? It has been recently appreciated that a common characteristic of members of the inwardly rectifying K+ (Kir) channel family is that they are all activated by phospatidylinositol-bis-phosphate (PIP2). Differences in channel-PIP2 interactions have been described among specific Kir members, both biochemically (Huang et al., 1998) and functionally (Huang et al., 1998; Zhang et al., 1999; Lopes et al., 2002). There seem to be Kir channels that show either relatively strong, intermediate or weak interactions with PIP2. Certain Kir channels are inhibited by PIP2 hydrolysis. The strength of channeI-PIP2 interactions correlates with the extent of channel activity and the degree of channel inhibition caused by signals that lead to PIP2 hydrolysis. Thus, channels that interact weakly with PIP2 are inhibited the most by PIP2 hydrolysis, while channels that interact strongly with PIP2 are not inhibited by PIP2 hydrolysis. Channel modulation by PKC affects channel activity in a manner dependent on channel-PIP2 interactions. Thus, in a wild-type channel showing PKC-mediated inhibition of activity, mutations strengthening channel-PIP2 interactions can attenuate or abolish the effect of PKC. Similarly, in a channel that interacts strongly with PIP2 and therefore lacks PKC-dependent inhibition, mutations that weaken channeI-PIP2 interactions can render the channel inhibitable by activated PKC. The PKC-mediated inhibition could be obtained in heterologous systems such as in Xenopus oocytes but not in others, such as the CHO mammalian cells. The current proposal aims to find answers to the following two major questions. (a) Are there specific PKC isoforms that are needed to reconstitute the PKC-mediated current inhibition in certain cell systems where the effect is absent? Are there specific PKC adaptor proteins involved in mediating the PKC effects? (b) To what extend is the muscarinic induced inhibition of Kir currents due to PKC mediated effects? Does PKC modulation of Kir channel activity weaken channel-PIP2 interactions? The current proposal expands and enhances the parent grant HL59949. In the parent grant our emphasis is to identify the PKC-dependent phosphorylation sites either on the channel protein itself or associated proteins and to study their relationship to channeI-PIPz interacting sites. In the current grant the focus is to identify the specific PKC isoforms and/or adaptor proteins involved in the PKC-mediated inhibition and to determine the relative contribution to the ACh-induced current inhibition by PKC versus the decrease in direct channel-PIP2 interactions that result due to the PIP2 hydrolysis. ? ? ?
