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Interactions of KChIP4a and its mutants with Ca2+ or Kv4.3 N-terminus by affinity capillary electrophoresis

Li, Meina, Lei, Lei, Jia, Linghan, Ling, Xiaomei, Zhang, Jianmei, Zhao, Yiran, Wang, KeWei
Analytical biochemistry 2014 v.449 pp. 99-105
calcium, capillary electrophoresis, chemical structure, mutants, profits and margins, proteins
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 Ca²⁺ 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)×10⁶Lmol–¹ and (5.26±0.71)×10⁶Lmol–¹, respectively. In addition, in the presence of calcium (10μmolL–¹), the binding constant of KChIP4a/KvN increased to (6.72±1.66)×10⁷Lmol–¹. In addition, the binding constant of KChIP4a with Ca²⁺ was (7.1±1.5)×10⁷Lmol–¹. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca²⁺, and the integrity of the molecular structure of KChIP4a was important for Ca²⁺ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.