Room 6C/6E The transient association between MARCKS and the cytoplasmic side of the apical membrane governs the PIP2 dependent regulation of the epithelial sodium channel

Friday, October 12, 2012: 8:00 PM
6C/6E (WSCC)
Abdel Alli, PhD/MPH , Physiology, Emory University School of Medicine, Atlanta, GA
Hui-Fang Bao, MD , Physiology, Emory University School of Medicine, Atlanta, GA
He-Ping Ma, MD , Physiology, Emory University School of Medicine, Atlanta, GA
Bing-Chen Liu, MD , Physiology, Emory University School of Medicine, Atlanta, GA
Ling Yu, PhD , Physiology, Emory University School of Medicine, Atlanta, GA
Douglas Eaton, PhD , Physiology, Emory University School of Medicine, Atlanta, GA
Phosphatidylinositol phosphates (e.g. PIP2) regulate the activity of ENaC at the apical membrane.  Here, we investigate a role for protein kinase C (PKC) and calmodulin (CaM) in the PIP dependent regulation of ENaC by MARCKS.  After treating Xenopus 2F3 cells with PMA, immunoblot studies showed an increase in MARCKS phosphorylation, confocal microscopy revealed displacement of MARCKS from the apical membrane, and amiloride-sensitive transepithelial current decreased significantly.  Single-channel patch clamp studies showed that ENaC activity increased after treating the apical side of Xenopus 2F3 cells with a pharmacological inhibitor of PKC (GF109203X).  We mutated the CaM binding site within the basic effector domain of MARCKS by site-directed mutagenesis and co-transfected the wildtype or mutant MARCKS CFP-tagged construct and an ENaC gamma GFP-tagged construct into Xenopus 2F3 cells.  Live cell imaging showed that in response to an increase in calcium, CaM translocated to the apical membrane and MARCKS translocated to the cytoplasm in cells transfected with wildtype MARCKS, but not with mutant MARCKS.  We show by single-channel patch clamp studies that application of the calmodulin inhibitor, calmidazolium, to the apical side of Xenopus 2F3 cells causes an increase in the open probability of ENaC.  The findings presented here suggest that the cycling of MARCKS between the apical membrane and cytoplasm is critical for the PIP2 dependent regulation of ENaC.