Baik Julia Young, Park Eunice Yon June, So Insuk
Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Korea.
Korean J Physiol Pharmacol. 2020 May 1;24(3):277-286. doi: 10.4196/kjpp.2020.24.3.277.
Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.
多囊肾病2样蛋白1（PKD2L1），也被称为多囊蛋白-L或TRPP3，是一种调节细胞内钙浓度的非选择性阳离子通道。钙调蛋白（CaM）是一种钙结合蛋白，由N端叶和C端叶组成，每个叶中有两个钙结合EF手结构域。在之前的研究中，我们证实CaM与PKD2L1的脱敏有关，并且特别涉及CaM N端叶和PKD2L1的EF手结构域。然而，PKD2L1的钙调蛋白结合结构域（CaMBD）及其抑制机制尚未确定。为了确定PKD2L1的钙调蛋白结合锚定残基，构建了假定的CaMBD单突变体和EF手结构域缺失突变体。用全细胞膜片钳记录突变体的电流变化。在不同细胞内浓度下使用钙调蛋白抑制剂氯丙咪嗪（CMZ）。在与PKD2L1野生型表现出相似或更高基础电流的突变体中，L593A在CMZ诱导的电流中变化很小。L593A与CaM共表达减弱了CaM对PKD2L1的抑制作用。在之前的研究中推断，在16 nM钙浓度下，CaM C端叶通过与PKD2L1结合抑制通道，在100 nM时CaM N端叶起作用。基于16 nM钙浓度条件下的结果，本研究表明CaM C端叶与亮氨酸-593结合，亮氨酸-593可能是CaM C端叶的锚定残基，从而调节通道活性。综上所述，我们的结果为CaM调节PKD2L1通道活性提供了一个模型。
