Identification and characterization of a cell membrane nucleic acid channel
Identification and characterization of a cell membrane nucleic acid channel
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DOI：
10.1073/pnas.95.4.1921
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发表时间：
1998-02-17
影响因子：
11.1
通讯作者：
Klotman, PE
中科院分区：
文献类型：
--
作者：
Hanss, B;Leal-Pinto, E;Klotman, PE
我们已经确定了一个45 kDa的蛋白质纯化大鼠肾刷状缘膜，结合短的单链核酸序列。在电压钳实验中，重组的45-kDa蛋白作为门控通道，允许核酸通过。加入寡核苷酸后立即观察到通道活性。通道活性没有观察到纯化的蛋白质或寡核苷酸的情况下，或当蛋白质热灭活之前，形成脂蛋白体。在对称缓冲溶液和寡核苷酸的存在下，电流在测试的保持电位范围内线性通过，电导为10.4 +/- 0.4皮西门子（pS），反转电位为0.2 +/- 1.7 mV。磷酸二酯和硫代磷酸寡核苷酸之间的通道电导或反转电位没有差异。离子取代实验证明，只有当建立了寡核苷酸的浓度梯度时，反转电位才发生变化，表明寡核苷酸单独的运动是电流的原因。通过使用P-32标记的寡核苷酸证实了寡核苷酸穿过双层的运动。硫酸乙酰肝素存在时，通道开放概率显著降低，这些研究为细胞表面传导核酸的通道提供了证据。
We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers, In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids, Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested, Conductance was 10.4 +/- 0.4 picosiemens (pS) and reversal potential was 0.2 +/- 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ion-substitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current, Movement of oligonucleotide across the bilayer was confirmed by using P-32-labeled oligonucleotides, Channel open probability decreased significantly in the presence of heparan sulfate, These studies provide evidence for a cell surface channel that conducts nucleic acids.