Specht Dana, Wu Shu-Biao, Turner Paul, Dearden Peter, Koentgen Frank, Wolfrum Uwe, Maw Marion, Brandstätter Johann Helmut, tom Dieck Susanne
Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Erlangen, Germany.
Invest Ophthalmol Vis Sci. 2009 Feb;50(2):505-15. doi: 10.1167/iovs.08-2758. Epub 2008 Oct 24.
Photoreceptor ribbon synapses translate light-dependent changes of membrane potential into graded transmitter release via L-type voltage-dependent calcium channel (VDCC) activity. Functional abnormalities (e.g., a reduced electroretinogram b-wave), arising from mutations of presynaptic proteins, such as Bassoon and the VDCCalpha1 subunit Cacna1f, have been shown to altered transmitter release. L-type VDCCalpha1 subtype expression in wild-type and mutant mice was examined, to investigate the underlying pathologic mechanism.
Two antisera against Cacna1f, and a Cacna1f mouse mutant (Cacna1fDeltaEx14-17) were generated. Immunocytochemistry for L-type VDCCalpha1 subunits and additional synaptic marker proteins was performed in wild-type, BassoonDeltaEx4-5 and Cacna1fDeltaEx14-17 mice.
Active zone staining at photoreceptor ribbon synapses with a panalpha1 antibody colocalized with staining for Cacna1f in wild-type mouse retina. Similarly, in the BassoonDeltaEx4-5 mouse, residual mislocalized staining for panalpha1 and Cacna1f showed colocalization. Unlike the presynaptic location of Cacna1f and panalpha1 antibody staining, the skeletal muscle VDCCalpha1 subunit Cacna1s was present postsynaptically at ON-bipolar cell dendrites, where it colocalized with metabotropic glutamate receptor 6 (mGluR6). Surprisingly, Cacna1s labeling was severely downregulated in the BassoonDeltaEx4-5 and Cacna1fDeltaEx14-17 mutants. Subsequent analyses revealed severely reduced ON-bipolar cell dendritic expression of the sarcoplasmic reticulum Ca(2+) ATPase Serca2 in both mouse mutants and of mGluR6 in the Cacna1fDeltaEx14-17 mutant.
Presynaptic mutations leading to reduced photoreceptor-to-bipolar cell signaling are associated with disturbances in protein expression within postsynaptic dendrites. Moreover, detection of Cacna1s and Serca2 in ON-bipolar cell dendrites in wild-type animals suggests a putative role in regulation of postsynaptic Ca(2+) flux.
光感受器带状突触通过L型电压依赖性钙通道（VDCC）的活性将膜电位的光依赖性变化转化为分级递质释放。已证明，由突触前蛋白（如巴松管和VDCCα1亚基Cacna1f）突变引起的功能异常（如视网膜电图b波降低）会改变递质释放。研究野生型和突变型小鼠中L型VDCCα1亚型的表达，以探究潜在的病理机制。
制备了两种针对Cacna1f的抗血清以及一种Cacna1f小鼠突变体（Cacna1fDeltaEx14 - 17）。对野生型、巴松管DeltaEx4 - 5和Cacna1fDeltaEx14 - 17小鼠进行了L型VDCCα1亚基和其他突触标记蛋白的免疫细胞化学检测。
在野生型小鼠视网膜中，用泛α1抗体对光感受器带状突触的活性区染色与Cacna1f染色共定位。同样，在巴松管DeltaEx4 - 5小鼠中，泛α1和Cacna1f残留的错误定位染色也显示共定位。与Cacna1f和泛α1抗体染色的突触前位置不同，骨骼肌VDCCα1亚基Cacna1s存在于ON双极细胞树突的突触后，在那里它与代谢型谷氨酸受体6（mGluR6）共定位。令人惊讶的是，在巴松管DeltaEx4 - 5和Cacna1fDeltaEx14 - 17突变体中，Cacna1s标记严重下调。随后的分析显示，在这两种小鼠突变体中，肌浆网Ca(2+)ATP酶Serca2在ON双极细胞树突中的表达严重降低，而在Cacna1fDeltaEx14 - 17突变体中mGluR6的表达也严重降低。
导致光感受器到双极细胞信号传导减少的突触前突变与突触后树突内蛋白质表达的紊乱有关。此外，在野生型动物的ON双极细胞树突中检测到Cacna1s和Serca2，提示其在调节突触后Ca(2+)通量中可能发挥作用。
