Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Colin Nichols

Abstract

Inward rectifying potassium: Kir) channels are important in regulating cellular excitability in organs such as the heart, brain and pancreas. Prokaryotic KirBac channels are structurally similar to eukaryotic Kir channels, but distantly related and of unknown function. The goal of this thesis has been to investigate the functional properties of KirBac1.1 and to relate these findings to eukaryotic Kir channels. The approach was to use recombinantly-expressed, purified K+ channels--KirBac1.1, KcsA, Kir2.1 and Kir2.2--in order to integrate findings from functional studies, using liposomal flux assays and patch-clamping, with high-resolution crystal structures. By reconstituting KirBac1.1 into giant liposomes for patch-clamping, I show that this channel is an inward rectifier, sensitive to spermine block when a pore-lining residue, 138, is mutated to an aspartate. However, unlike eukaryotic Kir channels, KirBac1.1 is inhibited by PIP2 and single channel currents exhibit multiple conductance states. These findings inform the use of KirBac1.1 as a structural model for eukaryotic Kir channels. A general feature of Kir channels is that K+-selectivity is dependent on the integrity of a highly conserved salt bridge behind the selectivity filter. To explore the role of this interaction further, I examined the prokaryotic K+ channel, KcsA, and found that the E71A mutation, which disrupts an equivalent molecular interaction, reduces K+-selectivity by maintaining a conductive selectivity filter in the presence of Na+ and absence of K+. By reconstituting purified KirBac1.1 and human Kir2.1 and Kir2.2 proteins into liposomes, I definitively and quantitatively show that PIP2 directly regulates human in addition to bacterial Kir channels. These findings demonstrate an absolute requirement of Kir2.1 and Kir2.2 activity for PIP2 as well as a non-specific secondary requirement for anionic phospholipids. The secondary requirement represents a distinct mode of lipid regulation implying that phosphatidylinositol phosphates: PIPs) can have dual regulatory effects on channel activity. Comparing functional properties of bacterial and human inward rectifying potassium channels not only informs the structural basis of these properties but raises new hypotheses about how these properties may have evolved.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7GX48KB

Download

Share

COinS