title: Interactions of KChIP4a and its mutants with Ca2+ or Kv4.3 N-terminus by affinity capillary electrophoresis.
abstract: The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca(2+) for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32±1.66)×10(6) L mol(-1) and (5.26±0.71)×10(6) L mol(-1), respectively. In addition, in the presence of calcium (10 μmol L(-1)), the binding constant of KChIP4a/KvN increased to (6.72±1.66)×10(7) L mol(-1). In addition, the binding constant of KChIP4a with Ca(2+) was (7.1±1.5)×10(7) L mol(-1). Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca(2+), and the integrity of the molecular structure of KChIP4a was important for Ca(2+) binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs and Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China.
authors.forename: Meina
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Molecular and Cellular Pharmacology and State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Lei
authors.affiliation: Department of Molecular and Cellular Pharmacology and State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China.
authors.forename: Linghan
authors.initials: L
authors.lastname: Jia
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs and Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China. Electronic address: lingxm@bjmu.edu.cn.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Ling
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs and Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China.
authors.forename: Jianmei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs and Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China.
authors.forename: Yiran
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Molecular and Cellular Pharmacology and State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People's Republic of China. Electronic address: wangkw@bjmu.edu.cn.
authors.forename: KeWei
authors.initials: K
authors.lastname: Wang
mesh_terms: D002118:Calcium / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D051605:Kv Channel-Interacting Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D051672:Shal Potassium Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051605:Kv Channel-Interacting Proteins; D051672:Shal Potassium Channels; D002118:Calcium
keywords: ACE; Ca(2+); Interaction; KChIPs; KvN