Bariloche protein symposium argentine society for biochemistry and molecular biology

35
BIOCELL, 27 (Suppl. I), 2003
S13.
INTRASTERIC REGULATION OF THE Ca
2+
TRANSPORTER FROM PLASMA MEMBRANES
Hugo P. Adamo.
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB),
Facultad de Farmacia y Bioquímica (UBA). Argentina. E-mail:
[e-mail]
The term intrasteric regulation was introduced to describe
autoinhibition of protein kinases and phosphatases by internal
sequences acting directly at the active site. it is now clear that
this powerful mechanism of control extends to diverse enzymes
including the Ca
2+
 transporter from plasma membranes (PMCA)
that is responsible of fine tuning the cytosolic Ca
2+
 concentration
in animal cells. The PMCA is a  highly regulated ion transport P-
ATPase, with a well characterized calmodulin binding-
autoinhibitory segment located at the C-terminal end of the
molecule. It has been proposed that in the apoPCMA (without
calmodulin) the autoinhibitory segment blocks the access of
substrates to the catalytic site. Calmodulin binding to a sequence
partially overlapping the autoinhibitory segment would result in
not yet well defined conformational changes that disengage the
inhibitor from the active site. We have been able to detect
calmodulin dependent changes in FRET between the blue and
green fluorescent proteins fused to both ends of the PMCA which
are indicative of important rearrangements of the protein upon
activation. however different intermediate states seem possible
as suggested by the fact that substitution of aspartate 170 by
asparagine activates the enzyme without completely separate the
autoinhibitory sequence from the catalytic core.
Supported by ANPCYT, UBA and CONICET.
S14.
REGULATION AND FUNCTIONAL ROLE OF
AQUAPORIN WATER CHANNELS IN HEPATOCYTES
Raúl A. Marinelli.
Instituto de Fisiología Experimental, IFISE (CONICET-UNR)
Rosario, Argentina. E-mail: [e-mail]
Hepatocytes express aquaporin water channels, a family of integral
proteins that increase cell membrane water permeability,
facilitating the osmotically driven movement of water. The water
channel aquaporin-8 is located primarily within the interior of
hepatocytes in a vesicular compartment (1,2). Glucagon, via the
cAMP-dependent protein kinase A signal transduction pathway,
stimulates the microtubule-mediated polarized trafficking of
aquaporin-8 vesicles to the canalicular plasma membrane domain
(i.e., the bile secretory pole of hepatocytes) (3). Water transport
studies support the corresponding hormone-induced increase of
membrane water permeability (3,4). The hepatocyte water channel
aquaporin-9 is present exclusively in sinusoidal membranes not
undergoing regulated trafficking (2). Thus, hepatocytes seem to
control their canalicular water permeability by changing the
number of aquaporin molecules. This mechanism would allow
efficient coupling of osmotically active solutes and water transport
during canalicular bile formation.
References:
1. García et al. J Biol Chem 276:12147-52, 2001.
2. Huebert et al. J Biol Chem 277:22710-17, 2002.
3. Gradilone et al. Hepatology 37:1435-41, 2003.
4. Marinelli et al. J Biol Chem 2003 (in press).
S15.
NICOTINIC RECEPTORS OF COCHLEAR AND
VESTIBULAR SENSORY SYSTEMS: FROM MOLECULAR
STRUCTURE TO FUNCTION
Ana Belén Elgoyhen.
Instituto de Investigaciones en Ingeniería Genética y Biología
Molecular, INGEBI (CONICET-UBA). Argentina. E-mail:
[e-mail]
The sensory epithelia of organs responsible for hearing (the
cochlea) and balance (vestibular labyrinth) share a unique subset
of hair cells which transduce mechanical stimuli into electrical
signals. These cells are under the influence of efferent fibers
originating in the brain, which modulate the dynamic range of
afferent fibers. Acetylcholine is the principal neurotransmitter
released by efferent axons. The existent data suggest a central
role for an atypical, nicotinic subtype of receptors (nAChRs)
located at the synapse between efferent fibers and hair cells. Over
the recent years we have cloned two novel nAChR genes involved
in hair cell physiology: 
α9 and α10. We demonstrate the existence
of two functional nAChRs: homomeric 
α9 and heteromeric α9
α10. While both α9 and α9α10 nAChRs exhibit similar
pharmacological profiles, the presence of 
α10 modifies key
biophysical characteristics of 
α9 nAChRs. Both α9 and α10
transcripts are observed in adult cochlear outer hair cells and
sensory epithelia of the otolithic organs and the semicircular canals.
However, while cochlear inner hair cells express 
α9 from
embryonic through adult stages, 
α10 transcripts are only observed
during early development, before the onset of hearing. Our results
indicate that efferent modulation of vestibular and outer hair cell
function occurs via heteromeric nAChRs assembled from both 
α9
and 
α10 subunits.
S16.
MOLECULAR BASIS OF CHANNEL GATING OF CYS-
LOOP RECEPTORS
Cecilia Bouzat.
Inst. Invest. Bioquímicas, UNS-CONICET, Bahía Blanca,
Argentina. E-mail: [e-mail]
Cys-loop receptors of the ligand-gated ion channel superfamily
play key roles in synaptic transmission. Their strategic positions
in the pathway of information flow makes them molecular targets
for drugs and neurological diseases. Neurotransmitters interact
with a ligand-binding site triggering a conformational change in
the protein that results in the opening of an ion channel. The
detailed structural mechanism of this process, which is known as
gating, remains a mystery. The cys-loop channels are pentameric
proteins composed of homologous subunits. Each subunit contains
an amino terminal extracellular domain which carries the signature
disulfide loop and the binding sites and four transmembrane
domains (M1-M4). Our goal has been to understand the molecular
arrengements underlying channel gating. To this end, we have
combined site-directed mutagenesis and chimeric subunits with
single-channel and macroscopic current recordings. We identified
residues in the less studied M1 and M3 domains as well as in the
lipid-exposed M4 domain of the nicotinic receptor (AChR) that
are involved in channel gating and described their mechanistic
contributions. Our results revealed that these domains govern
opening and closing rates in a subunit-selective manner. We also
clarified the fundamental mechanistic steps that are altered in
several myasthenic syndromes associated with mutations in the
AChR. We constructed a chimeric subunit linking the soluble ACh
binding protein (AChBP) to the channel of the 5-HT
3
 receptor.
Replacement of domains in AChBP until gating is achieved is our
current strategy to identify residues involved in channel activation.

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