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, KCa 2) are expressed in human atrial myocytes and are 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. We have previously demonstrated that the C- and N-termini of SK2 channels interact with the actin-binding proteins α-actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using total internal reflection fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (KCa 2.2) channels. When SK2 channels were co-expressed with filamin A or α-actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, 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 provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α-Actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A probably aids the recycling of SK2 channels from recycling endosomes.
离子通道的正常功能主要取决于精确的亚细胞定位和细胞表面膜上通道蛋白的数量。小电导,Ca2+激活K+通道(SK,KCa2)在人心房肌细胞中表达,并负责塑造心房动作电位。了解SK通道运输的机制可能为控制心房肌细胞复极化的调节提供新的见解。我们先前已经证明SK2通道的C和N末端与肌动蛋白结合蛋白α-肌动蛋白2和丝素A相互作用,分别。然而,相互作用蛋白在SK2通道运输中的作用仍未完全了解。使用全内反射荧光(TIRF)显微镜,我们研究了SK2(KCa2.2)通道的表面膜定位机制。当SK2通道与丝素A或α-肌动蛋白2共表达时,SK2通道的膜荧光强度显着增加。我们接下来测试了伯氨喹和dynasore对SK2通道表达的影响。用伯氨喹处理显著降低了SK2通道的膜表达。相比之下,dynasore处理未能改变SK2通道的表面膜表达。使用组成型活性或显性阴性形式的RabGTP酶的进一步研究提供了对两种细胞骨架蛋白在核内体SK2通道再循环过程中的不同作用的进一步见解。α-Actinin2促进了SK2通道从早期和再循环内体的再循环,而丝状蛋白A可能有助于SK2通道从再循环内体的再循环。