Structural and functional studies on the regulation of potassium channels by local anesthetics and intracellular auxiliary subunits

K+ channels, directly involved in regulating membrane potential, play important roles in many biological activities, e.g. rhythm in cardiacmyocytes, information integration in neurons, metabolic state in pancreatic beta-cells. K+ channels all have the conserved tetrameric transmembrane pore forming subunit (2TM/P in each subunit: 2 transmembrane helices/1 pore), which is composed of outer transmembrane helix (M1 or S5), pore helix, selectivity filter and inner transmembrane helix (M2 or S6). The diverse functions of K+ channels come from other domains, e.g. transmembrane voltage sensor domain (S1∼S4) in voltage-gated K + (Kv) channels, cytoplasmic T1 tetrameric assembly domain in Kv channels, cytoplasmic pore forming domain in inwardly rectifying K+ (Kir) channels. Kv (6TM/P) and Kir channels (2TMIP) are two main K+ channel subfamilies. In the early part of my studies, I studied regulation of Kir channels by small molecules. In the later part, I studied regulation of Kv channels by cytoplasmic proteins.; Kir channels have their functions modulated by different cellular signaling factors or drugs through the sub-domains or auxiliary subunits. 7 subfamilies (Kir1∼7) are found in Kir channel family. Bupivacaine, a widely used local anesthetic to block the signal transduction by Na+ channels, also work on other channels like G protein-gated K+ channels (Kir3). Bupivacaine inhibits GIRK channels to cause cardiac arrhythmia and nervous system malfunction. However, bupivacaine doesn't have inhibitory effect on IRK (Kir2) and ROMK (Kir1) channels, which are closely related to GIRK channels. By using electrophysiology and mutagenesis methods, we identified the molecular mechanisms underlying the GIRK channel specific inhibition by bupivacaine. Phosphatidylinositol 4,5-bisphosphate (PIP2 ) is known to be essential in the Kir channel activity. Bupivacaine inhibits GIRK channels by antagonizing the coupling between PIP2 and the channel opening.; Kv channels have four main subfamilies: Kv1 (Shaker), Kv2 (Shah), Kv3 (Shaw) and Kv4 (Shal). N-terminal cytoplasmic domain of Kv4 channels, involved in channel assembly, trafficking and gating, is composed of two sub-domains, T1 and alpha1. We studied the structure of T1 domain of Kv4.2 (Kv4.2T1) and identified the key interface residues involved in the Kv4-specific tetrameric channel assembly. We also revealed the mechanisms underlying the specific interaction between Kv channel-interacting proteins (KChIP) and the N-terminal alpha1 domain (residues 1∼20) of Kv4.2. The structure of KChIP consists of 4 EF-hands, like other neuronal calcium sensors, but showed a distinct mode of assembly upon binding the alpha1 of Kv4.2.