Ricard Masia
Research
My work focuses on the regulation of the ATP-sensitive Potassium
(KATP) channel by the Sulfonylurea Receptor (SUR), a member
of the ATP-Binding Cassette (ABC) transporter family of proteins.
SUR mediates the stimulation of the KATP channel by MgADP,
which allows the channel to couple changes in cellular metabolism
to membrane excitability. SUR possesses two cytoplasmic domains,
Nucleotide Binding Folds 1 and 2 (NBF1 and 2), which interact
with nucleotides and provide the basis for MgADP stimulation
of the channel. There are three main aspects of SUR structure-function
that I am interested in:
Differential channel regulation by tissue-specific SUR isotypes.
By performing patch-clamp experiments in a heterologous expression
system, I have shown that the pancreatic ?-cell SUR isotype,
SUR1, results in higher MgADP stimulation of the channel than
the ventricular cardiomyocyte isotype, SUR2A. This difference
correlates with, and is a potential mechanistic explanation
for, physiological differences observed at the tissue level
between pancreatic KATP channels (dynamically active over
a wide range of metabolic conditions) and cardiac KATP channels
(essentially always closed except under severe metabolic inhibition
of the cell).
Biochemical basis of SUR-dependent MgADP stimulation. Prior
evidence indicates that ATP hydrolysis at the NBFs is essential
for MgADP stimulation of KATP channels. I have measured the
in vitro ATPase activity of isolated NBF constructs and shown
that NBF2 of SUR1 is significantly more active than NBF2 of
SUR2A, which may provide an explanation for the greater MgADP
stimulation that SUR1 confers to the channel. I am now testing
the effects of NBF ATP-binding site mutations on in vitro
ATPase activity and on MgADP stimulation of the channel, in
order to establish a correlation between the two.
Dimerization of SUR NBFs. Crystallographic studies have shown
that bacterial NBFs form dimers, with the two nucleotide binding
sites located at the dimer interface, such that they interact
with amino acid from both NBFs. This dimeric configuration
corresponds to the hydrolytically active species. In order
to examine the possibility of SUR NBF dimerization, and its
effect on KATP channel function, Decha Enkvetchakul and I
have constructed a homology model of SUR1 NBF1 and NBF2 as
a heterodimer, based on the crystal structure of the bacterial
NBF, MJ0796 (Fig. 1). In order to test the dimer interface
predicted by this model, I am examining the effects on MgADP
stimulation of introduced pairs of cysteines, one in each
NBF, expected to lead to disulfide bond formation and thus
stabilization of the hypothetical NBF dimer.
Fig. 1. Homology model of SUR1 NBF1 and NBF2 as a heterodimer.
The two ATP molecules (shown here in space fill) are located
at the dimer interface, forming contacts with amino acids
from both NBFs.
Publications:
Flagg TP, Remedi MS, Masia R, Gomes J, McLerie M, Lopatin
AN, Nichols CG.
Transgenic
overexpression of SUR1 in the heart suppresses sarcolemmal
K(ATP).
J Mol Cell Cardiol 2005
Masia R, Enkvetchakul D, Nichols CG.
Differential
nucleotide regulation of K(ATP) channels by SUR1 and SUR2A.
J Mol Cell Cardiol 2005
Loussouarn G, Phillips LR, Masia R, Rose T, Nichols CG.
Flexibility
of the Kir6.2 inward rectifier K(+) channel pore.
Proc Natl Acad Sci U S A 2001
Masia R, Aneshansley D, Nagel W, Nachman RJ, Beyenbach KW.
Voltage
clamping single cells in intact malpighian tubules of mosquitoes.
Am J Physiol Renal Physiol 2000
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