칼슘 분비를 조절하는 전압 의존 칼슘 채널 beta3 subunit 압타머의 발견 및 기능 연구
- 칼슘 분비를 조절하는 전압 의존 칼슘 채널 beta3 subunit 압타머의 발견 및 기능 연구
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- In the pancreatic β cell, Ca2+ is the final factor for insulin secretion and it is regulated by several Ca2+ channels. Although VDCC β3 subunit is known for inhibiting regulator of calcium release in the β cell, working mechanism of β3 subunit and modulating reagent has not been known. In this study, we developed β3 subunit aptamers by SELEX procedure with benzyl and naphthyl modified aptamer library and then we got 10 clones of aptamer. Using a pull-down assay, we proved that only N5 apatmer bound to mammalian expressed β3 subunit. Then we transfected the N5 aptamer to the MIN6 cells and check the calcium regulation property. When MIN6 cell were stimulated with carbamylcholine (CCh), Ca2+ release in N5 aptamer transfected cell was higher than control and B1 aptamer transfected cell. Following [Ca2+]i increase, CaMKII phosphorylation was also increased in the N5 aptamer transfected cell. As well as insulin secretion is higher in the high glucose concentration. To find binding site of N5 aptamer for β3 subunit, we prepared three kinds of β3 subunit truncate. As a result, we revealed that this aptamer strongly bound to C-terminal GK domain in β3 subunit. Although we need to discover the molecular mechanism how N5 aptamer regulate GK domain of β3 subunit and modify the aptamer for medication, function of N5 aptamer Ca2+ regulation will be an interesting implication for the treatment of type 2 diabetes mellitus.
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