erwin-shibatauiowaedu

The primary focus in my laboratory is to understand the regulatory mechanism(s) of ion channels in excitable cells. Specifically, we are interested in the mechanism by which β-adrenergic and muscarinic receptor agonists modulate ionic currents. Previous studies have suggested that in addition to the phosphorylation-dependent effects on ion channels, Gαs has an additional “direct” effect on the increase of cardiac Na+ current that is independent but concurrent with phosphorylation effects. Our results strongly suggest that the number of functional Na+ channels increases in the membrane. We showed that the new channels are in caveolae (Yarbrough et al., 2002). Caveolae are dynamic ω-shaped structures whose membrane fusion and fission mechanisms are virtually unknown. These channel proteins do not migrate out of the caveolar membrane domain. Na+ channels within the caveolae membrane become functional only when the caveolae “neck” opens to establish electrical continuity between the extracellular space and the intra-caveolae compartment.

Our studies focus on determining the co-localization of Na+, Ca2+, and K+ channels in caveolae and the role of Gαs in the regulation of caveolae. Specifically, we propose to address the following questions:
Are Na+, Ca2+, and K+ channels sorted to the same or different caveolae? What is the functional role of the N-terminus of the Gαs protein in the regulation of caveolae? We showed that Gαs can enhance the size of Na+ current in a cAMP-independent fashion and is mimicked with application of GTPγS-activated Gαs but not with application of GDP β S (Matsuda et al., 1992). Lu et al., (1999) showed that a short N-terminal peptide of Gαs (a.a. 27-42) is able to mimic the effects of increasing the Na+ current. Using the Na+ current as our assay, we will examine the functional effects of the N-terminal of Gαs using Gαs/Gαt and Gαs/Gαi chimeras and short amino-terminal Gαs oligomers. We will also test the involvement of membrane-associated proteins in caveolar docking and/or fusion events. We will probe for the substrate(s) that interacts with Gαs to regulate the opening/closing of caveolae necks. Measuring the opening and closing of the caveolar neck will be accomplished by ultra-high resolution measurements of the added capacitance from the caveolar membrane.

Our animal models include single myocytes from rabbit, rat and human hearts. We use several types of approaches to test our hypothesis. These techniques include Western blot analysis, immunoprecipitation, confocal immunofluorescence, immuno-electron microscopy, and patch-clamping. The patch-clamp technique is used to determine changes in the biophysical properties of Na+, Ca2+, and K+ currents as well as ultra-high resolution capacitance measurements.

These studies will provide new information into the fusion mechanism of caveolae in heart and direct pathways for pharmacological interventions and therapeutic modalities.