Regulation of Voltage-gated Ion Channels by Calmodulin and Calneuron 1

• Main Authors: Chih-Hung Chi, 紀志弘
• Other Authors: 潘建源
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/4n5dq6

博士 === 國立臺灣大學 === 生命科學系 === 106 === The Ca2+ influx through the voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major extracellular factor responsible for the elevation in intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. The inactivation phase of CaVs determines the amount of Ca2+ that enters the cell, and calmodulin (CaM) is known to be involved in the Ca2+-dependent inactivation process. However, how CaM modulates CaV2.2 is still unclear. Here, we expressed CaV2.2 with CaM or CaM mutants in HEK293T cells and measured the currents to characterize the inactivation. The results showed that CaV2.2 had a fast inactivation with Ca2+, but not Ba2+, as the charge carrier; when it was co-expressed with CaM mutants with a Ca2+-binding deficiency, the levels of inactivation decreased. Using GST-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of CaV2.2 in the presence or absence of Ca2+. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. The mutations in the CaM-binding site greatly reduced the current of CaV2.2 but could be rescued by CaM12 (Ca2+-binding deficiency at the N-lobe) overexpression; in addition, CaM12 enhanced the total expression level of CaV2.2, but the ratios between the membrane and total fractions remained unchanged. Together, our data suggest that CaM not only modulates the inactivation of CaV2.2 but also regulates its expression to control [Ca2+]i elevation for physiological activities. Previous studies in our lab showed that calneuron 1 (Caln1), a Ca2+ binding protein with structure similar to CaM, inhibits the inward current of CaVs in chromaffin cells and interacts with Scyl1 verified by yeast-2-hybrid. Here we showed Caln1 inhibited the inward currents of CaV1.3, 2.2, 3.1, and NaV1.4 expressed in HEK293T cells, also, its overexpression suppressed neuronal activities in primary cultured cortical neurons. Caln1 did not directly interact with CaV2.2 and CaV3.1, however, the chemical LTP decreased the presence of channel proteins on the plasma membrane. Caln1 also can be upregulated in primary cortical neuron. Both Caln1 and Scyl1 has been reported to regulate the vesicle tracking from Golgi to plasma membrane and ER to Golgi, respectively. Here, the patch clamping results showed that Scyl1 inhibited the currents of CaV2.2 with Caln1. In the immunostaining experiment, Scyl1 and Caln1 colocalized and accumulated at the intracellular vesicles, considered to be the Golgi apparatus, however, Scyl1ΔKLHT, lacking kinase function and self-associated manner, eliminated this appearance. Therefore, Caln1 is important in the trafficking of CaV2.2 and inward current regulation of VGICs (voltage-gated ion channels), however, the correlation between trafficking and modulation of VGICs in controlling neuronal activities still remains the open questions.