Regulatory Partners Involvement in Sodium Channel Trafficking in Cardiomyocytes

Kir2.x potassium channels and Nav1.5 sodium channel are key players in cardiomyocytes (CMs) excitability. In inherited arrhythmias, mutations in genes encoding ion channels affect either their biophysical properties or their functional expression. Moreover, in acquired arrhythmias, ion channel dysfunctions are often the consequence of structural remodeling of CMs. These observations point out the major role of both CMs architecture's integrity and dynamic regulation of ion channels expression, from intracellular trafficking to membrane anchoring. Do ion channel partners participate in CMs organization by their ability to regulate current properties and by their predisposition to localize into specific membrane domains? To achieve this study, real-time trafficking and targeting in CMs microdomains of Nav1.5 and its partners, would be investigated with Retention Using Selective Hooks (RUSH) system. The RUSH is based on the expression of two proteins: the human invariant chain of the major histocompatibility complex Ii fused to streptavidin as hook in the Endoplasmic Reticulum (ER) and downstream of an Internal Ribosome Entry Site, the protein reporter fused to Streptavidin Binding Protein in N-terminus and to eGFP or mCherry in C-terminus. After several sub-cloning for RUSH tools preparation, our preliminary data showed the RUSH system permits retention and release of Nav1.5 and two transmembrane partners, Kir2.1 and Connexin-43 successfully, in Madin-Darby Canine Kidney cells. These cells have been transfected with two RUSH plasmids coding for Nav1.5 and Kir2.1 or Connexin-43, tagged with eGFP and mCherry respectively. This method enables the trapping of these proteins into the ER with streptavidin, their subsequent synchronous release after addition of biotin and their co-trafficking followed by fluorescence. As expected, 24 hours post-transfection, the proteins localized to the ER without biotin, whereas biotin addition at the time of transfection redistributes them to the cell periphery, of which a fraction localized at the membrane. The present study may help to understand the molecular basis of the complex interaction between structural and electrical polarity in CMs. We will use our RUSH constructs into adult polarized cardiomyocytes of Rat to test the hypothesis of a sorting hub at early stages of trafficking where interactions between Nav1.5 channels and its regulatory partners orientate the final targeting of channels into membrane microdomains.