Bariloche protein symposium argentine society for biochemistry and molecular biology

160
BIOCELL, 27 (Suppl. I), 2003
CA-P13.
DESENSITIZATION OF ACETYLCHOLINE RECEPTOR
BY THE LOCAL ANESTHETIC PROADIFEN
Guillermo Spitzmaul, Fernanda Gumilar, James Dilger and Cecilia
Bouzat.
Instituto de Investigaciones Bioquímicas. UNS/CONICET. 8000
Bahía Blanca. Argentina. E-mail: [e-mail]
The nicotinic receptor (AChR) of adult muscle is composed of
five subunits in the order 
α
2
βεδ. Non competitive inhibitors (NCI)
decrease the probability of channel opening by different
mechanisms. The binding site for proadifen has been located at
the extracellular end of the M2 domain. To elucidate the
mechanism of action of proadifen on the AChR we made single-
channel and macroscopic current recordings. Single-channel
recordings reveal a decrease in the frequency of openings events
without significant changes in the mean open time of the channel.
This observation is compatible with an increase in desensitization
rate from the open or closed state. At high ACh concentrations
clusters of openings in the presence of proadifen were similar to
those of the control, suggesting that proadifen is affecting the closed
state. We also studied macroscopic currents activated by rapid
application of ACh. Proadifen decreases the peak current without
changing the decay rate due to desensitization. Preincubation with
proadifen is necessary for its maximal pharmacological  effect.
Our results reveal that proadifen desensitizes AChRs that are in
the closed state and neither affects open AChRs nor acts as an
open-channel blocker.
CA-P14.
IDENTIFICATION OF KEY FUNCTIONAL DOMAINS IN
CYS-LOOP RECEPTORS
Gumilar F, Rayes D, Sine S, and Bouzat C.
Inst. Invest Bioq., UNS-CONICET. B. Bca, Argentina. E-mail:
[e-mail]
The AChR, 5-HT
3
 and GABA
A
 receptors are members of the  cys-
loop superfamily of ligand-gated ion channels that  mediate rapid
synaptic transmission throughout the nervous system.
Neurotransmitters interact with a ligand-binding site in these
channels triggering a conformational change in the protein that
results in the opening of an ion channel. The structure of an
homopentameric soluble protein (AChBP) from Lymnaea
stagnalis, which is secreted by snail glial cells into cholinergic
synapses,  has been recently described at a high resolution. AChBP
binds agonists and competitive antagonists of the AChR and its
sequence is 20-24% identical to aligned sequences of the amino-
terminal, extracellular halves of AChR subunits. To ascertain
whether AChBP exhibits the potential to function as the
extracellular region of a LGIC, and to identify extracellular
domains involved in coupling the recognition of agonist to the
opening of the channel, we constructed a chimeric AChBP-5HT
3
subunit and expressed it in mammalian cells. The chimeric receptor
shows high expression and the same pharmacological profile as
that of the soluble AChBP, indicating that the overall structure of
the binding domain is conserved. However, results from
macroscopic current recordings show that channel opening is not
efficient in this receptor. Exchanging the cys-loop of AChBP by
that of 5-HT
3 
leads to the lack of expression, suggesting that this
domain is essential for appropriate protein folding. The
replacement of domains in AChBP until gating is achieved is our
current strategy to identify residues involved in channel activation.
CA-P15.
AChR DISTRIBUTION AFTER CERAMIDE TREATMENT
OF CHO-K1/A5 CELLS
Pediconi MF, Gallegos CE, de los Santos EB, Cutini P, and
Barrantes FJ.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca. E-mail: [e-mail]
With the aim to study the mechanisms involved in the regulation
of acetylcholine receptor (AChR) targeting to the plasma
membrane, we studied the effect promoted by short (C6)- or long
brain ceramides on AChR expression, metabolism, and cellular
distribution. C6-Cer reduced by 30-80% the amount of [
125
I]
α-
BTX AChR detected at the cell surface of CHO-K1/A5 cells.  This
decrease was a dose- and time-dependent effect, augmenting also
the ligand affinity for [
125
I] 
α-BTX.  The half-life of surface AChR
was 5.7 and 8.3 h for control and treated cells, respectively. C6-
Cer produced intracellular accumulation of AChR (72
±15% vs.
49
±3 in control cells) without apparent cell damage. Fluorescence
microscopy using Alexa
594
α-BTX labeling showed that brain-Cer
and C6-cer decreased the amount of AChR localized at the
plasmalemma. Whereas C6-Cer induced intracellular AChR
accumulation in a vesicle-like compartment, brain Cer increased
the amount of intracellular AChR with a more homogeneous
distribution.
Work supported in part by grants from UNS and FONCYT.
CA-P16.
MUTATION OF CONSERVED RESIDUES IN THE M2
DOMAIN YIELDS A 
α9α10 NICOTINIC RECEPTOR WITH
A GAIN OF FUNCTION
Plazas PV
1
, Katz E
2
 and Elgoyhen AB
1
1
INGEBI, UBA-CONICET, Buenos Aires, Argentina, 
2
Depto. de
Biología, FCEyN, UBA, Buenos Aires, Argentina. E-mail:
[e-mail]
Nicotinic acetylcholine receptors (nAChRs) form part of a gene
superfamily, which includes GABA
A
, GABA
c
, serotonin type 3
and glycine receptors. The putative channel-forming M2 domains
of these receptors contain two highly conserved residues: a leucine
(L9´) and a valine (V13´), which are postulated to form a
constricting hydrophobic girdle in the middle of the ion pathway.
The aim of the present work was to study the role of these residues
in the 
α9α10 nAChR function. cDNAs for rat α9 and α10 subunits,
where the amino acids L9´ or V13´ were mutated to threonine (T),
were expressed in Xenopus laevis oocytes and agonist-evoked
currents were measured under two-electrode voltage-clamp. When
compared to wild type receptors, ACh-evoked currents through
α9α10(L9´T) and α9α10(V13´T) nAChRs exhibited much lower
desensitization kinetics and an increase in their apparent affinity
for this agonist. Choline, a weak partial agonist of the wild type
receptor, behaved as a full agonist of the mutant receptors.
Furthermore, nicotine, muscarine and ICS-205 930, antagonists
of the wild type receptor, elicited ionic currents in oocytes
expressing these mutants. A constitutive activation of a fraction
of mutant receptors was observed, even in the absence of the
agonist. Our results suggest that the sites where these two residues
are located are structurally critical for opening-closing transitions
of the 
α9α10 nAChR.
Supported by ANPCyT and HHMI.

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