Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.
Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.
复制标题
DOI：
10.1085/jgp.119.2.147
复制
发表时间：
2002-02
影响因子：
3.8
通讯作者：
Valiunas, Virginijus
中科院分区：
文献类型：
--
作者：
Valiunas, Virginijus
用转染小鼠Cx45的人HeLa细胞和转染鸡Cx45的大鼠RIN细胞研究了Cx45缝隙连接半通道的电学和通透性。在没有细胞外钙离子的情况下，全细胞记录显示在两个转基因细胞系中都有从半管产生的电流。多通道电流表现出对电压Vm敏感的时间依赖性激活或失活，未转染细胞不存在这些电流。细胞外钙离子浓度升高或CO2酸化可抑制半通道电流。单位电导表现出Vm依赖性(即γHc，主要随超极化/去极化而增大/减小)。外推到Vm=0 mV导致了γHc，主为57ps，大约是完整Cx45间隙结通道电导的两倍。开放通道概率Po依赖于Vm，在负Vm时下降(Po<；0.11，Vm<；−50 mV)，在正Vm时增加(Po∼0.76，Vm>；50 mV)。此外，当细胞外钙离子水平降低时，Cx45非连接半通道似乎介导了从外液中摄取荧光黄(LY)和碘化丙啶(PI)染料。染料摄取量与功能半通道的数量成正比。未转染细胞中未检测到明显的染料摄取。Cx45基因转染的HeLa和RIN细胞在预先负载LY后在无钙外液中孵育时也可使染料渗出，而在2 mM外液中孵育时染料渗漏很少或没有。完整的Cx45缝隙连接通道允许LY或PI染料通过，但它们各自的通量速率不同。比较了LY在Cx45半通道和完整的缝隙连接通道中的扩散，发现前者具有更强的渗透性，这表明缝隙连接通道的孔对染料分子的变构限制比未对置的半通道更大。这些数据表明，当外界钙离子浓度降低时，脊椎动物细胞中的Cx45非连接半通道开放。
Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca2+, whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V m. These currents did not occur in nontransfected cells. The hemichannel currents were inhibited by raising extracellular Ca2+ or by acidification with CO2. The unitary conductance exhibited V m dependence (i.e., γhc,main increased/decreased with hyperpolarization/depolarization). Extrapolation to V m = 0 mV led to a γhc,main of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P o, was V m-dependent, declining at negative V m (P o < 0.11, V m < −50 mV), and increasing at positive V m (P o ∼0.76, V m > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca2+ level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in nontransfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca2+-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca2+. Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca2+ concentration is reduced.