This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. KCNQ1 is expressed abundantly in epithelium with various KCNE family members at either the basolateral or apical surfaces depending on the cell type. The mechanism by which KCNQ1 is trafficked to different membrane subdomains in epithelial cells and its physiological role and modulation understood. Evidence suggests that KCNQ1 and the cystic fibrosis transmembrane conductance regulator (CFTR), which can both regulated by cAMP, may act in concert to regulate electrolyte and water balance in epithelial cells. We hypothesize that PKA modulation of KCNQ1 activity in airway epithelial cells, as in heart, is largely mediated via A-kinase anchoring proteins (AKAPs), and that AKAPs play a role in trafficking of KCNQ1 to the plasma membrane. Whole cell patch-clamp analysis revealed that Calu-3 cells, an airway epithelial cell line, exhibit a forskolin-activated current that is blocked by XE-991, a specific KCNQ1 inhibitor. This current was absent when Calu-3 cells were dialyzed with St-Ht31, a peptide that disrupts the association of the PKA RII subunit with AKAPs. Activation of PKA by treatment with ?-adrenergic agonists or forskolin resulted in increased expression of KCNQ1 at the plasma membrane of Calu-3 cells. The increased trafficking of KCNQ1 to the plasma membrane was blocked by pretreatment with St-Ht31. Further, the PKA-RII subunit and KCNQ1 were co-immunoprecipitated from both rat lung and Calu-3 cells. Interestingly, the XE-991-sensitive current was absent in IB-3 cells, a human airway cell line that carries the delta-508 CFTR mutation. Moreover, KCNQ1 was not detected by immunocytochemistry or RT-PCR in IB-3 cells. These results suggest that CFTR and KCNQ1 are tightly associated in epithelial cells. Currently, we are pursuing the mechanism by which KCNQ1 and CFTR are regulated in epithelial cells.
Showing the most recent 10 out of 206 publications
