Student Theses and Dissertations

Vanessa Ruta

Date of Award

2005

Document Type

Thesis

RU Laboratory

MacKinnon Laboratory

Keywords

voltage-dependent ion channels, voltage-dependent gating, KvAP channels, potassium channel

Abstract

Voltage-dependent ion channels are finely tuned to open and allow ion conduction in response to changes in the membrane voltage, a function that lies at the heart of nerve impulse generation and propagation. These channels contain transmembrane voltage-sensing domains that contain highly conserved cationic amino acids known as "gating charges." The gating charge residues move under the influence of the membrane's electric field, a voltage dependent conformational change that induces pore opening. In this thesis, I explore the structural basis of gating charge movement through crystallographic, biochemical and functional studies of KvAP, a voltage-dependent K+(Kv) channel from a thermophilic archeabacterium. I show that KvAP channels possess all the functional properties of eukaryotic Kv channels responsible for nerve impulses. This functional similarity arises from fundamental conservation of the voltage sensor structure as demonstrated by KvAP's sensitivity to tarantula toxins that bind a receptor on its voltage sensor—toxins that evolved to inhibit the voltage-dependent channels of the eukaryotic prey of these spiders. I present low-resolution crystal structure of the KvAP channel and compare it with a previous structure of the channel crystallized with monoclonal Fab fragments bound to its voltage sensors. In both structures, w e find a canonical K+ selective pore surrounded by voltage sensors that contain "voltage-sensor paddles"— hydrophobic helix-turn-helix structures located on the channel's outer perimeter. The gating charge residues are embedded in a helical segment of each voltage-sensor paddle. The crystal structures suggest that the voltage-sensor paddles are attached to the channel through flexible hinges and move as rigid units in response to membrane voltage changes, carrying their gating charges through the lipid. I used tethered biotin and avidin as a molecular ruler to measure the depth of residues in KvAP relative to the plane of the membrane and evaluate the gating motions of the voltage-sensor paddles. From this study, I conclude that the voltage-sensor paddles are a uniquely mobile part of the channel and translate the gating charge residues a -15-20 A perpendicular to the plane of the membrane to induce pore opening.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/238

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