Diapositiva 1

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C5a Receptor Activation GENETIC IDENTIFICATION OF
CRITICAL RESIDUES IN FOUR TRANSMEMBRANE
HELICES Thomas J. Baranski et al. THE JOURNAL OF
BIOLOGICAL CHEMISTRY Vol. 274, No. 22, Issue of
May 28, pp. 1575715765, 1999 Hormones and
sensory stimuli activate serpentine
receptors, transmembrane switches that relay
signals to heterotrimeric guanine
nucleotide-binding proteins (G proteins). To
understand the switch mechanism, we subjected 93
amino acids in transmembrane helices III, V, VI,
and VII of the human chemoattractant C5a
receptor to random saturation mutagenesis. Twenty-
one of the 121 mutant receptors exhibit
constitutive activity. Amino acids
substitutions in these activated receptors
predominate in helices III and VI other
activating mutations truncate the receptor near
the extracellular end of helix VI. These results
identify key elements of a general mechanism
for the serpentine receptor switch.
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Gai Is Not Required for Chemotaxis Mediated by
Gi-coupled Receptors Enid R. Neptune et al. THE
JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 5,
Issue of January 29, pp. 28242828,
1999 Pertussis toxin inhibits chemotaxis of
neutrophils by preventing chemoattractant
receptors from activating trimeric G proteins in
the Gi subfamily. In HEK293 cells expressing
recombinant receptors, directional
migration toward appropriate agonist ligands
requires release of free G protein bg subunits
and can be triggered by agonists for receptors
coupled to Gi but not by agonists for receptors
coupled to two other G proteins, Gs and Gq.
Because activation of any G protein
presumably releases free Gbg, we tested the
hypothesis that chemotaxis also requires
activated a subunits (Gai) of Gi
proteins. Because chemotaxis is mediated by Gbg
subunits released when Gi-coupled receptors
activate Gaqz5, but not when Gq- or Gs-coupled
receptors activate their respective G proteins,
we propose that Gi-coupled receptors transmit a
necessary chemotactic signal that is independent
of Gai.
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A PtdInsP3- and Rho GTPase-mediated positive
feedback loop regulates neutrophil polarity Orion
D. Weiner et al. NATURE CELL BIOLOGY VOL 4 JULY
2002 http//cellbio.nature.com When presented
with a gradient of chemoattractant, many
eukaryotic cells respond with polarized
accumulation of the phospholipid PtdIns(3,4,5)P3.
This lipid asymmetry is one of the earliest
readouts of polarity in neutrophils, Dictyostelium
discoideum and fibroblasts. However, the
mechanisms that regulate PtdInsP3 polarization
are not well understood. Using a cationic lipid
shuttling system, we have delivered exogenous
PtdInsP3 to neutrophils. Exogenous PtdInsP3
elicits accumulation of endogenous PtdInsP3 in a
positive feedback loop that requires endogenous
phosphatidylinositol-3-OH kinases (PI(3)Ks) and
Rho family GTPases. This feedback loop is
important for establishing PtdInsP3 polarity in
response to both chemoattractant and to exogenous
PtdInsP3 it may function through a
self-organizing pattern formation system.
Emergent properties of positive and negative
regulatory links between PtdInsP3 and Rho family
GTPases may constitute a broadly conserved module
for the establishment of cell polarity during
eukaryotic chemotaxis.
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A biosensor that uses ion-channel switches B. A.
Cornell, et al. NATURE VOL 387 5 JUNE
1997 Biosensors are molecular sensors that
combine a biological recognition mechanism with a
physical transduction technique. They provide a
new class of inexpensive, portable instrument
that permit sophisticated analytical measurements
to be undertaken rapidly at decentralized
locations1. However, the adoption of biosensors
for practical applications other than the
measurement of blood glucose is currently limited
by the expense, insensitivity and inflexibility
of the available transduction methods. Here
we describe the development of a biosensing
technique in which the conductance of a
population of molecular ion channels is switched
by the recognition event. The approach mimics
biological sensory functions2,3 and can be used
with most types of receptor, including antibodies
and nucleotides. The technique is very flexible
and even in its simplest form it is sensitive to
picomolar concentrations of proteins. The sensor
is essentially an impedance element whose
dimensions can readily be reduced to become an
integral component of a microelectronic circuit.
It may be used in awide range of applications and
in complex media, including blood. These uses
might include cell typing, the detection of large
proteins, viruses, antibodies, DNA, electrolytes,
drugs, pesticides and other low-molecular-weight
compounds.
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USE OF A CHANNEL BIOSENSOR FOR THE ASSAY OF
PARALYTIC SHELLFISH TOXINS BYEUNG SOO CHEUN et
al. Toxicon Vol. 36, No. 10, pp. 13711381,
1998 Gonyautoxin (GTX), saxitoxin (STX) and
tetrodo- toxin (TTX), also known as paralytic
shellsh poisons (PSP), block Na channels,
including those in the frog bladder membrane. A
tissue biosensor has been developed, consisting
of a Na electrode covered with a frog blad- der
membrane integrated within a flow cell. The
direction of Na transfer, investigated in the
absence of Na channel blockers, established that
active transport of Na occurs across the frogs
bladder membrane from the in- ternal to the
external face. Transfer was shown to be TTX
sensitive. The tis- sue sensor response to each
of the dierent PSP was recorded and the results
compared with toxicities determined by the
standard mouse bio-assay. Using high
concentrations of TTX from the puer sh Takifugu
niphobles, a linear correlation was found between
the results from the two assay systems. However,
the tissue biosensor system was also able to
detect very low concentrations of TTX in samples
from two species of puffer fish (Takifugu
niphobles and Takifugu pardalis) at
concentrations below the detection limit of the
mouse bio-assay.
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Anthrax Biosensor, Protective Antigen Ion
Channel Asymmetric Blockade Kelly M. Halverson et
al. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280,
NO. 40, pp. 3405634062, October 7, 2005 The
significant threat posed by biological agents
(e.g. anthrax, tetanus, botulinum, and diphtheria
toxins) requires innovative technologies and
approaches to understand the mechanisms of toxin
action and to develop better therapies. Anthrax
toxins are formed from three proteins secreted by
fully virulent Bacillus anthracis, protective
antigen (PA, 83 kDa), lethal factor (LF, 90 kDa),
and edema factor (EF, 89 kDa). Here we present
electrophysiological measurements demonstrating
that full-length LF and EF convert the
current-voltage relationship of the heptameric
PA63 ion channel from slightly nonlinear to
highly rectifying and diode-like at pH 6.6. This
effect provides a novel method for
characterizing functional toxin interactions.
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Detection of DNA via an Ion Channel Switch
Biosensor Sally Wright Lucas and Margaret M.
Harding Analytical Biochemistry 282, 7079
(2000) Detection of DNA by an ion channel switch
biosensor has been demonstrated in a model
system, using single-stranded oligonucleotide
sequences of 5284 bases in length. Two different
biotinylated probes are bound, via streptavidin,
either to the outer region of a gramicidin ion
channel dimer or to an immobilized membrane
component. The ion channels are switched off upon
detection of DNA containing complementary epitopes
to these probes, separated by a
nonbinding region, at nanomolar levels. The DNA
cross-links the ion channel to the immobilized
species, preventing ions passing through the
channel. Addition of DNase I after the target DNA
has been added switches the ion channels on.
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Recombinant maxi-K channels on transistor, a
prototype of iono-electronic interfacing Bernhard
Straub, Elisabeth Meyer, and Peter
Fromherz NATURE BIOTECHNOLOGY VOL 19 FEBRUARY
2001 http//biotech.nature.com We report on the
direct electrical interfacing of a recombinant
ion channel to a field-effect transistor on a
silicon chip. The ion current through activated
maxi-KCa channels in human embryonic kidney
(HEK293) cells gives rise to an extracellular
voltage between cell and chip that controls the
electronic sourcedrain current. A comparison
with patch-clamp recording shows that the
channels at the cell/chip interface are fully
functional and that they are significantly
accumulated there. The direct coupling
of potassium channels to a semiconductor on the
level of an individual cell is the prototype for
an ionoelectronic interface of ligand-gated or G
protein-coupled ion channels and the development
of screening biosensors with many transfected
cells on a chip with a large array of transistors.
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Figure 1. Celltransistor junction. (A) Cross
section. The silicon chip with source, drain, and
open gate area of a field-effect transistor
is insulated by silicon dioxide and coated with
collagen. The width of the cleft between HEK293
cell and chip is 4565 nm. The diameter of
the contact area is 1020 µm. (B) Equivalent
circuit. Ionic and capacitive currents through
the membrane are driven by the intracellular
voltage VM. They give rise to an extracellular
voltage profile in the cleft that functions as a
gate voltage for the current between source and
drain. The response of the transistor is
calibrated in terms of an effective voltage VJ
that is applied to the gate without a cell.
Source, bulk silicon, and drain are kept at
positive bias voltages with respect to the bath
at ground potential. The intracellular voltage is
controlled by a patch-pipette that is used to
measure the current IM through the whole cell
membrane.
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Movement of gating charge is coupled to ligand
binding in a G-protein-coupled receptor Yair
Ben-Chaim, Baron Chanda, Nathan Dascal, Francisco
Bezanilla, Itzchak Parnas Hanna Parnas NATURE
Vol 444 106-109 (2006) Activation by agonist
binding of G-protein-coupled receptors (GPCRs)
controls most signal transduction processes1.
Although these receptors span the cell membrane,
they are not considered to be voltage sensitive.
Recently it was shown that both the activity of
GPCRs25 and their affinity towards agonists6 are
regulated by membrane potential. However, it
remains unclear whether GPCRs intrinsically
respond to changes in membrane potential. Here we
show that two prototypical GPCRs, the m2 and m1
muscarinic receptors (m2R and m1R), display
charge-movement-associated currents analogous to
gating currents of voltage-gated channels. The
gating chargevoltage relationship of m2R
correlates well with the voltage dependence of
the affinity of the receptor for acetylcholine.
The loop that couples m2R and m1R to their G
protein has a crucial function in coupling
voltage sensing to agonist- binding affinity. Our
data strongly indicate that GPCRs serve as
sensors for both transmembrane potential and
external chemical signals.