Rectification ratio based determination of disulfide bonds of ?2 extracellular loop of BK channel.

ABSTRACT: Large-conductance Ca2+-activated K+ (BK) channels are composed of a pore-forming ? and a variable number of auxiliary ? subunits and play important roles in regulating excitability, action potential waveforms and firing patterns, particularly in neurons and endocrine and cardiovascular cells. The ?2 subunits increase the diversity of gating and pharmacological properties. Its extracellular loop contains eight cysteine residues, which can pair to form a high-order structure, underlying the stability of the extracellular loop of ?2 subunits and the functional effects on BK channels. However, how these cysteines form disulfide bonds still remains unclear. To address this, based on the fact that the rectification and association of BK ? to ?2 subunits are highly sensitive to disruption of the disulfide bonds in the extracellular loop of ?2, we developed a rectification ratio based assay by combining the site-directed mutagenesis, electrophysiology and enzymatic cleavage. Three disulfide bonds: C1(C84)-C5(C113), C3(C101)-C7(C148) and C6(C142)-C8C(174) are successfully deduced in ?2 subunit in complex with a BK ? subunit, which are helpful to predict structural model of ?2 subunits through computational simulation and to understand the interface between the extracellular domain of the ? subunits and the pore-forming ? subunit.

SUBMITTER: Guo X 

PROVIDER: S-EPMC6298698 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature