Gincel Dan, Shoshan-Barmatz Varda
Department of Life Sciences and the Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Biophys J. 2002 Dec;83(6):3223-9. doi: 10.1016/S0006-3495(02)75324-1.
The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.
突触小泡蛋白突触素可通过相对低浓度的 Triton X-100（0.2%）从大鼠脑突触体中溶解出来，并使用羟基磷灰石/硅藻土树脂上的快速单步色谱法进行高度纯化（纯度超过 95%）。将纯化后的突触素重组到平面脂质双分子层中，并对突触素的通道活性进行表征。在不对称 KCl 溶液（顺式 300 mM/反式 100 mM）中，突触素在 +60 mV 时形成一个电导为 414±13 pS 的快速波动通道。突触素通道的开放概率在去极化时降低，并且发现通道具有阳离子选择性。突触素通道对 K⁺ 的选择性高于 Cl⁻（P(K⁺)/P(Cl⁻) > 8），并且优先选择 K⁺ 而非 Li⁺、Na⁺、Rb⁺、Cs⁺ 或胆碱⁺。突触素通道对 Ca²⁺ 不可渗透，Ca²⁺ 对其通道活性没有影响。本研究是对纯化的突触素通道活性的第二次证明，但首次对其通道特性进行了生物物理表征。大量纯化的突触素及其特征性通道特性的可用性可能有助于确定突触素在突触传递中的作用。
