The Exploration And Functional Study Of Large Conductance Calcium Activated Potassium Channels(BK Channels)Activatior

The BK channels are the large-conductance calcium-activated potassium channels(BK channels)which are widely expressed in various cells and tissues of mammals except myocardial cells,and participate in many physiological processes.BK channels are potential drug targets for the treatment of hypertension,urinary incontinence,asthma,coronary heart disease and ischemic stroke,et al.Exploring BK channel activators with high activity,high selectivity,excellent cell permeability and good drug-likeness will not only help to elucidate the mechanism of action of BK channels in physiological and pathological conditions,but also lay the foundation for the drug researches on the treatment of cardiovascular and cerebrovascular diseases.HNTX-? and HNTX-?? has been purified and characterized from the venom of the Chineses spider Haplopema hainanum.The HNTX-? is composed of 33 amino acid residues,including six cysteines,three of the two disulfide bonds,with a mass of 3828.7 Da.The complete sequence of HNTX-? is.ECRYWLGTCSKTGDCCSHLSCS PKHGWCVWDWT.It suggests that HNTX-? may be inhibitor cysteine knot(ICK)toxin.The HNTX-?? is composed of 34 amino acid residues,including eight cysteines,four of the two disulfide bonds,with a mass of 3799.7 Da.The complete sequence of HNTX-?? is SCAKPRENCNRMNILCCRGECVCPTFGDCFCYGD.And the molecular structure of HNTX-?? is still unclear.In this study,we chosen HNTX-? to conduct futher research.In this study,by using reversed-phase chromatography and patch clamp electrophysiology experiment techniques,we foud that 1?M HNTX-? activated iberiotoxin-sensitive(strongly blocked by 100 nM IbTx)BK channel(mSlo)currents by 6.46%,and the dose-response curve of HNTX-? activated BK(mSlo)currents was fitted by the Hill equation,and the ECSO value is 2.09 pM.While,1?M HNTX-?activated iberiotoxin-sensitive(strongly blocked by 100 nM IbTx)BK channel(mSlo+?2)currents by 22.16%,and the dose-response curve of HNTX-? activated BK(mSlo+?1)currents was aslo fitted by the Hill equation,and the EC50 value is 1.07?M,suggesting that HNTX-?displayed a higher preference with BK channel(mSlo+?1)over BK channel(mSlo)by about 1.95 folds.The results implied that when(mSlo+?1)channel coexpessed in HEK 293T cells,suggesting that the role of the ?1 subunit is probably to facilitate HNTX-? binding.Therefore HNTX-? can as a more efficient tool for studying the physiological and pathological mechanism of hypertension,epilepsy,and cardiovascular diseases.What's more,1?M HNTX-? activated iberiotoxin-insensitive(slightly blocked by 100 nM IbTx)BK channel(mSlo+?3)currents by 4.86%,but the iberiotoxin-sensitive BK channel(mSlo+?2)and iberiotoxin-insensitive BK channel(mSlo+?4)were almost insensitive to HNTX-?.The results implied that can as a invaluable tool for discriminating most BK channel subtypes.While,1?M HNTX-? not shift in the conductance-voltage curve for BK(mSlo+?1)currents,which rules out the possibility that HNTX-?interacts directly with the voltage sensor.In this study,we used the electrophysiological technique to detect whether HNTX-? effects calcium-activated potassium channels expressed HEK 293T cells.The result show that HNTX-? has no obvious inhibition on the intermediate-conductance calcium-activated K+channels(IK)and small-conductance calcium-activated K+channels(SK)besides large-conductance calcium-activated potassium channels(BK).Hence,these results strongly implied that HNTX-? is a strongly specificity activator.