Studies On Dynamic Ca²⁺ Sensitivity And Interaction Mechanisms Between α/β Subunits Of Bk Channel

Large-conductance Ca2+-and voltage-activated potassium (MaxiK or BK) channel is a kind of potassium channel that possesses the largest single channel conductance (about300pS) and uniquely responses to both membrane potential and intracellular Ca2+. BK channels are tetramers, composed of four functional a subunits and0-4auxiliary β subunits. Each a subunit has seven transmembrane segments S0-S6with an extracellular N-terminus and a large cytoplasmic C-terminus that contains a mechanical linker following the S6pore region and two Rossmann-fold RCK (Regulator of Conductance of K+) domains with two Ca2+binding sites (Slo1(D362,D367) and Ca2+bowl) required for activation by calcium. Native BK-type channels exhibit numerous modified properties after associating with tissue-specific auxiliary β1-β4subunits. Among the β subunits, β2and β3b subunits not only modulate activation of BK channels, but also inactivate its currents. As a voltage and calcium sensor, BK channels play critical roles in modulating many physiological activities, including neurotransmitter release and endocrine secretion in neurons or endocrine cells, contraction in smooth muscle cells and frequency tuning in hair cells.Studies on dynamic calcium sensitivity and interaction mechanisms between α/β subunits of BK channels have been conducting for many years. However, all of these questions remain elusive due to the limitations on technical or methods. Intensive study of them is of far-reaching practical significance for revealing the mechanism of diseases and looking for the therapeutic methods.The research works of this article mainly consist of two parts:In the first part, we combined conventional patch-clamp technique with calcium release photolysis technique to study the BK channels response to fast changing calcium signal and the calcium binding rate constant. In most studies, we usually apply a constant intracellular calcium concentration before onsetting the voltage. However, calcium signals are always changing during the actual physiological activities, the study on BK channels response to fast changing calcium signal is an original but important project. Moreover, the main method to monitor the calcium signals during physiological activities until now is dependent on fluorescence method to get the global intracellular calcium signal as an "average" concentration, no one knows the exact local calcium signal, which triggers certain physiological activities more accurately and opportunely. The main results and conclusions of this section are as follows:(1) With the sub-millisecond calcium changing during the uncaging photolysis, we determined the BK calcium binding rate constant is1.8*108M-s-1, which indicated that the BK can ultrafast follow the sub-millisecond calcium signal change without delay.(2) Took BK channel as a Ca2+sensor, we measured the local Ca2+signal during pseudo-action potential:the local calcium concentration increased to≥20μM during the rise and early fall phases of action potential and then decreased and stabilized at around4μM along with the fall phases of action potential.(3) We examined the response to the changing Ca2+signal from Cav1.2calcium channels of a BK channel mutant D369G, which is associated with epilepsy in rat. We found that D369G response faster not only under steady-state calcium concentration but also to the changing calcium signal, which will help to better understand of epilepsy caused by BK channel.In the second part, with combo double-mutant cycle method, the rectifier test, immunofluorescence resident method and structural biology, we mainly studied interactions between function a subunit and auxiliary β2subunit of mSlol/β2type BK channel. We know the specific expression of β subunits in different tissues will produce different BK channel functions, but by limitation to the lack of Slol/βx structure and methods for explore the protein-protein interaction, these problems have haunted numerous BK channel researchers for years. In this part, our main findings and conclusions are as follows:(1) We determined the pre-inactivation site and the pre-inactivation mechanism: C←→O←→O*←→1, the β2(E16,D17) on the N-terminal of β2will bind to mSlo1(K330,K331) on the mouth of the channel before inactivation, which will form the PI site and O*state. (2) We determined the enhanced activation site, the β2(D44,E45) on the N-terminal of β2bound to mSlol(K392,R393) on the a subunit, which has an important influence on the binding and expression of a and P subunits.(3) We further determined the the enhanced activation originated from ECaB site (ECa2+bowl site), which formed by the binding between β2(K33,R34,K35) and the Ca2+bowl. And we thus untied the enhanced activation pathway of BK (β2) channel:it is generated from EcaB site, and then E site served as a pulley to optimize the gating force to open the door easier.(4) We have therefore not only established a feasible and reliable method to determine the interaction between subunits of BK channel, but also provide an important reference for studying interactions of other channels.In summary, the article is devoted to study the dynamic calcium sensitivity and interaction mechanisms between α/β subunits of BK channels and successfully solved a few problems of public concern, which paves solid foundation for a clearer understanding of the BK channels and other basic research and related pathology research.