The major regulator of CFTR and ENaC in human airway epithelia is cAMP. In vivo, CFTR mediated Cl- conductance is approximately 50% active even at rest, and maximal activation can be attained when there is no detectable increase in total intracellular cAMP. These observations strongly suggest that protein kinase I (PKA) must be held within the apical plasma membrane in close proximity to CFTR and/or ENaC. Sequestration of PKA within very precise subcellular locations and in apposition with channels and transporter in not an unusual phenomenon. In fact, a diverse family of A- Kinase Anchoring Proteins (AKAPS) have been show to localize PKA in subcellular compartments in neurons, muscle and germ cells. Preliminary data for this application implicate AKAPS in the regulation of CFTR and ENaC in airway epithelia. Completion of these studies is Aim 1 of this application. However, the molecular identity of relevant AKAPS and their precise mechanisms of action promise enormous new insight into the regulation of apical membrane ion conductances. Therefore, in Aim 2, we will clone airway epithelial AKAPS by a well established and highly specific RII interaction strategy.
In Aim 3 we will determine which airway epithelial AKAPS are concentrated at the apical cell membrane. We will use site-directed mutagenesis to identify RII binding sites and domains responsible for the apical localization. We will design cDNAs encoding mutant AKAPS that will exert dominant-negative effects when expressed in airway epithelial cell lines. These cell lines will be used to test the role of specific apical membrane AKAPS in the regulation of CFTR under basal and stimulated conditions. Finally, we will determine if the same apical membrane AKAPS that affect CFTR regulate ENaC activity or are involved in CFTR-mediated negative modulation of ENaC. The proposed studies will reveal how PKA is compartmentalized in the apical pole of airway epithelial cells, and will assess the significance of such localization for basal and stimulated function of apical membrane ion conductances.
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