Fritzius Thorsten, Turecek Rostislav, Seddik Riad, Kobayashi Hiroyuki, Tiao Jim, Rem Pascal D, Metz Michaela, Kralikova Michaela, Bouvier Michel, Gassmann Martin, Bettler Bernhard
Department of Biomedicine, Institute of Physiology, University of Basel, 4056 Basel, Switzerland.
Institute of Experimental Medicine, ASCR, 14220 Prague 4-Krc, Czech Republic, and.
J Neurosci. 2017 Feb 1;37(5):1162-1175. doi: 10.1523/JNEUROSCI.2181-16.2016. Epub 2016 Dec 21.
GABA receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABA receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K-channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABA receptors also associate with hetero-oligomers of KCTD subunits. Coimmunoprecipitation experiments indicate that two-thirds of the KCTD16 proteins in the hippocampus of adult mice associate with KCTD12. We show that the KCTD proteins hetero-oligomerize through self-interacting T1 and H1 homology domains. Bioluminescence resonance energy transfer measurements in live cells reveal that KCTD12/KCTD16 hetero-oligomers associate with both the receptor and the G-protein. Electrophysiological experiments demonstrate that KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties on G-protein-activated Kir3 currents. During prolonged receptor activation (one min) KCTD12/KCTD16 hetero-oligomers produce moderately desensitizing fast deactivating K currents, whereas KCTD12 and KCTD16 homo-oligomers produce strongly desensitizing fast deactivating currents and nondesensitizing slowly deactivating currents, respectively. During short activation (2 s) KCTD12/KCTD16 hetero-oligomers produce nondesensitizing slowly deactivating currents. Electrophysiological recordings from hippocampal neurons of KCTD knock-out mice are consistent with these findings and indicate that KCTD12/KCTD16 hetero-oligomers increase the duration of slow IPSCs. In summary, our data demonstrate that simultaneous assembly of distinct KCTDs at the receptor increases the molecular and functional repertoire of native GABA receptors and modulates physiologically induced K current responses in the hippocampus.
The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABA receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABA receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K current responses in the brain.
γ-氨基丁酸（GABA）受体是大脑中主要抑制性神经递质GABA的G蛋白偶联受体。已表明GABA受体与辅助性KCTD8、KCTD12、KCTD12b和KCTD16亚基的同型寡聚体相关联（以其T1钾通道四聚化结构域命名），这些亚基调节该受体的G蛋白信号传导。在此，我们提供证据表明GABA受体也与KCTD亚基的异型寡聚体相关联。免疫共沉淀实验表明，成年小鼠海马中三分之二的KCTD16蛋白与KCTD12相关联。我们表明，KCTD蛋白通过自身相互作用的T1和H1同源结构域形成异型寡聚体。活细胞中的生物发光共振能量转移测量表明，KCTD12/KCTD16异型寡聚体与受体和G蛋白均相关联。电生理实验证明，KCTD12/KCTD16异型寡聚体赋予G蛋白激活的Kir3电流独特的动力学特性。在受体长时间激活（1分钟）期间，KCTD12/KCTD16异型寡聚体产生适度脱敏的快速失活钾电流，而KCTD12和KCTD16同型寡聚体分别产生强烈脱敏的快速失活电流和非脱敏的缓慢失活电流。在短暂激活（2秒）期间，KCTD12/KCTD16异型寡聚体产生非脱敏的缓慢失活电流。KCTD基因敲除小鼠海马神经元的电生理记录与这些发现一致，并表明KCTD12/KCTD16异型寡聚体增加了缓慢抑制性突触后电流（sIPSCs）的持续时间。总之，我们的数据表明，在受体处不同KCTD的同时组装增加了天然GABA受体的分子和功能多样性，并调节海马中生理诱导的钾电流反应。
KCTD蛋白8、12和16是GABA受体的辅助亚基，它们以不同方式调节该受体的G蛋白信号传导。一般认为KCTD蛋白作为同型寡聚体发挥作用。在此，我们表明KCTD蛋白也以所有可能的双重组合形成异型寡聚体。活细胞实验表明，KCTD异型寡聚体至少形成四聚体，并且这些四聚体直接与受体和G蛋白相互作用。KCTD12/KCTD16异型寡聚体赋予异源细胞中GABA受体诱导的Kir3电流独特的动力学特性。KCTD12/KCTD16异型寡聚体在海马中丰富，在那里它们延长了锥体细胞中缓慢抑制性突触后电流的持续时间。因此，我们的数据支持KCTD异型寡聚体调节大脑中生理诱导的钾电流反应。
