Date of Award
The activity of voltage-gated cation channels underlies the action potentials that allow for neuronal signaling and muscle contraction. The canonical family of voltage-gated K+, Na+ and Ca2+ channels has been the subject of extensive electrophysiological, biophysical, genetic, biochemical and structural characterization since the 1950s. These channels all share a conserved six-transmembrane helix topology (S1-S6) in which the first four transmembrane helices (S1-S4) form the regulatory voltage-sensor domain and the last two transmembrane helices (S5 and S6) comprise the ion-conducting pore domain. It was thought that all voltage-gated cation channels shared this conserved domain architecture. However, this scheme was challenged by the discovery of the gene for the voltage-gated H+ channel. This voltage-gated cation channel has a four transmembrane helix topology that is homologous to the voltage-sensor domain of the canonical voltage-gated cation channels alone, without a separate pore domain. In this thesis, I present my work, which constitutes the first ever biochemical characterization of the human voltage-gated H+ channel (hHV1). First, I demonstrate by site-specific cross-linking that hHV1 is a dimer in the membrane and define the oligomerization interface. Then, by developing methods for the heterologous expression, purification and reconstitution of hHV1, I establish that the four transmembrane helix voltage-sensor-domain-like putative channel protein is in fact responsible for H+ conduction. Next, I present my work on the structural characterization of hHV1 by X-ray crystallography. I solved a low-resolution structure of a chimeric voltage-gated proton channel but then demonstrated that although this channel is functional in a membrane, the conformation seen in the crystal is non-native. Finally, I present my work on the analysis of hHV1 by solution state NMR in detergent micelles. This technique allowed us to define the secondary structure of the channel for the first time but full three-dimensional structural characterization was determined to be unfeasible. From these studies, I conclude that the HV channel structure is dependent on the constraints imposed by the lipid bilayer and is destabilized upon detergent solubilization. Future structural studies of HV channels will have to focus on channels imbedded within a membrane-like environment.
Letts, James Anthony, "Functional and Structural Studies of the Human Voltage-Gated Proton Channel" (2014). Student Theses and Dissertations. 177.