The Change Of Function And Structure On BK Channel Conferred By Loop Segment Of β Subunit

The presence of cell membrane helps cells form the stably internal state.But the cell is not an enclosed system,which can perform material and energy exchange with surrounding environment.Ion channels are one of the important functional proteins that can pass several ions through the membrane.There are hundreds of ion channels in the world and they can be distinguished based upon their gating mechanism.All the ion channels can be divided into four subtypes—voltage gated channels,ligand gated channels, pH gated channels and mechanically gated channels.BK channel is a kind of large-conductance,calcium and voltage activated potassium channel that is found in all excitable and unexcitable cells except heart myocytes.The conductance of BK channel is ten times more than general potassium channels,so it plays the key role in physiological process.In different kinds of cells,BK channel always show dissimilar characteristic of macroscopic current.People have found the association with its auxiliary subunits is one of the reasons.In this study,using patch clamp,molecular biology construction, site-mutation,eukaryotic cell culture and computer 3D modeling,we study the change of function and structure conferred by loop segment ofβ2 andβ4 modified subunit.Main results of the study are as follows:(1) Besides altering the time constant of activation and deactivation,β2 subunit bring BK channel a fast and complete inactivation and change the sensitivity to Ca²⁺.In addition, the channel displays the outward-rectification current when it associates withβ2 subunit. Through electrophysiological and site-mutation methods,we find the rectification phenomena at high voltages are mainly generated by four positive charged amino acids in loop segment ofβ2 subunit.After analysis,we believe that the loop segment ofβ2 subunit will take place above the pore of BK channel to form a "helmet".Several positive charged lysines—K137,K141,K147 and K150 gather above the pore of BK channel and form a strong positive charge field,thereby preventing the potassium ions from entering cells,so they produced the outward rectification.(2)β4 subunit mainly expresses in nervous system,especially in brain that can slow the activation and deactivation of BK channel.Currently known,the most important nature that is brought byβ4 subunit is to make the channel to be resistant to some special toxins,such as charybdotoxin(CTX) and iberiotoxin(IbTX).After association withβ4 subunit,the block rate of the toxin reduces over 1000 time and our nervous system will be kept form destroying.Through experiments,we discover the resistance to some toxin relates to a tyrosine in unique position and three positive charged amino acids.The tyrosine in 100 or so is the binding site.Toxin can be easily close to the pore of channel if there is such a tyrosine.The sequence of CTX and IbTX are both rich in positive charged amino acids.The positive charged amino acids inβ4 loop segment and toxin produce mutex,thereby preventing toxin from getting near to the pore of channel.Becauseβ4 subunit has no special tyrosine for toxin binding,its ability of resisting toxin becomes stronger.(3) Through the two above experiments,we know loop segment ofβsubunits will form a "helmet" after association withαsubunit and bring BK channel the structural and functional changes.It is difficult to get crystal structure of ion channel proteins at present. So we apply computer modeling to forecast the structure of the protein complex.After analysis,we find that the linker between S5 transmembrane segment and Pore-loop decentralizes from the center when there is noβsubunit that is different from previous published Kcsa crystal structure.Followed by association withβsubunit,the S5-P linker centralizes toward the pore of channel.After formation of the complex structure,three positive charged amino acids locate just above the mouth of the channel and assist resisiting positive charged toxins.Positive charged amino acids ofβ2 subunit migrate in sequence,compared toβ4 subunit.Therefore,its resistance to toxin is weak.Butβ2 subunit makes BK channel to produce outward rectification.