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.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1623 |
| Date | 30 May 2012 |
| Creators | Mruk, Karen |
| Publisher | eScholarship@UMMS |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | GSBS Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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