Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels.

KCNQ1 is a homotetrameric voltage-gated potassium channel expressed in cardiomyocytes and epithelial tissues. However, currents arising from KCNQ1 have never been physiologically observed. KCNQ1 is able to provide the diverse potassium conductances required by these distinct cell types through coassembly with and modulation by type I transmembrane β-subunits of the KCNE gene family. KCNQ1-KCNE K+ channels play important physiological roles. In cardiac tissues the association of KCNQ1 with KCNE1 gives rise to IKs, the slow delayed outwardly rectifying potassium current. IKs is in part responsible for repolarizing heart muscle, and is therefore crucial in maintaining normal heart rhymicity. IKschannels help terminate each action potential and provide cardiac repolarization reserve. As such, mutations in either subunit can lead to Romano-Ward Syndrome or Jervell and Lange-Nielsen Syndrome, two forms of Q-T prolongation. In epithelial cells, KCNQ1-KCNE1, KCNQ1-KCNE2 and KCNQ1-KCNE3 give rise to potassium currents required for potassium recycling and secretion. These functions arise because the biophysical properties of KCNQ1 are always dramatically altered by KCNE co-expression. We wanted to understand how KCNE peptides are able to modulate KCNQ1. In Chapter II, we produce partial truncations of KCNE3 and demonstrate the transmembrane domain is necessary and sufficient for both assembly with and modulation of KCNQ1. Comparing these results with published results obtained from chimeric KCNE peptides and partial deletion mutants of KCNE1, we propose a bipartite modulation residing in KCNE peptides. Transmembrane modulation is either active (KCNE3) or permissive (KCNE1). Active transmembrane KCNE modulation masks juxtamembranous C-terminal modulation of KCNQ1, while permissive modulation allows C-terminal modulation of KCNQ1 to express. We test our hypothesis, and demonstrate C-terminal Long QT point mutants in KCNE1 can be masked by active trasnsmembrane modulation. Having confirmed the importance the C-terminus of KCNE1, we continue with two projects designed to elucidate KCNE1 C-terminal structure. In Chapter III we conduct an alanine-perturbation scan within the C-terminus. C-terminal KCNE1 alanine point mutations result in changes in the free energy for the KCNQ1-KCNE1 channel complex. High-impact point mutants cluster in an arrangement consistent with an alphahelical secondary structure, "kinked" by a single proline residue. In Chapter IV, we use oxidant-mediated disulfide bond formation between non-native cysteine residues to demonstrate amino acid side chains residing within the C-terminal domain of KCNE1 are close and juxtaposed to amino acid side chains on the cytoplasmic face of the KCNQ1 pore domain. Many of the amino acids identified as high impact through alanine perturbation correspond with residues identified as able to form disulfide bonds with KCNQ1. Taken together, we demonstrate that the interaction between the C-terminus of KCNE1 and the pore domain of KCNQ1 is required for the proper modulation of KCNQ1 by KCNE1, and by extension, normal IKs function and heart rhymicity.