Gravagna Nicole G, Knoeckel Christopher S, Taylor Alison D, Hultgren Barbara A, Ribera Angeles B
Department of Physiology and Biophysics, University of Colorado Denver and Health Sciences Center, Aurora, Colorado 80045, USA.
J Comp Neurol. 2008 Oct 10;510(5):508-24. doi: 10.1002/cne.21804.
Voltage-gated potassium (Kv) channels sculpt neuronal excitability and play important developmental roles. Kv channels consist of pore-forming alpha- and auxiliary subunits. For many Kv alpha-subunits, existing mRNA probes and antibodies have allowed analysis of expression patterns, typically during adult stages. Here, we focus on the Kv2.2 alpha-subunit, for which the mRNA shows broad expression in the embryo and adult. A lack of suitable antibodies, however, has hindered detailed analysis of Kv2.2 protein localization, especially during development. We developed an antibody that specifically recognizes Kv2.2 protein in Xenopus laevis, a vertebrate well suited for study of early developmental stages. The Kv2.2 antibody recognized heterologously expressed Kv2.2 but not the closely related Kv2.1 protein. Immunodetection of the protein showed its presence at St 32 in ventrolateral regions of the hindbrain and spinal cord. At later stages, several sensory tissues (retina, otic, and olfactory epithelia) also expressed Kv2.2 protein. As development progressed in the central nervous system, Kv2.2 protein distribution expanded in close association with the cytoskeletal marker alpha-tubulin, consistent with growth of neuronal tracts. We analyzed the subcellular distribution of Kv2.2 protein within single cultured neurons. In addition to a surface membrane presence, Kv2.2 protein also resided intracellularly closely associated with alpha-tubulin, as in vivo. Furthermore, in contrast to Kv2.1, Kv2.2 protein localized to long, axonal-like processes, consistent with its in vivo location in tracts. Despite their primary sequence similarity, the contrasting localizations of Kv2.1 and Kv2.2 support different roles for the two during development and neuronal signaling.
电压门控钾（Kv）通道塑造神经元兴奋性并发挥重要的发育作用。Kv通道由形成孔道的α亚基和辅助亚基组成。对于许多Kvα亚基，现有的mRNA探针和抗体已可用于分析表达模式，通常是在成年阶段。在这里，我们聚焦于Kv2.2α亚基，其mRNA在胚胎和成年期均呈现广泛表达。然而，缺乏合适的抗体阻碍了对Kv2.2蛋白定位的详细分析，尤其是在发育过程中。我们开发了一种抗体，该抗体能特异性识别非洲爪蟾（一种非常适合研究早期发育阶段的脊椎动物）中的Kv2.2蛋白。Kv2.2抗体识别异源表达的Kv2.2，但不识别密切相关的Kv2.1蛋白。对该蛋白的免疫检测显示其在胚胎发育第32阶段存在于后脑和脊髓的腹外侧区域。在后期阶段，几个感觉组织（视网膜、耳和嗅觉上皮）也表达Kv2.2蛋白。随着中枢神经系统发育的推进，Kv2.2蛋白的分布与细胞骨架标记物α微管蛋白密切相关地扩展，这与神经束的生长一致。我们分析了单个培养神经元内Kv2.2蛋白的亚细胞分布。除了存在于表面膜上，Kv2.2蛋白在细胞内也与α微管蛋白紧密相关，如同在体内一样。此外，与Kv2.1不同，Kv2.2蛋白定位于长的、类似轴突的突起上，这与其在体内神经束中的位置一致。尽管Kv2.1和Kv2.2的一级序列相似，但它们截然不同的定位支持了二者在发育和神经元信号传导过程中发挥不同的作用。
