Expression,Purification And Structure Study Of H?4 Subunits Of BK Channels And The Study About The Interaction Sites Of H?1 And ? Subunits

Large conductance voltage-dependent and calcium-activated potassium channels(Maxi-K or BK)control varieties of physiological process,including neurotransmitter release,smooth muscle contraction and hearing.Other than the pore-forming mSlo1 subunits,there are several assistant ? and y subunits,which greatly contribute to the diversified function of BK channels.This diversity is the basis of different function of BK channels in many tissues.So it is very important to study the regulatory mechanism and the unique structure of BK channel ? subunits.Here we tried to express,purify and crystallize the external loop of h?4 subunits and studied the interaction sites between h?1 and a subunit by molecular biology,biochemical methods and electrophysio logical techniques.(1)By fusing different protein-tags we find that the loop domain of h?4 can form a soluble expression in prokaryotic expression system and after a process of affinity chromatography,ion-exchange chromatography,gel filtration chromatography,We harvested some proteins of which the purity,stability and homogeneity is good enough for crystallizing.We also tried to express the other ? subunits loop domain and the denaturation and renaturation of h?2loop protein inclusions.(2)Compared to h?2 subunits,we explored the other ?'s interaction cite with mSlol,which is determined by a double mutation analysis.We identified the mutation h?1(E13,T14)which can significantly change the G-V curves of BK channel,and coupled with mSlol(K392,R393).We also revealed the interaction between h?1(Y105)and mSlo1(S202)and verified that h?1(L5,V6,M7)may have interaction with mSlol too.Although the ? subunits have great homology,the way they interact with mSlo1 is of great differences.(3)We also expressed and purified several kinds of toxins,including the scorpion toxin IBTX and the spider toxin spiToxin1 in prokaryotic expression system.The recombinant peptides have been identified about their bioactivity,and this study provided a reference for further study about design and obtention of peptides for medical applying and BK ? subunits research about how they change the channel's sensitivity to toxin.(4)we also studied the kinetics property of Nav1.5 channel,especially the slow inactivation and slow recovery,to provide the detailed information for better modeling.