Mullins L J, Whittembury J, Requena J
Department of Biophysics, University of Maryland School of Medicine, Baltimore.
Cell Calcium. 1989 Aug-Sep;10(6):401-12. doi: 10.1016/0143-4160(89)90031-6.
Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.
将水母发光蛋白和四乙铵注入鱿鱼巨大轴突，并用电极（对氢离子敏感、可测量电流和电压）刺入轴突。使用一种新设计的水平微量注射器来注入水母发光蛋白。它同时还用作电压钳制的电流和电压电极以及离子敏感微电极测量的参考电极。轴突通常浸浴在含有150 mM的Na⁺、K⁺以及一些惰性阳离子的溶液中，浴液中Ca²⁺浓度（[Ca²⁺]ₒ）可以是生理浓度或零浓度，并且离子电流被药物阻断。反复施加电压钳制脉冲，其幅度以诱导水母发光蛋白发光变化（轴浆中离子化Ca²⁺水平[Ca²⁺]ᵢ的一种度量）所需的程度为准。或者，将膜电位稳定保持在代表相对于在150 mM [K⁺]ₒ时观察到的静息膜电位的偏差的值（即约 -15 mV）。在没有[Ca²⁺]ₒ的情况下，电流引起的显著稳定去极化会增加[Ca²⁺]ᵢ（并使轴浆酸化）。由内部铂丝电流引起的内部氢活性[H⁺]ᵢ的变化限制了对[Ca²⁺]ᵢ进行有效测量的程度。然而，存在可归因于膜电位的对[Ca²⁺]ᵢ的影响。因此，在没有[Ca²⁺]ₒ的情况下，超极化可降低[Ca²⁺]ᵢ，这意味着Ca²⁺外流机制增强。还观察到去极化会增加[Ca²⁺]ᵢ。这些结果与鱿鱼巨大轴突中一种将Na⁺与Ca²⁺交换的电生机制的运作一致。
