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Iodide Channel
Golstein
Iodide channel of the thyroid: reconstitution of iodide conductance in proteoliposomes.
Golstein PE, Sener A, Colin F, Beauwens R.
Methods Enzymol. 1999;294:304-19. Review.
[citation only]
Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein.
Golstein PE, Sener A, Beauwens R.
Biochem J. 1995 Dec 1;312 ( Pt 2):543-8.
"A sensitive assay is
developed to assess the existence of an iodide channel in a fraction
of solubilized membrane proteins. This step is critical when
considering various procedures for purification of this channel.
Sodium cholate is used as a detergent as it does not denature the
iodide channel. A simple and rapid method involving gel-filtration
chromatography is used simultaneously to remove the detergent and to
adjust the buffer composition, before protein insertion into
liposomes. The presence of an iodide channel is investigated by
measuring the iodide conductance of these proteoliposomes at 4 degrees
C. An outward iodide gradient is set up across the proteoliposomal
membrane by anion-exchange chromatography, allowing uptake of
radiolabelled iodide. This uptake is conductive as it is abolished by
valinomycin in the presence of potassium. It is specifically mediated
by a thyroid plasma-membrane protein inserted into liposomes, as its
denaturation before insertion totally abolished uptake. It was
observed only within a well-defined fraction of thyroid membrane
proteins collected by size-exclusion chromatography (molecular mass
between 100 and 200 kDa). Furthermore, it was not observed with other
membrane proteins such as ileal brush-border-membrane proteins or
bacteriorhodopsin. Like many anion channels, this conductance was also
inhibited by N-phenylanthranilic acid. Optimization of the assay is
described, validating the measurement of conductive iodide uptake at
30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of
protein to 90 micrograms of lipid, with an outward iodide gradient (KI
15 mM inside and 1 microM outside). This assay provides a test of the
biological activity of the iodide channel at each step of the
purification; it can be applied to any anionic channel."
The iodide channel of the thyroid. II. Selective iodide conductance inserted into liposomes.
Golstein PE, Sener A, Beauwens R.
Am J Physiol. 1995 Jan;268(1 Pt 1):C111-8.
[abstract only]
"An iodide channel has been
previously identified in the plasma membrane of bovine throcytes
[Golstein et al., Am. J. Physiol. 263 (Cell Physiol. 32): C590-C597,
1992]. The plasma membrane proteins were solubilized and ultrafiltered,
and the protein fraction collected above 100 kDa was inserted in
liposomes. Voltage-sensitive uptake of radiolabeled I- by these
proteoliposomes was studied. To this end, an outward I- gradient was
set up by loading the proteoliposomes with KI and removing
extraliposomal I-. I- exit from the proteoliposome induces an inside
positive membrane potential, which leads to the uptake of 125I- added
to the incubation medium. This uptake was abolished by valinomycin,
which in the presence of K+ short circuits the liposomal membrane
potential, demonstrating the conductive nature of this uptake. A
double reciprocal plot of I- influx over I- concentration suggests the
existence of a single population of channels in these proteoliposomes
with a Michaelis-Menten constant for I- of approximately 9 microM.
When the proteoliposomes were loaded with KCl or KSCN instead of I-,
no conductive uptake occurred anymore, suggesting that these anions
are unable to diffuse through the I- conductance, hence do not
generate a diffusion potential. I- uptake by KI-loaded proteoliposomes
was not inhibited in the presence of a 1,000-fold excess of
extraliposomal Cl- but was completely inhibited by a 1,000-fold excess
of extraliposomal SCN-, indicating that Cl- does not permeate the I-
channel, whereas SCN- inhibits it. SCN- and flufenamate were both
shown to be competitive inhibitors of the I- channel with an inhibitor
constant of approximately 10 and 750 microM, respectively. (ABSTRACT
TRUNCATED AT 250 WORDS)"
The iodide channel of the thyroid: a plasma membrane vesicle study.
Golstein P, Abramow M, Dumont JE, Beauwens R.
Am J Physiol. 1992 Sep;263(3 Pt 1):C590-7.
[abstract only]
"The uptake of radioactive
iodide or chloride by plasma membrane vesicles of bovine thyroid was
studied by a rapid filtration technique. A Na(+)-I- cotransport was
demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4
degrees C and in the absence of Na+), an uptake of iodide above
chemical equilibrium could be induced, driven by the membrane
potential. The latter was set up by allowing potassium to diffuse into
the membrane vesicles in the presence of valinomycin and of an inward
K+ gradient. This potential difference (positive inside) induced the
uptake of iodide (or other anion present). The data support the
existence of two anionic channels. The first one, observed at low
near-physiological iodide concentration (micromolar range), which
exhibits a high permeability and specificity for iodide (hence called
the iodide channel), has a Km of 70 microM. The other one appears
similar to the epithelial anion channel as described by Landry et al.
(J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more
permeable to iodide than to chloride and presents a Km of 33 mM. Under
physiological conditions the latter channel would mediate chloride
transport, and the iodide channel, which is proposed to be restricted
to the apical plasma membrane domain of the thyrocyte, transports
iodide from the cytosol to the colloid space."