Chraïbi Ahmed, Horisberger Jean-Daniel
Institut de Pharmacologie et de Toxicologie, CH-1005 Lausanne, Switzerland.
J Gen Physiol. 2002 Aug;120(2):133-45. doi: 10.1085/jgp.20028612.
The regulation of the open probability of the epithelial Na(+) channel (ENaC) by the extracellular concentration of Na(+), a phenomenon called "Na(+) self inhibition," has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na(+) self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na(+) concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q(10)act = 1.5) activation rate and a strongly temperature-dependant (Q(10)inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na(+) self inhibition depended only on the extracellular Na(+) concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na(+) as well as a reduction of Na(+) outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na(+) self inhibition is an intrinsic property of sodium channels resulting from the expression of the alpha, beta, and gamma subunits of human ENaC in Xenopus oocyte. The extracellular Na(+)-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.
上皮钠通道（ENaC）的开放概率受细胞外Na⁺浓度调节，这种现象称为“Na⁺自抑制”，已在几种天然紧密上皮中得到充分描述，但其分子机制尚不清楚。我们研究了非洲爪蟾卵母细胞中表达的人ENaC上Na⁺自抑制的动力学。快速去除氨氯吡咪或使细胞外Na⁺浓度从1 mM迅速增加到100 mM会导致内向电流峰值，随后下降至较低的准稳态电流。电流下降速率和稳态水平与温度有关，电流瞬变可用双态（激活-失活）模型很好地解释，该模型具有弱温度依赖性（Q₁₀act = 1.5）的激活速率和强温度依赖性（Q₁₀inact = 8.0）的失活速率。失活速率对温度的强烈依赖性导致高温下稳态氨氯吡咪敏感电流出现反常下降。Na⁺自抑制仅取决于细胞外Na⁺浓度，而不取决于内向电流的幅度，并且在Na⁺的反转电位处表现为电导降低以及Na⁺外向电流减少。通过暴露于细胞外蛋白酶（一种已知可激活ENaC的处理方法）或用对氯汞苯甲酸处理可防止自抑制。蛋白酶处理后，氨氯吡咪敏感电流随温度升高呈现预期的增加。这些结果表明，Na⁺自抑制是钠通道的一种内在特性，是由人ENaC的α、β和γ亚基在非洲爪蟾卵母细胞中表达所致。细胞外Na⁺依赖性失活具有较大的激活能，可通过细胞外蛋白酶处理消除。
