| Large conductance, calcium-activated potassium (BK) channels are ubiquitously expressed and play important roles in many physiological processes, from muscle contraction, neural transmission to hearing. These physiological roles of the BK channel derive from its unique property; namely, it is synergistically activated by membrane voltage, intracellular Ca2+, and Mg 2+. As an example, my study shows that a mutation (D434G) in the cytoplasmic domain of the human BK channel increases its Ca2+ sensitivity, which is associated with generalized epilepsy and paroxysmal dyskinesia (GEPD). Previous studies show that sensors for voltage (VSD, voltage sensor domain) and intracellular Ca2+/Mg2+ (cytoplasmic domain) directly link to the ion conduction pore and regulate its gating. In addition, BK channel gating is modulated by its auxiliary beta subunits, which underlies its tissue-specific phenotypes. However, the molecular mechanism underlying these regulations is still not clear. I have studied voltage and Mg 2+ dependent activation and the results show that the interaction between the VSD and the cytosolic domain is important in BK channel activation. Intracellular Mg2+ activates the channel by mediating this interaction. Experimental results demonstrate that Mg2+ binds at the interface between the two sensory domains, coordinated by residues E374 and E399 in the cytoplasmic domain, and D99 and N172 in the VSD, and regulates the voltage sensor movement by an electrostatic interaction with R213 in the transmembrane segment S4. Interestingly, the beta1 subunit may enhance Ca2+ sensitivity also by altering the conformation and movements of the VSD because the perturbation of the VSD by mutations disrupts the ability of the beta1 subunit to enhance Ca2+ sensitivity. Taken together, my studies in this dissertation indicate that the direct interactions among different functional domains within the alpha subunit of BK channels and the protein-protein interactions between the alpha and beta subunits are critical to the gating properties of BK channels. |
