Tomlins B, Williams A J
Pflugers Arch. 1986 Sep;407(3):341-7. doi: 10.1007/BF00585312.
Sarcoplasmic reticulum (SR) membrane vesicles have been prepared from rabbit skeletal muscle and solubilised using K+ cholate. Solubilised membrane proteins were reconstituted into small asolectin liposomes by dialysis against cholate-free solution. Large liposomes were produced by freezing and thawing at -80 degrees C and room temperature, respectively. The liposomes were assayed for the SR K+ channel using the patch clamp technique. Channel density was modulated by varying protein:lipid ratios during reconstitution. Channels inserted into the membrane with a preferred orientation. The solubilised and reconstituted channel behaves ohmically over the holding potential range +/- 70 mV and has a conductance of 178.4 +/- 4.4 pS (mean +/- SE, n = 37) in 200 mM KCl. The channel has a selectivity sequence of K+ greater than NH4+ greater than Rb+ greater than Na+ and K+ conductance is blocked by hexamethonium and decamethonium. The opening probability of the reconstituted channel is voltage dependent. The conductance and gating characteristics displayed by the solubilised and reconstituted channel correlate well with those previously observed following the fusion of native SR membrane vesicles with planar phospholipid bilayers.
已从兔骨骼肌制备了肌浆网（SR）膜囊泡，并用钾胆酸盐使其溶解。通过对无胆酸盐溶液进行透析，将溶解的膜蛋白重构成小的大豆卵磷脂脂质体。分别通过在-80℃冷冻和室温解冻来产生大的脂质体。使用膜片钳技术对脂质体进行SR钾通道检测。在重构过程中通过改变蛋白质与脂质的比例来调节通道密度。通道以优先取向插入膜中。溶解并重构的通道在±70 mV的保持电位范围内呈欧姆特性，在200 mM KCl中电导为178.4±4.4 pS（平均值±标准误，n = 37）。该通道具有K +＞NH4 +＞Rb +＞Na +的选择性序列，并且K +电导被六甲铵和十甲铵阻断。重构通道的开放概率取决于电压。溶解并重构的通道所显示的电导和门控特性与先前在天然SR膜囊泡与平面磷脂双层融合后观察到的特性密切相关。
