Cheng Y M, Kelly T, Church J
Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
Neuroscience. 2008 Feb 19;151(4):1084-98. doi: 10.1016/j.neuroscience.2007.12.007. Epub 2007 Dec 8.
We examined the potential contribution of a voltage-gated proton conductance (gH+) to acid extrusion from cultured postnatal rat hippocampal neurons. In neurons loaded with Ca2+- and/or pH-sensitive fluorophores, transient exposures to 25-139.5 mM external K+ (K+o) or 20 microM veratridine in the presence of 2 mM Ca2+o (extracellular pH (pHo) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi), respectively. In contrast, under external Ca2+-free conditions, the same stimuli failed to affect [Ca2+]i but caused an increase in pHi, the magnitude of which was related to the [K+]o applied and the change in membrane potential. Consistent with the properties of gH+s in other cell types, the magnitude of the rise in pHi observed in the absence of external Ca2+ was not affected by the removal of external Na+ but was sensitive to external Zn2+ and temperature and was dependent on the measured transmembrane pH gradient (DeltapHmemb). Increasing DeltapH(memb) by pretreatment with carbonylcyanide-p-trifluoromethoxyphenylhydrazone augmented both the high-[K+]o-evoked rise in pHi and the Zn2+-sensitive component of the rise in pHi, suggestive of increased acid extrusion via a gH+. The inhibitory effect of Zn2+ at a given DeltapHmemb was further enhanced by increasing pHo from 7.35-7.8, consistent with a pHo-dependent inhibition of the putative gH+ by Zn2+. Under conditions designed to isolate H+ currents, a voltage-dependent outward current was recorded from whole-cell patch-clamped neurons. Although the outward current appeared to show some selectivity for protons, it was not sensitive to Zn2+ or temperature and the H+-selective component could not be separated from a larger conductance of unknown selectivity. Nonetheless, taken together, the results suggest that a Zn2+-sensitive proton conductive pathway is present in rat hippocampal neurons and contributes to H+ efflux under depolarizing conditions.
我们研究了电压门控质子电导（gH+）对培养的新生大鼠海马神经元酸外排的潜在作用。在装载了Ca2+和/或pH敏感荧光团的神经元中，在2 mM Ca2+o（细胞外pH（pHo）恒定为7.35）存在的情况下，短暂暴露于25 - 139.5 mM的细胞外K+（K+o）或20 microM藜芦碱，分别导致细胞内游离钙浓度（[Ca2+]i）可逆性升高和降低，以及细胞内pH（pHi）可逆性降低和升高。相比之下，在细胞外无Ca2+的条件下，相同的刺激未能影响[Ca2+]i，但导致pHi升高，其幅度与所施加的[K+]o以及膜电位变化有关。与其他细胞类型中gH+的特性一致，在无细胞外Ca2+时观察到的pHi升高幅度不受去除细胞外Na+的影响，但对细胞外Zn2+和温度敏感，并且依赖于所测量的跨膜pH梯度（DeltapHmemb）。用羰基氰化物 - p - 三氟甲氧基苯腙预处理增加DeltapH(memb)，增强了高[K+]o诱发的pHi升高以及pHi升高中对Zn2+敏感的成分，提示通过gH+增加了酸外排。在给定的DeltapHmemb下，将pHo从7.35提高到7.8进一步增强了Zn2+的抑制作用，这与Zn2+对假定的gH+的pHo依赖性抑制一致。在旨在分离H+电流的条件下，从全细胞膜片钳记录的神经元中记录到电压依赖性外向电流。尽管外向电流似乎对质子表现出一定的选择性，但它对Zn2+或温度不敏感，并且H+选择性成分无法与更大的未知选择性电导分离。尽管如此，综合来看，结果表明大鼠海马神经元中存在对Zn2+敏感的质子传导途径，并在去极化条件下有助于H+外流。
