Structure, Trafficking And Regulatory Mechanism Of Hβ2 Subunits Of BK Channels

Large conductance voltage-dependent and calcium-activated potassium channels (Maxi-K or BK) control a large variety of physiological process, including smooth muscle contraction, neurotransmitter release and hearing. Beside the pore-formingαsubunits, there are several auxiliaryβsubunits, which greatly contribute to the diversity of BK channels. This diversity is the fundament of different function of BK channels in many tissues. It is still very important to study the structure and regulatory mechanism of BK channelβsubunits nowadays. Here we show the detailed study of transmembrane structure, trafficking and regulatory properties of humanβ2 (hβ2) subunits by molecular biology, fluorescent imaging and electrophysiological techniques. And we have also tried to express, purify and crystallize the external loop of hβ2 and hβ4 subunits.We verified the transmembrane structure of hβ2 subunits which contains two transmembrane segments, a huge and hydrophobic external loop. We found that only if co-assembled withαsubunits to form BK channels can the hβ2 subunits reach the membrane. And when expressed alone, hβ2 subunits will reside in the endoplasmic reticulum (ER). We further demostrated that there is a retention signal located at the NH2 terminus of hβ2 subunits. This retention signal is a motif consisted of five residues "QKIRD" which is a portion of a 13-residueαhelix. The retention signal of hβ2 NH2 terminus may be masked when tightly co-assemble with BKαsubunits, releasing the hβ2 subunits from the ER.Immunofluorescent experiments revealed that the region around position 137 is the most hydrophilic region, and this region may reside very cosely the outer pore of the BK channels. We then found that lysines around position 137 of hβ2 form postitively charged rings to modify the BK channels conductance dramatically. These rings can prevent the K+ or positively charged ChTX from approaching the pore through electrostatic mechanism, decrease the extracellular K+ concentration, leading to the outward rectification and low ChTX affinity of BK channels. The reduction of inward current due to outward rectification of BK channels can facilitate the cellular repalorization.To further study the structure ofβsubunits, we expressed and purified the extracellular loop of hβ2 and hβ4, and tried to crystallize them. This work may provide us some clues to study the modulatory properties ofβsubunits and the interaction of a andβsubunits profoundly. It is also the basis for natural toxin exploitation and application, and the basis for drug design.