Identification of the role of PKA in Na+ channel modulation

Voltage-dependent sodium channels (VDSCs) are responsible for action potential generation and conduction in excitable cells. Superfusion of Xenopus laevis oocytes coexpressing beta2-adrenergic receptors and rat (SkM2) or human (hH1) cardiac alpha-subunit of the sodium channel with isoproterenol led to a modest increase in peak sodium inward current. Intracellular injection of cAMP and of the catalytic subunit of PKA reproduced this increase, demonstrating that this signal transduction pathway involves PKA dependent modulation. Mutations of the five major consensus PKA phosphorylation sites in the putative cytosolic loops of SkM2 did not abolish the observed effect. A set of chimerical sodium channels made from portions of the human hH1 alpha-subunit and the rat skeletal muscle SkM1 alpha-subunit (which is not affected by PKA stimulation) were used to localize the structural determinant in the hH1 sequence responsible for PKA modulation. Studies revealed that the large cytosolic loop interconnecting transmembrane domains I and II (IDI-II), which is not conserved among sodium channel subtypes accounts for the effects of PKA on hH1. Thus it is the aim of this project to investigate the mechanism by which cardiac sodium channels can be regulated via PKA activation. To testify the hypothesis that the bill alpha-subunit functions as a direct substrate for PKA, in-vitro and in-vivo phosphorylations of portions of the IDI-II loop and bill will be carried out. Furthermore, alpha-subunits of bill missing defined regions of IDI-il will be synthesized, overexpressed in Xenopus laevis oocytes and analyzed for PKA susceptibility. By looking for protein:protein interaction of IDI-II and cytosolic proteins, the question if there are sodium channel regulatory proteins (NaCRP) that function as transmitter between PKA and sodium channels will be answered. Recent studies revealed that the activation of PKA not only modulates the function but also the trafficking of hill to the plasma membrane. Therefore, hH1 will be tagged with GFP and expressed in oocytes and HEK cells to monitor the transport-characteristics of the alpha-subunit under the influence of activated PKA and/or substances that disrupt plasma membrane recycling. Results of these studies may contribute to a better understanding of how PKA acts on cardiac alpha-subunit sodium-channels.