Bariloche protein symposium argentine society for biochemistry and molecular biology

159
BIOCELL, 27 (Suppl. I), 2003
CA-P9.
A NEW INSIGHT INTO THE ATP HYDROLYSIS CYCLE
OF THE PLASMA MEMBRANE CALCIUM PUMP (PMCA)
Echarte M.M., Rossi R.C., Rossi J.P.F.C.
IQUIFIB. Facultad de Farmacia y Bioquímica, Junín 956. Buenos
Aires, Argentina. E-mail: [e-mail]
We have developed a microscale method to quantify phosphorylated
intermediates of PMCA, isolating it by SDS-PAGE (Echarte et al.
2001). Applying this technique, we have shown that EP steady-
state level at 4ºC depends on [ATP
] concentration in a complex
way. We also studied the effect of Mg
2+
 measuring EP with 1 mM
or 8 
µM Mg
2+
, and observed that EP dependence on [ATP
] can be
described by a third degree polynomial quotient. These results
suggest the participation of ATP in 3 steps of the reaction cycle, as
proposed by Rega and Garrahan (1986).
We have measured the Ca
2+
-ATPase activity (v), under the same
conditions that we achieved phosphorylation experiments, and
calculated EP turnover (kpEP) as the ratio v/EP. We observed that
when [Mg
2+
] is saturating (1 mM), kpEP increases hyperbolically
with [ATP
]. When [Mg
2+
] is limiting (8 µM), kpEP as a function
of [ATP
] follows a complex behavior that cannot be explained by
the accepted mechanism for ATP hydrolysis. We measured the rate
of EP desphosphorylation in the presence of both 8 
µM and 1 mM
Mg
2+
, and with 0.010 or 1 mM ATP. According to these results we
proposed a model for the reaction cycle, which considers that ATP
accelerates the conformational transition E
1
P 
→  E
2
P, through a
new reaction intermediate E
1
P.ATP. This model also explains the
behavior of the pump at 25ºC.
CA-P10.
RELATIONSHIP BETWEEN THE PRESENCE OF 
α7
NICOTINIC RECEPTOR AND APOPTOSIS OF HUMAN
LYMPHOCYTES
Esandi MC, De Rosa MJ, Garelli A, Rayes D, and Bouzat CB.
Instituto de Investigaciones Bioquímicas, UNS-CONICET, Bahía
Blanca, Argentina. E-mail: [e-mail]
Acetylcholine (ach) is well known as a neurotransmitter in both
the central and peripheral nervous systems. Ach receptors (achr)
have been recently identified in extraneuronal cells such as those
of the immune system. By using rt-pcr we have detected mrna of
α
αα
α
α7 nicotinic receptor (achr) in peripheral human lymphocytes after
incubation with different concentrations of nicotine (10-100 ì m).
To determine the role of 
α
αα
α
α7 in lymphocytes,  we measured the
degree of apoptosis in the presence of different nicotinic ligands.
The degree of apoptosis was determined by dna cleavage forming
a ladder pattern and by evaluating nuclear condensation with dapi
staining. Lymphocytes pretreated with nicotine (10-100 ì m) were
less susceptible to cortisol-induced apoptosis compared to
untreated cells. The inhibition of apoptosis was concentration-
dependent, reaching a value of 50% at 100 µm nicotine. Correlation
between the presence of 
α
αα
α
α7 mrna and protection from apoptosis
was always observed. Furthermore, apoptosis protection was also
observed in the presence of the antagonist 
α
αα
α
α-bungarotoxin. This
effect might be due either to the ability of both, the antagonist and
nicotine, to induce upregulation or to desensitize and block achrs.
Given that lymphocytes secrete ach, our results suggest that these
cells contain functional 
α
αα
α
α7 receptors.
Supported by ANPCYT, UNS, Ministerio de Salud, Fundación
Antorchas and TWASCA-P11.
CA-P11.
INHIBITION OF MUSCLE ACETYLCHOLINE
RECEPTOR BY THE ANTIEPILEPTIC DRUG
LAMOTRIGINE
Vallés AS, Garbus I, and Barrantes FJ.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca. E-mail: [e-mail]
Some forms of autosomal dominant nocturnal frontal lobe epilepsy
(ADNFLE) have been found to be associated with genes coding
for the 
α
4
β
2 
 neuronal acetylcholine receptor (AChR). Lamotrigine
(LTG) is a triazine compound chemically unrelated to other
antiepileptic drugs. Its major mechanism of action is blocking the
voltage-dependent sodium-channel conductance. Here we
characterized the effect of LTG on the muscle AChR heterologously
expressed in CHOK1/A5 cells by recording the single-channel
activity of the receptor using the patch-clamp technique. LTG was
found to alter AChR channel kinetics. Exposure to the drug 1)
diminished the channel amplitude; 2) caused shortening of the
channel open state; 3) increased burst duration, 4) increased the
duration and area of the briefest component of the channel closed
state. In the concentration range tested (50-150 
µM), the effects
did not depend on the dose. We propose that LTG blocks the AChR
allowing it to reopen quickly, through a mechanism that is
compatible with that of open channel blockers.
Work supported in part by grants from UNS and FONCYT.
CA-P12.
MODULATION OF NICOTINIC ACETYLCHOLINE
RECEPTOR SINGLE-CHANNEL BEHAVIOR UPON
MODIFICATION OF MEMBRANE CHOLESTEROL
Garbus I, White MM, and Barrantes FJ.
UNESCO Chair Biophys. & Molec. Neurobiol./INIBIBB,
B8000FWB Bahía Blanca, Argentina, and Drexell University
College of Medicine, Dept. of Pharmacology & Physiology, 19102-
1192 Philadelphia, U.S.A. E-mail: [e-mail]
The ability of the nicotinic acetylcholine receptor (achr) to conduct
cations across the membrane and undergo conformational changes
is highly dependent on the presence of cholesterol (chol). We
studied the effect of membrane chol enrichment or depletion on
single-channel kinetics of adult muscle-type achr in cho-k1/a5 cells
treated with methyl-
  β
  β
  β
  β
  β-cyclodextrin (cdx) and chol-cdx,
respectively. Chol depletion caused a lengthening of the achr
channel mean open time, whereas its enrichment diminished this
kinetic parameter. These changes were accompanied by significant
changes in the open probability of the channel, whereas the burst
duration and the number of events per burst remained unchanged.
No modifications of channel amplitude were observed. Under
hyperpolarizing conditions, the effect of chol depletion was no
longer observed. A plausible explanation of these results is that
regulation is exerted on endogenous chol at specific sites
presumably located at the achr-lipid interface.
Work supported in part by grants from UNS, FONCYT, AND FIRCA
1-RO3-TW01225-01 (NIH).

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