A-Actinin2 and Filamin a Cytoskeletal Interacting Proteins Facilitate SK2 Channels Recycling from Endosomes to the Surface Membrane

The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small-conductance, Ca2+-activated K+ channels (SK) are unique in that they are gated solely by changes in intracellular Ca2+. The channels are expressed in atrial cardiomyocytes and responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes.Surface membrane localization of SK2 channels were evaluated using Total Internal Reflection Fluorescence (TIRF) Microscopy. SK2 channels were tagged with Tomato fluorescent protein and expressed in HEK 293 cells. We have previously demonstrated that the C and N termini of SK2 channels interact with actin-binding proteins, α-actinin2 and filamin A, respectively. When SK2 channels were co-expressed with filamin A, the membrane fluorescence intensity of SK2 channels increased significantly. Similar findings were observed with the putative interacting cytoskeletal protein, α-actinin2. These observations supported our notion that filamin A and α-actinin2, facilitate the forward trafficking or decrease the retrograde trafficking. We next tested the effects of primaquine and dynasore. Primaquine has been shown to block the recycling pathway from endosomes while dynasore is known to be a specific inhibitor of dynamin which is responsible for endocytosis. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. While treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively-active or dominant-negative forms of Rab GTPases provide additional insights into the distinct roles of the two putative cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α-actinin2 and filamin A facilitates SK2 channels recycling to the surface membrane through different endosomal pathways.