 Wayland Chang
Research
KirBac1.1 is a
prokaryotic homolog of eukaryotic inward rectifier potassium (Kir)
channels. The crystal structure of KirBac1.1 and related KirBac3.1 have
been used extensively to generate in silico models for eukaryotic Kir
channels, but functional analysis has been limited to Rb flux experiments
and no voltage-clamp analysis has been available. I have expressed pure,
full-length, His-tagged KirBac1.1 protein in E. coli, and obtained
voltage-clamp recordings of recombinant channel activity in excised
membrane patches from giant liposomes. I have shown from these recordings
that KirBac1.1 behaves functionally as a bona fide Kir channel, opening
the way for combined biochemical, structural and electrophysiological
analysis of a tractable model Kir channel, as has been successfully
achieved for the archetypal K+ channel KcsA.
I am now functionally characterizing other recombinant channel proteins,
drawing comparisons from the KirBac1.1 structural model. This has led me
to pursue two veins of investigation.
- As part of a collaborative effort, we have
successfully expressed and purified eukaryotic Kir2.1 and Kir2.2
protein from S. cerevisia, and have shown it to be
functional. My plan is to characterize the lipid dependence of
activity for these channels by reconstitution in liposomes. In
particular, the goal of this study is to explore the molecular basis
for why PIP2 activates eukaryotic Kir channels, but
inhibits KirBac1.1.
- I have found that disruption of a conserved
H-bond located behind the selectivity filter in KcsA (E71A) leads to
significant permeation of Na and Li. I plan to continue exploring
the effect of this mutation on selectivity through functional
studies and crystallography. In KirBac1.1, I hypothesize that the
equivalent mutation should have effects on selectivity and/or inward
rectification. Ultimately, these studies will provide insights into
the structural basis of ion selectivity, inward rectification, and
lipid regulation of gating.
GRADUATE PUBLICATIONS
Cheng WW, Enkvetchakul D, Nichols CG. KirBac1.1: it's an
inward rectifying potassium channel. J Gen Physiol 2009 133(3):295-305.
PMC2654083. PMID: 19204189.
Cheng WW, Tong A, Flagg TP, Nichols CG. Random assembly
of SUR subunits in K(ATP) channel
complexes. Chanels (Austin)
2008 2(1):34-8. PMID: 18690055.
Kurata HT, Cheng WW, Arrabit C,
Slesinger PA, Nichols CG. The role of cytoplasmic pore in inward
rectification of Kir2.1 channels. J Gen Physiol 2007 130(2):145-55.
PMC2151631. PMID: 17635958.
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