind to monophosphoinositols such as phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 5-phosphate (PI5P). The C-terminals of Kir3.2 and 3.4 did not show any binding. However, the addition of the N-terminal cytoplasmic domain of Kir3.4 to the corresponding C-terminal resulted in significant binding to monophosphoinositols similar to that observed with the Kir3.1 C-terminus. Within the Kir6.x family only the C-terminus of Kir6.1 was observed to bind PI4P and PI5P, no binding of Kir6.2 to any phospholipids was observed. On the other hand, the binding profile observed for KCNQ1 was different in that it bound most of the phosphatidylinositols, including PI4,5P₂ and phosphatidylinositol 3,4,5-triphosphate (PI3,4,5P₃). Interestingly, weak or no binding of Kir3.x or Kir6.x domains to PI4,5P₂ was detected.