Komwatana P, Dinudom A, Young J A, Cook D I
Department of Physiology, University of Sydney, NSW, Australia.
J Membr Biol. 1996 Mar;150(2):133-41. doi: 10.1007/s002329900038.
We have previously reported that intralobular salivary duct cells contain an amiloride-sensitive Na+ conductance (probably located in the apical membranes). Since the amiloride-sensitive Na+ conductances in other tight epithelia have been reported to be controlled by extracellular (luminal) Na+, we decided to use whole-cell patch clamp techniques to investigate whether the Na+ conductance in salivary duct cells is also regulated by extracellular Na+. Using Na(+)-free pipette solutions, we observed that the whole-cell Na+ conductance increased when the extracellular Na+ was increased, whereas the whole-cell Na+ permeability, as defined in the Goldman equation, decreased. The dependency of the whole-cell Na+ conductance on extracellular Na+ could be described by the Michaelis-Menten equation with a K(m) of 47.3 mmol/1 and a maximum conductance (Gmax) of 2.18 nS. To investigate whether this saturation of the Na+ conductance with increasing extracellular Na+ was due to a reduction in channel activity or to saturation of the single-channel current, we used fluctuation analysis of the noise generated during the onset of blockade of the Na+ current with 200 mumol/l 6-chloro-3,5-diaminopyrazine-2-carboxamide. Using this technique, we estimated the single channel conductance to be 4 pS when the channel was bathed symmetrically in 150 mmol/l Na+ solutions. We found that Na+ channel activity, defined as the open probability multiplied by the number of available channels, did not alter with increasing extracellular Na+. On the other hand, the single-channel current saturated with increasing extracellular Na+ and, consequently, whole-cell Na+ permeability declined. In other words, the decline in Na+ permeability in salivary duct cells with increasing extracellular Na+ concentration is due simply to saturation of the single-channel Na+ conductance rather than to inactivation of channel activity.
我们之前曾报道，小叶内涎腺导管细胞含有一种对氨氯吡咪敏感的Na⁺电导（可能位于顶端膜）。由于据报道其他紧密上皮中的氨氯吡咪敏感Na⁺电导受细胞外（管腔）Na⁺调控，我们决定采用全细胞膜片钳技术来研究涎腺导管细胞中的Na⁺电导是否也受细胞外Na⁺调控。使用无Na⁺的移液管溶液，我们观察到当细胞外Na⁺增加时，全细胞Na⁺电导增加，而根据戈德曼方程定义的全细胞Na⁺通透性降低。全细胞Na⁺电导对细胞外Na⁺的依赖性可用米氏方程描述，米氏常数（K(m)）为47.3 mmol/1，最大电导（Gmax）为2.18 nS。为研究随着细胞外Na⁺增加，Na⁺电导的这种饱和是由于通道活性降低还是单通道电流饱和所致，我们利用200 μmol/l 6 - 氯 - 3,5 - 二氨基吡嗪 - 2 - 甲酰胺阻断Na⁺电流起始阶段产生的噪声进行波动分析。使用该技术，当通道在150 mmol/l Na⁺溶液中对称浸泡时，我们估计单通道电导为4 pS。我们发现，定义为开放概率乘以可用通道数的Na⁺通道活性并不随细胞外Na⁺增加而改变。另一方面，单通道电流随着细胞外Na⁺增加而饱和，因此全细胞Na⁺通透性下降。换句话说，随着细胞外Na⁺浓度增加，涎腺导管细胞中Na⁺通透性的下降仅仅是由于单通道Na⁺电导的饱和，而非通道活性的失活。
Zotero 插件