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

水平细胞（HCs）对视锥光感受器的反馈在视网膜神经元的中心-周围感受野组织中起着关键作用。使用含有GABA拮抗剂（100 μM微微毒素）的超液，采用全细胞膜片钳技术获得了蝾螈视网膜切片锥体光感受器的记录。感受野周围光照增加了视锥细胞中电压依赖性钙电流（I_<Ca>），并将I_<Ca>的激活电压移至负电压。外碱化使锥体I_< ca>升高，激活电压向负电压偏移。细胞外溶液pH缓冲能力的增强增加了锥体I_<Ca>，并阻断了周围光照下锥体I_<Ca>的任何额外增加。谷氨酸受体拮抗剂对HCs的超极化增强了锥体I_<Ca>，而盐对HCs的去极化抑制了锥体I_<Ca>。根据这些结果，我们认为pH值的变化更多地发生在突触间隙中，而突触间隙与细胞的膜电压密切相关，并介导细胞向锥状光感受器的反馈。为了验证去极化hc释放质子的想法，我们通过荧光比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值（pH_a）。当用5-hexadecanoylaminofluorescein（一种ph敏感的亲脂性染料）染色的hc通过盐酸盐或高k ^+培养液去极化时，pHa降低。pH_s的变化量单调地依赖于去极化量，100 mV （100 mM K^+诱导）的去极化可达0.21±0.05 pH单位。液泡型氢离子泵（v - atp酶）的特异性抑制剂400 nM巴菲霉素Al抑制了酸化，表明质子通过电压敏感的氢离子泵去极化从hc中释放出来。免疫细胞化学分析，使用a和V-ATPase抗体，发现V-ATPase存在于解离的hc质膜上。这些结果证实了质子从hc中释放出来，并支持了hc对锥体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