Neurotransmitter release at retinal ribbon-style synapses utilizes a specialized t-SNARE protein called syntaxin3B (STX3B). In contrast to other syntaxins, STX3 proteins can be phosphorylated in vitro at T14 by Ca^2+/calmodulin-dependent protein kinase II (CaMKII). This modification has the potential to modulate SNARE complex formation required for neurotransmitter release in an activity-dependent manner. To determine the extent to which T14 phosphorylation occurs in vivo in the mammalian retina and characterize the pathway responsible for the in vivo phosphorylation of T14, we utilized quantitative immunofluorescence to measure the levels of STX3 and STX3 phosphorylated at T14 (pSTX3) in the synaptic terminals of mouse retinal photoreceptors and rod bipolar cells (RBCs). Results demonstrate that STX3B phosphorylation at T14 is light-regulated and dependent upon the elevation of intraterminal Ca²⁺. In rod photoreceptor terminals, the ratio of pSTX3 to STX3 was significantly higher in dark-adapted mice, when rods are active, than in light-exposed mice. By contrast, in RBC terminals, the ratio of pSTX3 to STX3 was higher in light-exposed mice, when these terminals are active, than in dark-adapted mice. These results were recapitulated in the isolated eyecup preparation, but only when Ca²⁺ was included in the external medium. In the absence of external Ca²⁺, pSTX3 levels remained low regardless of light/dark exposure. Using the isolated RBC preparation, we next showed that elevation of intraterminal Ca²⁺ alone was sufficient to increase STX3 phosphorylation at T14. Furthermore, both the non-specific kinase inhibitor staurosporine and the selective CaMKII inhibitor AIP inhibited the Ca²⁺-dependent increase in the pSTX3/STX3 ratio in isolated RBC terminals, while in parallel experiments, AIP suppressed RBC depolarization-evoked exocytosis, measured using membrane capacitance measurements. Our data support a novel, illumination-regulated modulation of retinal ribbon-style synapse function in which activity-dependent Ca²⁺ entry drives the phosphorylation of STX3B at T14 by CaMKII, which in turn, modulates the ability to form SNARE complexes required for exocytosis.

视网膜带状突触的神经递质释放利用一种特殊的t-SNARE蛋白，称为syntaxin3B（STX3B）。与其他语法素相反，STX3蛋白可以被磷酸化体外在T14时，Ca ^{2 + /}钙调蛋白依赖性蛋白激酶II（CaMKII）。该修饰具有以活性依赖性方式调节神经递质释放所需的SNARE复合物形成的潜力。确定T14磷酸化发生的程度体内 在哺乳动物的视网膜中，并描述了负责 体内磷酸化的T14，我们利用定量免疫荧光法测量了小鼠视网膜感光器和杆状双极细胞（RBCs）的突触末端中T14（pSTX3）磷酸化的STX3和STX3的水平。结果表明，T14处的STX3B磷酸化受到光调节，并取决于末端内Ca ²⁺的升高。在杆感光细胞末端，当杆处于活动状态时，暗适应小鼠中pSTX3与STX3的比例明显高于光照小鼠。相比之下，在RBC终端中，当这些终端处于活动状态时，pSTX3与STX3的比例在受光照的小鼠中要比适应黑暗的小鼠更高。这些结果在隔离的眼罩制备中进行了概括，但仅当Ca ²⁺包含在外部媒体中。在没有外部Ca ²⁺的情况下，无论光照/黑暗暴露如何，pSTX3的水平均保持较低。使用分离的RBC制备物，我们接下来表明，单独升高末端内Ca ²⁺足以增加T14处STX3的磷酸化。此外，非特异性激酶抑制剂星形孢菌素和选择性CaMKII抑制剂AIP均可抑制分离的RBC末端中pSTX3 / STX3比值的Ca ²⁺依赖性增加，而在平行实验中，AIP抑制RBC去极化引起的胞吐作用，使用膜电容测量。我们的数据支持一种新颖的，照明调节的视网膜带状突触功能调节，其中活性依赖的Ca ²⁺ 进入驱动CaMKII在T14处STX3B的磷酸化，进而调节形成胞吐作用所需的SNARE复合物的能力。