Bariloche protein symposium argentine society for biochemistry and molecular biology

158
BIOCELL, 27 (Suppl. I), 2003
CA-P5.
D170N SUBSTITUTION ACTIVATES THE PMCA
WITHOUT FULLY  DISENGAGING  THE  C-TERMINAL
AUTOINHIBITORY DOMAIN FROM  THE CATALYTIC
CORE
Luis M. Bredeston and Hugo P. Adamo.
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB),
Facultad de Farmacia y Bioquímica (UBA). E-mail:
[e-mail]
At low Ca
2+
 the interaction of a C-terminal inhibitory domain with
the catalytic core keeps the Plasma Membrane Calcium ATPase
(PMCA) in a basal state of low activity. Binding of Ca-Calmodulin
to the autoinhibitory domain release the active site and activates
the enzyme. We have previously reported that substitution of
aspartic 170 by asparagine results in PMCA mutant with high
Vmax and high Ca
2+
 affinity in the absence of calmodulin (SAB
2001) suggesting that the mutation disrupts the interaction between
the autoinhitory region and the catalytic core. The exposure of the
C-terminal inhibitory domain was assessed by measuring the
apparent affinity for calmodulin and the sensibility of the C-
terminal segment to proteolysis. We found that the concentration
of calmodulin for half maximal stimulation was 12.9 nM for D170N
and 16.4 nM for WT.  On the other hand both  the D170N and WT
proteins were cut by chymotrypsin at a similar rate and calmodulin
produced a similar increase in  the rate of  proteolysis. These
results suggest that in D170N the autoinhibitory region is still in
a closed conformation. Thus the activation induced by D170N may
be  bypassing the need for the complete disengagement of the
autoinhibitory region from the catalytic core.
With grants from ANPCyT, UBA and CONICET.
CA-P6.
SOME PROPERTIES OF A SOLUBLE CYTOSOLIC
FACTOR REQUIRED FOR Mg.ATP STIMULATION OF
Na
+
/Ca
2+
 EXCHANGE IN SQUID NERVE
Centanni C, Roberts G, Dipolo R, Berberián G, and Beaugé L.
Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra,
Córdoba, Argentina. E-mail: [e-mail]
Squid giant axons subjected to prolonged dialysis and isolated
nerve membrane vesicles (Loligo pealei) need a soluble cytosolic
protein (SCP, Mr around 13 kDa), for MgATP stimulation of the
Na/Ca exchanger. In this work we optimised the purification of
that protein to have enough material for its characterization. We
used two species of squid: (i) Loligo pealei and  (ii) Illex argentinus.
The starting material was the 400.000 g supernatant of the optic
ganglia. The activity was recovered in a 30 kDa filtrated and was
retained in 10 kDa filter. Proteins isolated from both squids
promoted Mg.ATP stimulation of Na/Ca exchange in nerve vesicles
from Loligo pealei. The activity remained in the flow-through of
an anionic resin (1x8-400 Dowex) and was retained in a cationic
resin (50x 8-400 Dowex). The 13 kDa protein from both species
was phosphorylated by [32-P]-
γ-ATP when they were incubated
in the presence of active membrane nerve vesicles in conditions
for Mg.ATP stimulation of Na/Ca exchange (1 uM Ca
2+
, 3 mM
Mg
2+
 and 0.2 mM vanadate). The protein from Loligo was isolated
and sequenced by mass spectrometry. Peptide analysis indicates
it is a de novo protein.
Supported by FONCYT-Argentina (PICT99 05-05158) and Agencia
Córdoba Ciencia (181/01).
CA-P7.
IDENTIFICATION OF RESIDUES IN THE M1 DOMAIN
OF THE NICOTINIC RECEPTOR THAT CONTRIBUTE TO
CHANNEL GATING
Jeremías Corradi, Guillermo Spitzmaul and Cecilia Bouzat.
Instituto de Investigaciones Bioquímicas. UNS/CONICET. 8000
Bahía Blanca. Argentina. E-mail: [e-mail]
The nicotinic receptor (AChR) is a pentamer of homologous
subunits with an 
α
2
βεδ composition in adult muscle. Each subunit
contains four transmembrane domains (M1-M4). We have
combined site directed mutagenesis and construction of chimeric
subunits with expression in cells and single-channel recordings
to study the contribution of M1 to AChR activation. This segment
shows highly conserved residues (positions 1’, 2’ and 14’) or
differentially conserved residues between 
α and non-α subunits
(positions 3’, 4’, 13’ and 15´). Our results showed that positions
3’ and 14’ slightly affect single channel kinetics but positions 1’,
2’, 4’ and 13’ are not involved in channel gating.  In contrast,
mutations at position 15´ significantly increase the rate of channel
opening and decrease the closing rate. When the entire 
αM1
domain is replaced by that of the neuronal 
α7 AChR profound
kinetic changes are observed. The duration of channel openings
increase 10-fold, openings appear in clusters at low ACh
concentrations, and the dissociation constant is reduced about 3-
fold. Our results demonstrate that M1 segment contributes to
channel gating and that the degree of conservation of residues is
not strictly correlated with their functional roles.
CA-P8.
CONFORMATIONAL CHANGES OCURRING DURING
THE ACTIVATION OF THE PMCA BY CALMODULIN.
INTRAMOLECULAR FRET IN A BFP-PMCA-GFP FUSION
CONSTRUCT
Gerardo R. Corradi and Hugo P. Adamo.
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB),
Facultad de Farmacia y Bioquímica (UBA). E-mail:
[e-mail]
A BFP-PMCA-GFP fusion protein suitable for intramolecular
FRET measurements has been constructed by adding the blue
(BFP) and green (GFP) fluorescent proteins at the N and C
terminus of the plasma membrane Ca
2+
 transporter (PMCA). The
recombinant protein purified from S. cerevisiae was a functional
PMCA. When the purified protein was suspended in a media
containing 500 
µM EGTA and excited at 387 nm (BFP excitation
maxima) the BFP-PMCA-GFP exhibited a major emission peak
at 409 nm corresponding to BFP and a minor one at 509 nm due to
GFP emission by FRET (Ida/Ia 0.2). Ca
2+ 
did not significantly affect
FRET. The removal of the fluorescent proteins from the PMCA by
trypsin cleavage lead to a progressive disappearance of the peak
at 509 nm (Ida/Ia 0.16). A similar drop in FRET was observed
after addition of Ca-CaM. Adding enough EGTA to reduce the
Ca
2+
 
to less than 0.01 
µM recovered Ida/Ia to a value close to that
observed in the absence of CaM. These results are indicative of a
conformational change occurring upon calmodulin binding and
activation of the PMCA and suggest that the C terminal
autoinhibitory region is moving apart or reorienting with respect
to the N terminus which is part of the catalytic core of the enzyme.

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