Kir channels serve important functional roles in cellular physiology: Kir6 channels control insulin secretion, myocardial resistance to hypoxia and protection against hypoxia-induced generalized seizure; Kir3 channels mediate the vagal control of heart rate and contribute to the G protein mediated regulation of neurotransmitter release in the brain; Kirl channels control K+ secretion in kidney; Kir4 channels in glial cells and inner ear control K+ homeostasis and excitability. In humans there are three major diseases linked so far to mutations in a Kir channel or an associated protein: persistent hyperinsulinimic hypoglycemia of infancy (Kir6,2), Barter's syndrome (Kir1.1), and Andersen's syndrome (Kir2.1). In addition, the weaver phenotype, a neurological disorder in mice has also been associated with mutations in a Kir3 channel. A common characteristic of members of the inward rectifier K+ (Kir) channel family is that they are all activated by the plasma membrane phospholipid PIP2, phosphatidylinositol-bis-phosphate. Differences in channel-PIP2 interactions have been described among specific Kir members both biochemically and functionally. Channels that experience strong interactions with PIP2 are highly active and are not normally inhibited by PIP2 hydrolysis, presumably because the PIP2 that interacts with these channels is protected from hydrolyzing enzymes (e.g. Kir2.1). Channels that experience intermediate strength of interaction with PIP2 show also intermediate levels of activity relative to other family members and they can indeed be inhibited by signals that lead to PIP2 hydrolysis (e.g. Kir2.3). Finally, channels that interact weakly with PIP2 require a gating molecule to strengthen channel-PIP2 interactions in order to be activated. These channels are highly susceptible to inhibition by signals that lead to PIP2 hydrolysis (e.g. Kir3.4). Regulation of channel activity by phosphorylation depends on channel-PIP2 interactions. Several of the mutations that result in disease involve PIP2-interacting residues. Since the activity and its regulation is so critically dependent on interactions with PIP2 in Kir channels, and since mutations that affect channel-PIP2 interactions result in disease, it is clear that understanding in detail the mechanisms responsible for channel-PIP2 interactions and their regulation is of paramount importance in molecular medicine. The current proposal aims to find answers to the following three questions. (a) What are the general and specific structural determinants that dictate the strength of interactions of each Kir family member with PIP2? (b) What are the sites of action of PKA/PKC-dependent phosphorylation and what is their relation to specific channel-PIP2 interacting sites? and (c) Which are the structural determinants that explain differences in stereospecific interactions of each Kir family member with PIP2?
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