Evidence for the Interaction of Endophilin A3 with Endogenous Kca2.3 Channels in PC12 Cells
Evidence for the Interaction of Endophilin A3 with Endogenous Kca2.3 Channels in PC12 Cells
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DOI：
10.1159/000363016
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发表时间：
2014-01-01
影响因子：
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通讯作者：
Jaeger，Heike
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文献类型：
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作者：
Janbein，Malika;Quader，Mohamed Abo;Jaeger，Heike
小电导钙激活（SK）通道通过控制可兴奋细胞的后超极化而发挥重要作用。这些通道的表达水平和密度是控制不同细胞功能的重要因素。一些研究表明，在不同的组织中的共定位的KCa 2.3通道和Endophilin A3。Endophilin A3属于一个家族的BAR和SH3结构域包含的蛋白质，结合到发动蛋白，并参与在网格蛋白介导的内吞作用的囊泡断裂的过程中。MethodsUsing酵母双杂交系统和GST下拉分析，我们证明，Endophilin A3与K Ca 2.3通道的N-末端部分相互作用。此外，我们研究了这种相互作用对通道活性的影响，通过膜片钳测量在表达内源性KCa 2.3通道的PC12细胞。结果转染EndophilinA3的PC12细胞的全细胞测量结果显示，KCa2.3特异性Cs+通道电流减少，而Ca2+通道电流则减少。电流表明，在哺乳动物细胞中也发生了内皮素A3与KCa 2.3通道的相互作用，这种相互作用对流经KCa 2.3通道的电流具有功能性影响渠道由于KCa 2.3特异性电流可以在用具有DC-EBIO（30 μ M）的Endophilin A3转染的PC12细胞中增加，DC-EBIO是一种已知的SK通道激活剂，这些数据还表明，EndophilinA3并没有显著地从膜上去除KCa2.3通道，但改变了通道对Ca2+的敏感性，这可以被DC-EBIO.结论这种相互作用似乎是重要的K-Ca 2.3通道的功能，因此可能发挥重要作用的情况下，通道激活是关键的细胞功能。
Background/AimsSmall-conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of K Ca 2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR-and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis.MethodsUsing the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of K Ca 2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous K Ca 2.3 channels. K Ca 2.3 currents were activated by using pipette solutions containing 1 µM free Ca 2+.ResultsWhole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of K Ca 2.3 specifc Cs+ currents indicating that the interaction of Endophilin A3 with K Ca 2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through K Ca 2.3 channels. Since K Ca 2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM), a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove K Ca 2.3 channels from the membrane but changed the sensitivity of the channels to Ca 2+ which could be overcome by DC-EBIO.ConclusionThis interaction seems to be important for the function of K Ca 2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function.