Proton transport mechanism of retinal horizontal cells mediating pH changes in the synaptic cleft of cone photoreceptor terminal

水平细胞对视锥细胞的反馈在视网膜神经元的中心-周围-感受野结构中起着关键作用。通过全细胞膜片钳技术，使用含有GABA拮抗剂（100 μM印防己毒素）的灌流液，获得蝾螈视网膜切片中锥体光感受器的记录。感受野周围光照可使视锥细胞电压依赖性钙电流（<Ca>I_1）增加，并使I_1的激活电压<Ca>向负电压方向移动。外碱化也增加了视锥细胞的I_<Ca>，使其激活电压向负电位移动。细胞外溶液的pH缓冲能力的富集增加了锥I_<Ca>，并且通过周围照明阻断了锥I_的任何额外增加<Ca>。谷氨酸受体拮抗剂对HC的超极化增强了锥I_<Ca>，而红藻氨酸对HC的去极化则抑制了锥I_<Ca>。 关于我们 在与毛细胞膜电压密切相关的突触裂隙中，介导毛细胞向视锥光感受器的反馈。为了验证去极化HC释放质子的观点，我们用荧光比率成像技术测量了从鲤鱼或金鱼视网膜分离的HC的直接外表面（pH_a）。当用5-十六烷酰氨基荧光素（一种对pH敏感的亲脂性染料）染色的毛细胞在红藻氨酸盐或高K ^+介质中去极化时，pH值降低。pH_s的变化量与去极化量呈单调关系，100 mV去极化（由100 mM K^+诱导）可达0.21±0.05 pH单位。400 nM巴弗洛霉素Al（一种液泡型H^+泵（V-ATP酶）的特异性抑制剂）可抑制酸化作用，这表明质子是通过电压敏感的H^+泵去极化从HC中释放出来的。免疫细胞化学分析，使用和V-ATP酶抗体，显示存在的V-ATP酶的游离HC的质膜上。这些结果证实了质子从HC释放，并支持从HC到视锥细胞的反馈可能是质子介导的假设。少

Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround-receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 μM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (I_<Ca>) in the cones, and shifted the activation voltage of I_<Ca> to negative voltages. External alkalinization also increased cone I_<Ca> and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone I_<Ca>, and blocked any additional increase in cone I_<Ca> by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist augmented cone I_<Ca>, whereas depolarization of the HCs by kainate suppressed cone I_<Ca>.From these results, we propose that pH changes … More in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. To verify the idea that depolarized HCs released protons, we measured, by a fluorescence ratio imaging technique, the pH of the immediate external surface (pH_a) of HCs isolated from the carp or goldfish retina. When HCs stained by 5-hexadecanoylaminofluorescein, a pH-sensitive lipophilic dye, were depolarized by bath-application of kainate or high-K^+ medium, pHa was lowered. The amount of pH_s change was monotonically dependent on the amount of depolarization, as much as 0.21±0.05 pH unit by 100 mV depolarization (induced by 100 mM K^+). Acidification was suppressed by 400 nM bafilomycin Al, a specific inhibitor of vacuolar type H^+ pump (V-ATPase), suggesting that protons were released from HCs by the voltage-sensitive H^+ pump by depolarization. Immunocytochemical analysis, using an and V-ATPase antibody, revealed the existence of the V-ATPase on the plasma membrane of dissociated HCs. These results confirm that protons are released from HCs and support the hypothesis that the feedback from HCs to cones could be proton-mediated. Less