Harley Takatsuna Kurata
Harley Kurata's Curriculum Vitae
My studies in the lab revolve around two main objectives.
The first is to determine the detailed molecular mechanism
of polyamine block in inwardly-rectifying K+ channels. In
this vein, my most recent projects have involved ‘blocker
protection’ assays, in which I have characterized the
effects of pore occupancy by spermine (or other various polyamine
analogues) on the rate of modification of specific introduced
cysteine residues by MTSEA. Based on the results of these
experiments, and previously published work from the laboratory,
we have proposed that spermine binds at a deep location in
the Kir pore, between the ‘rectification controller’
residue (equivalent to D172 in Kir2.1) and the selectivity
filter, and possibly even within the selectivity filter of
the channel (see Figure 1). A second set of experiments involves
the characterization of the role of negatively charged residues
in the inner cavity of Kir2.1 in regulating the kinetic and
equilibrium properties of channel blockade by various polyamines.
Crystallization of the cytoplasmic domains of Kir2.1 has led
to the identification of several acidic residues in the pore
of this channel that can dramatically alter the kinetics of
spermine block, and we are carefully examining the molecular
basis for this effect.
Figure 1.
The second general theme in my experiments is the characterization
of the gating mechanisms of the ATP-dependent K+ channel (KATP).
Through a glutamate scan of the Kir6.2 pore, I have identified
several mutant channels that exhibit voltage-dependent gating
(see Figure 2). This contrasts with WT Kir6.2 channels, in
which the macroscopic conductance is essentially independent
of voltage. Interestingly, in these voltage-dependent Kir6.2
mutants, voltage-dependent gating allosterically influences
gating of the channel by its physiological ligands ATP and
PIP2. For example, voltage-dependent opening of the channel
mimics the effects of PIP2 application – reducing the
apparent sensitivity of the channel to ATP. We are using these
voltage-dependent Kir6.2 mutants to gain insights into the
mechanisms of regulation of the KATP channel complex.
Figure2.
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