Structural and Functional Studies of SLO K+ Channels: Mechanisms of Gating by Intracellular Signaling

<p>Eukaryotic K⁺ channels from the SLO family (SLO1, SLO2 and SLO3) provide a link between intracellular signaling and the electrical activity of a cell. The opening and closing (gating) of the three different SLO homologs is controlled by the synergistic action of membrane voltage and specific intracellular cues: Ca²⁺ binding in SLO1, Na⁺ binding in SLO2 and pH increase in SLO3. It is known that intracellular signals activate SLO channels by acting on the large cytoplasmic domains (CTDs) of these proteins, which follows the transmembrane ionconduction pore. However, a molecular description of the mechanisms of intracellular gating in SLO channels is still lacking. In this thesis, I present biochemical, structural and functional studies aiming at understanding how the activity of SLO1 and SLO3 channels is controlled by intracellular Ca²⁺ binding and pH increase, respectively. First, I describe recombinant methods for the large-scale expression and purification of functional SLO channels, paving the way for a more complete biochemical and structural analysis of these proteins. Then, I report the crystal structures of the large cytoplasmic domains (CTDs) from two different SLO1 channels. Structures of the Ca²⁺-bound CTDs from human and zebrafish SLO1 channels define the precise molecular architecture of SLO1's Ca²⁺-sensing module: CTDs from the four subunits of a tetrameric SLO1 channel assemble in a so-called gating ring structure at the intracellular face of the membrane. In conjunction with other studies, these results describe how Ca²⁺ binding affects the conformation of one layer of the SLO1 gating ring, which can explain the Ca²⁺-driven opening of SLO1's ion conduction pore. Next, I present the crystal structure of the human SLO3 gating ring. A comparison with the SLO1 structures suggests that the hSLO3 structure represents the open conformation of the hSLO3 gating ring. Finally, I describe functional mutagenesis studies on the mouse SLO3 ortholog, which reveal a possible mechanism for pH sensing in the mouse SLO3 channel. Surprisingly, the mechanism I propose appears not to be conserved in SLO3 channels from other species. This could be a dramatic example of how new functional mechanisms can easily evolve within the very versatile scaffold of a gating ring structure. Altogether, the results presented in this thesis provide a molecular framework to understand the mechanisms of intracellular gating in SLO channels.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

2012

Author dc:creator

• Leonetti, Manuel D

Contributors dc:contributor

• Roderick MacKinnon

• SLO channels
• intracellular gating
• Ca2+ sensing
• pH regulation
• gating ring structure
• structural biology
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/240

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1244