ANTISENSE DEPLETION OF BETA-SUBUNITS MODULATES THE BIOPHYSICAL AND PHARMACOLOGICAL PROPERTIES OF NEURONAL CALCIUM CHANNELS
ANTISENSE DEPLETION OF BETA-SUBUNITS MODULATES THE BIOPHYSICAL AND PHARMACOLOGICAL PROPERTIES OF NEURONAL CALCIUM CHANNELS
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DOI：
10.1113/jphysiol.1995.sp020534
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发表时间：
1995-02-01
影响因子：
5.5
通讯作者：
DOLPHIN, AC
中科院分区：
文献类型：
--
作者：
BERROW, NS;CAMPBELL, V;DOLPHIN, AC
1.在体外培养的大鼠背根神经节神经元中研究了电压依赖性钙通道（VDCC）β亚基的作用。一种抗肽抗体产生了，它识别了许多制备物中对应于β亚基的蛋白质。VDCC β亚基在DRG中的免疫反应性集中在质膜的内侧，但也存在于细胞质中。将与所有已发表的VDCC β亚基序列具有同源性的26聚体反义寡核苷酸显微注射到单个细胞中，108 h后观察到VDCC β亚基免疫反应性的最大消耗，表明β亚基周转的半衰期大于50 h。用无义阿糖胞苷没有获得耗竭。VDCC β亚基免疫反应性耗竭对这些细胞中钙通道电流的影响是最大电流幅度降低约47%，电流激活的电压依赖性偏移约+7 mV。这些效应与在卵母细胞和其他表达系统中共表达克隆的β-和α（1）-亚基后观察到的效应匡威。1，4-二氢吡啶（DHP）激动剂Bay K 8644增强钙通道电流的能力在β亚基免疫反应性耗尽后大大降低。这一结果与在几个系统中的发现一致，即β亚基与α（1）亚基的共表达导致DHP结合位点的数量增加。这些结果表明，钙通道β亚基形成天然神经元钙通道的一部分，并改变其生物物理和药理学性质。
1. The role of the voltage-dependent calcium channel (VDCC) beta-subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta-subunits in a number of preparations. Immunoreactivity for the VDCC beta-subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm.2. A twenty-six-mer antisense oligonucleotide with homology to all published VDCC beta-subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta-subunit immunoreactivity was observed after 108 h suggesting a half-life for the turnover of the beta-subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide.3. The effect of depletion of VDCC beta-subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co-expression of cloned beta- with alpha(1)-subunits in oocytes and other expression systems.4. The ability of the 1,4-dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta-subunit immunoreactivity. This result is in agreement with the finding in several systems that co-expression of the beta-subunit with alpha(1)-subunits results in an increased number of DHP binding sites.5. These results show that calcium channel beta-subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.