Data_Sheet_2_Changes in H+, K+, and Ca2+ Concentrations, as Observed in Seizures, Induce Action Potential Signaling in Cortical Neurons by a Mechanism That Depends Partially on Acid-Sensing Ion Channels.pdf

Acid-sensing ion channels (ASICs) are activated by extracellular acidification. Because ASIC currents are transient, these channels appear to be ideal sensors for detecting the onset of rapid pH changes. ASICs are involved in neuronal death after ischemic stroke, and in the sensation of inflammatory pain. Ischemia and inflammation are associated with a slowly developing, long-lasting acidification. Recent studies indicate however that ASICs are unable to induce an electrical signaling activity under standard experimental conditions if pH changes are slow. In situations associated with slow and sustained pH drops such as high neuronal signaling activity and ischemia, the extracellular K+ concentration increases, and the Ca2+ concentration decreases. We hypothesized that the concomitant changes in H+, K+, and Ca2+ concentrations may allow a long-lasting ASIC-dependent induction of action potential (AP) signaling. We show that for acidification from pH7.4 to pH7.0 or 6.8 on cultured cortical neurons, the number of action potentials and the firing time increased strongly if the acidification was accompanied by a change to higher K+ and lower Ca2+ concentrations. Under these conditions, APs were also induced in neurons from ASIC1a–/– mice, in which a pH of ≤ 5.0 would be required to activate ASICs, indicating that ASIC activation was not required for the AP induction. Comparison between neurons of different ASIC genotypes indicated that the ASICs modulate the AP induction under such changed ionic conditions. Voltage-clamp measurements of the Na+ and K+ currents in cultured cortical neurons showed that the lowering of the pH inhibited Na+ and K+ currents. In contrast, the lowering of the Ca2+ together with the increase in the K+ concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na+ channels. We conclude that the ionic changes observed during high neuronal activity mediate a sustained AP induction caused by the potentiation of Na+ currents, a membrane depolarization due to the changed K+ reversal potential, the activation of ASICs, and possibly effects on other ion channels. Our study describes therefore conditions under which slow pH changes induce neuronal signaling by a mechanism involving ASICs.

酸敏感离子通道（Acid-sensing ion channels, ASICs）可被细胞外酸化激活。由于ASIC电流具有瞬时性，这类通道堪称检测快速pH变化起始的理想传感器。ASICs参与缺血性脑卒中后的神经元死亡过程，以及炎性疼痛的感知通路。缺血与炎症往往伴随缓慢发生且持续时间较长的细胞外酸化现象。然而近期研究显示，若pH变化速率较为缓慢，在标准实验条件下，ASICs无法诱导电信号活动。在伴随缓慢且持续性pH下降的场景中，例如神经元高活动状态与缺血状态，细胞外钾离子（K+）浓度会升高，而钙离子（Ca2+）浓度则会降低。我们提出假说：氢离子（H+）、钾离子与钙离子的协同变化，或许能够介导依赖ASICs的持续性动作电位（action potential, AP）信号产生。我们在体外培养的皮层神经元中开展实验，当pH从7.4降至7.0或6.8时，若酸化过程伴随钾离子浓度升高与钙离子浓度降低，神经元的动作电位数量与放电时长均显著提升。在此类条件下，即便在ASIC1a基因敲除（ASIC1a–/–）的小鼠神经元中——这类小鼠的神经元需要pH≤5.0才能激活ASICs——也可诱导产生动作电位，这表明本次动作电位的诱导并不依赖ASIC激活。对比不同ASIC基因型的神经元可见，在上述离子环境改变的条件下，ASICs可对动作电位的诱导过程起到调控作用。对培养的皮层神经元进行钠离子（Na+）与钾离子电流的电压钳（voltage-clamp）检测后发现，pH降低会抑制钠、钾离子电流。与之相反，钙离子浓度降低结合钾离子浓度升高，会使电压门控钠离子通道的激活电压依赖性产生超极化偏移。我们最终得出结论：神经元高活动期间出现的离子变化，可通过多种途径介导持续性动作电位的产生，包括增强钠离子电流、改变钾离子逆转电位引发膜去极化、激活ASICs，以及可能对其他离子通道产生的影响。综上，本研究阐明了在特定条件下，缓慢的pH变化可通过依赖ASICs的机制诱导神经元信号活动。