Bariloche protein symposium argentine society for biochemistry and molecular biology

161
BIOCELL, 27 (Suppl. I), 2003
CA-P17.
MECHANISM BY WHICH p-CHLOROMERCURIPHENYL
SULPHONIC ACID  BLOCKS ATP  STIMULATION  OF
MAMMALIAN HEART Na
+
/Ca
2+ 
EXCHANGE
Posada V, Beaugé L, and Berberián G.
IMMF-INIMEC, Córdoba, Argentina.
E-mail:  [e-mail]
The Na
+
/Ca
2+
 exchanger of mammalian heart (NCX1) is activated
by ATP through an increase of the phosphatidylinositol-4,5-
biphosphate (PtdIns-4,5-P
2
) bound to the exchanger. (Asteggiano
et al., Eur. J. Biochem., 268: 437-442, 2001). A preliminary report
(V. Posada, et. al., SAB-2002) indicated that p-
chloromercuriphenyl sulphonic acid (PCMBS) blocks ATP effect
on the NCX1. This result could be due to a direct effect of PCMBS
on the PtdIns-4,5-P
2
 binding to NCX1, on the synthesis of
phosphoinositides or both. In order to test these possibilities we
studied the effect of PCMBS on the novo synthesis of PtdIns-4-P
and PtdIns-4,5-P
2
 in plasma membrane vesicles of bovine heart
using [32P]-
γ- ATP as substrate. The results showed that 0.1 mM
PCMBS (15 min preincubation) under conditions that blocked ATP
stimulation (1 uM Ca
2+
, 3 mM Mg
2+
, 0.3 mM Vanadate),
significantly reduced the net production of PtdIns-4-P
 
and abolished
the net production of PtdIns-4,5-P
2
. An important additional
observation was that exogenous  PtdIns-4,5-P
2
 stimulated the Na
+
-
gradient-dependent Ca
2+
 uptake in PCMBS pretreated vesicles.
This PtdIns-4,5-P
2
 stimulation was similar to that seen with ATP
in vesicles without PCMBS. The results suggest that the effect of
PCMBS on the ATP activation of NCX1 follows an impairment in
the pathway/s involved in PtdIns-4-P and PtdIns-4,5-P
2
 production.
Supported by FONCYT-Argentina (PICT99 05-05158) and Agencia
Córdoba Ciencia (181/01).
CA-P18.
PHOSPHOARGININE UP REGULATION OF Na
+
/Ca
2+
EXCHANGE IN SQUID OPTIC NERVE VESCICLES
Raimunda D, Roberts G, Dipolo R, Beaugé L, and Berberián G.
Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra,
Córdoba, Argentina. E-mail: [e-mail]
Phosphoarginine (PA) is a phosphagen present in the cytosol of
invertebrate cells. We had demonstrated that millimolar [PA]
stimulates Na
+
/Ca
2+
 exchange in squid axon following a metabolic
pathway different from that of ATP [DiPolo, R. and Beaugé, L.,
Biochim. Biophys. Acta, 1422: 57-71, 1999]. In order to further
characterize this PA stimulation we used vesicles of squid optic
nerve performing, in parallel, transport and phosphorylation of
membrane proteins. The results show that: (i) PA stimulates a
Na
+
-gradient-dependent Ca
2+
 uptake, even in absence of the
cytosolic protein required for ATP stimulation of the Na
+
/Ca
2+
exchanger; (ii) the apparent K
m 
for PA is in the millimolar range
(about 4 mM), similar to that observed in dialyzed squid axons (7
mM); (iii) the effects produced by PA and ATP are additives; (iv)
the pattern of [
32
P]PA phosphorylation, analysed in SDS-PAGE,
was different from that see with [
32
P]ATP. Two phosphorylated
bands, that were exclusives of PA, were sequenced. The results
showed that they correspond to the NF 60 and NF 70 kDa subunits
of the neurofilament protein.
Supported by FONCYT-Argentina (PICT99 05-05158) and Agencia
Córdoba Ciencia (181/01).
CA-P19.
MUTATIONS IN THE CHRNA7 GENE IN A FAMILY WITH
JUVENILE MYOCLONIC EPILEPSY
Rauschemberger MB, Vecchi C, and  Barrantes FJ.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca, Argentina. E-mail: [e-mail]
Juvenile Myoclonic Epilepsy (JME) is an idiopathic generalized
epilepsy that affects up to 26% of all individuals with idiopathic
generalized epilepsy. Approximately a third of JME patients have
a positive family history of epilepsy. Strong evidence for linkage
was found to polymorphic loci encompassing the region containing
the gene that encodes the neuronal AChR
 α7 subunit, which maps
to 15q14. Elmslie et al. (1997) suggested that this major locus
(EJM2) contributes to genetic susceptibility to JME in the majority
of the families studied, but no precise localization was achieved.
We studied a family with clinically diagnosed JME, and found  a
mutation at position 269 of the neuronal
 α7 AChR subunit. This
corresponds to the M2 transmembrane segment, lining the walls
of the AChR channel. The mutation consisted of the replacement
of codon TTA by the codon ACA, resulting in the substitution
Leu
269
Thr.
Work supported in part by grants from UNS and FONCYT.
CA-P20.
EFFECT OF THE ANTHELMINTIC AGENT LEVAMISOLE
ON MAMMALIAN NICOTINIC RECEPTORS
Rayes D, De Rosa MJ, Bartos M, and Bouzat C.
Instituto de Investigaciones Bioquímicas, UNS-CONICET, Bahía
Blanca, Argentina. E-mail: [e-mail]
Levamisole is an anthelmintic agent that exerts its therapeutic
effects by acting as a full agonist of nicotinic receptors (AChR) of
muscle nematodes. We explore at the single-channel and
macroscopic-current levels the action of levamisole on mammalian
muscle AChR. Levamisole is capable of activating mammalian
AChRs. However, single-channel openings do not appear in clearly
identifiable clusters at high concentrations. In addition, levamisole
activation is not enough to elicit macroscopic currents.  Both
findings suggest that levamisole acts as a weak agonist of
mammalian AChRs. In the presence of levamisole the channel
mean open time decreases as a function of concentration (7-fold
at 100 µM), indicating an additional open-channel blockade (Kd
140 µM at –70mV). Sequence alignment of mammalian and
nematode AChR shows several non conserved residues which
might be candidates for the differential activation by levamisole.
The replacement of the conserved glycine at position 153 in the 
α
subunit by its homologous in the parasite (
αG153E) allows
levamisole to produce identifiable clusters, thus making
mammalian AChRs more sensitive to levamisole activation. We
show that levamisole is a weak agonist and an open-channel blocker
of mamalian AChRs and that the glutamic acid at position 153 is
important for the behavior of the anthelmintic on the parasite
AChR.

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