Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation.
Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation.
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Connexin46 半通道制剂揭示了 pH 对连接蛋白的快速而直接的影响。
DOI：
10.1085/jgp.113.5.721
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Verselis,VK
中科院分区：
文献类型：
--
作者：
Trexler,EB;Bukauskas,FF;Bennett,MV;Bargiello,TA;Verselis,VK
pH 是间隙连接 (GJ) 介导的细胞间通讯的有效调节剂。提出的通过酸化关闭 GJ 通道的机制包括 H+ 对 GJ 蛋白的直接作用和可溶性中间体介导的间接作用。在此，我们报告酸化对 Neuro-2a 细胞中表达的连接蛋白 (Cx)46 细胞间通道和爪蟾细胞中表达的 Cx46 半通道的影响。酸化对半通道的影响通过宏观和切除的斑块进行了检查，这些斑块允许在暴露的半通道表面快速（<1毫秒）和均匀的pH变化。两种类型的 Cx46 通道都被发现对细胞质 pH 敏感，并且有两种明显的影响。短时间暴露于低pH值时可重复地引起快速且可逆的闭合，而较长时间暴露时会发生可逆性较差或不可逆的损失。我们将前者归因于 pH 门控，后者归因于 pH 失活。半通道中 pH 门控开放概率的半最大降低发生在 pH 6.4 时。当切除并且细胞质 [Ca2+] 维持在接近静息 (∼10−7M) 水平时，半通道仍然对细胞质 pH 敏感。因此，Cx46 半通道 pH 门控不依赖于细胞质中间体或 [Ca2+] 的增加。对开放半通道的细胞质面快速施加低 pH 值，导致关闭的最小潜伏期接近于零，表明 Cx46 半通道直接感知 pH 值。应用于闭合半通道延长了其闭合时间，表明 pH 传感器可从闭合半通道的细胞质侧接近。当对细胞外表面施加低pH值时，观察到快速闭合且敏感性显着降低，但这可以通过H + 渗透穿过孔到达内部位点来解释。 pH 值闭合取决于电压，并且具有相同的极性，且任何一侧都施加低 pH 值。这些数据表明 pH 传感器直接位于细胞质侧孔入口附近的 Cx46 上。
pH is a potent modulator of gap junction (GJ) mediated cell–cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell–cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed inXenopusoocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting (∼10−7M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.