Yang Qian, Hu Lan, Lawson-Qureshi Dorian, Colbran Roger J
Department of Molecular Physiology and Biophysics.
Vanderbilt Brain Institute.
bioRxiv. 2025 Jan 8:2025.01.08.631979. doi: 10.1101/2025.01.08.631979.
Neuronal excitation-transcription (E-T) coupling pathways can be initiated by local increases of Ca concentrations within a nanodomain close to the L-type voltage-gated Ca channel (LTCC). However, molecular mechanisms controlling LTCC organization within the plasma membrane that help creation these localized signaling domains remain poorly characterized. Here, we report that neuronal depolarization increases Ca1.3 LTCC clustering in cultured hippocampal neurons. Our previous work showed that binding of the activated catalytic domain of Ca/calmodulin-dependent protein kinase II (CaMKII) to an RKR motif in the N-terminal cytoplasmic domain of Ca1.3 is required for LTCC-mediated E-T coupling. We tested whether multimeric CaMKIIα holoenzymes can bind simultaneously to co-expressed Ca1.3 α1 subunits with two different epitope tags. Co-immunoprecipitation assays from HEK293T cell lysates revealed that CaMKIIα assembles multimeric Ca1.3 LTCC complexes in a Ca/calmodulin-dependent manner. CaMKII-dependent assembly of multi-Ca1.3 complexes is further facilitated by co-expression of the CaMKII-binding LTCC β2a subunit, relative to the β3 subunit, which cannot bind directly to CaMKII. Moreover, clustering of surface localized Ca1.3 α1 subunits in intact HEK293 cells was increased by pharmacological LTCC activation, but only in the presence of co-expressed wild-type CaMKIIα. Moreover, depolarization-induced clustering of surface-expressed Ca1.3 LTCCs in cultured hippocampal neurons was disrupted by suppressing the expression of CaMKIIα and CaMKIIβ using shRNAs. The CaMKII-binding RKR motif is conserved in the N-terminal domain of Ca1.2 α1 subunits and we found that activated CaMKIIα promoted the assembly of Ca1.2 homomeric complexes, as well as Ca1.3-Ca1.2 heteromeric complexes . Furthermore, neuronal depolarization enhanced the clustering of surface-expressed Ca1.2 LTCCs, and enhanced the colocalization of endogenous Ca1.2 LTCCs with surface-expressed Ca1.3, by CaMKII-dependent mechanisms. This work indicates that CaMKII activation-dependent LTCC clustering in the plasma membrane following neuronal depolarization may be essential for the initiation of a specific long-range signal to activate gene expression.
神经元兴奋-转录（E-T）偶联途径可由靠近L型电压门控钙通道（LTCC）的纳米域内钙浓度的局部升高引发。然而，控制质膜内LTCC组织以帮助形成这些局部信号域的分子机制仍不清楚。在此，我们报告神经元去极化会增加培养的海马神经元中Ca1.3 LTCC的聚集。我们之前的工作表明，LTCC介导的E-T偶联需要钙/钙调蛋白依赖性蛋白激酶II（CaMKII）的活化催化结构域与Ca1.3 N端胞质结构域中的RKR基序结合。我们测试了多聚体CaMKIIα全酶是否能同时与带有两种不同表位标签的共表达Ca1.3 α1亚基结合。从HEK293T细胞裂解物中进行的共免疫沉淀分析表明，CaMKIIα以钙/钙调蛋白依赖性方式组装多聚体Ca1.3 LTCC复合物。相对于不能直接与CaMKII结合的β3亚基，CaMKII结合性LTCC β2a亚基的共表达进一步促进了多Ca1.3复合物的CaMKII依赖性组装。此外，药理学上的LTCC激活增加了完整HEK293细胞中表面定位的Ca1.3 α1亚基的聚集，但仅在共表达野生型CaMKIIα的情况下。此外，使用短发夹RNA（shRNA）抑制CaMKIIα和CaMKIIβ的表达可破坏培养的海马神经元中去极化诱导的表面表达Ca1.3 LTCC的聚集。CaMKII结合的RKR基序在Ca1.2 α1亚基的N端结构域中保守，我们发现活化的CaMKIIα促进了Ca1.2同聚体复合物以及Ca1.3-Ca1.2异聚体复合物的组装。此外，神经元去极化通过CaMKII依赖性机制增强了表面表达的Ca1.2 LTCC的聚集，并增强了内源性Ca1.2 LTCC与表面表达的Ca1.3的共定位。这项工作表明，神经元去极化后质膜中CaMKII激活依赖性的LTCC聚集可能对于启动特定的长距离信号以激活基因表达至关重要。
