Whole Cell experiments in CaCo2 cells' Currents stimulated by cAMP IBMX' Almost totally inhibited by

1
WHAT IS THE ROLE OF ATP HIDROLYSIS ON THE KINETIC
MECHANISM OF CFTR?
The protein encoded by the CFTR gene is known to
be a chloride channel, whose dysfunction is
associated with the development of cystic
fibrosis. The mechanism by which the CFTR
promotes the transport of chloride is not yet
completely understood as an example, it is not
known the way by which ATP hydrolysis in the NBD
domain regulates the CFTR activity. The different
kinetic models for the proteins activity are
distinguishable in those that propose distinct
functions for the two NBDs and those that assume
the same function for both NBDs. In the present
work, three kinetic models for CFTR were
analysed. The first model (1) proposes that the
ligation and hydrolysis of ATP in NBD1 is
responsible for the beginning of a burst of
activity that reaches its end when hydrolysis
occurs in NBD2. The second model (2) is based on
the hypothesis that the ligation of ATP to both
NBDs gives rise to a burst of activity that ends
with the hydrolysis of ATP. The third model (3)
proposes that the ligation of ATP to any of the
NBDs causes the opening of the channel inducing
the burst of activity. The ATP hydrolysis is
associated with the transition between two
distinct open states one that is ligated to ATP
and participates in the beginning of the burst
and another that occurs after the ATP hydrolysis
being responsible for the transition that ends
the burst. The activity of a single channel as
predicted by the three models was simulated using
a probabilistic method. The simulation results
were compared with published experimental data.
2.
a)
b)
c)
1.
3.
Model 1.
5.
a)
a) and b) are typical simulation results for the
normal CFTR these patterns were constant for all
the parameter values tried in these simulations
the results for models 2 and 3 are similar to
experimental data, while model 1 results were
always different. Besides normal CFTR, two well
known mutants of CFTR were also simulated. These
mutants have lower ATP hidrolysis rates in NBD1
and NBD2, respectively. To simulate them the kh1
or kh2 (according to the mutant) values were
reduced to very small values. Amongst other
experimental results, both mutants showed reduced
PA (open probability) values. Models 2 and 3
were insenitive to these parameter changes. c)
shows the mutants simulation results for model 1.
It is easily seen that the results for the mutant
with low NBD2 activity do not agree with
experimental data.
4.
a)
b)
b)
Model 2 and 3.
Fluxogram showing the strategy used in
these simulations it is a probabilistic method
based on a Markov model in continuous time it
generates open and closed time distributions that
follow an exponencial rule, as it is observed
experimentaly.
Model 1.
c)
a) Scheme of an hypothetical simulation output.
The open state (A) alternates with the closed
state (F) of the channel. tA represents the open
time and tF the closed time. tb is the burst
duration, tib is the interburst duration and tFb
is the closed time during a burst, the average
values of these variables were calculated and are
represented by lttAgt, lttFgt, lttbgt, lttibgt and lttFbgt.
b) Example of a simulation output obtained with
Berkeley Madonna, Version 8.0.2a1
(http//www.berkeleymadonna.com).
Conclusions None of the three kinetic models
analysed were able to simulate entirely the
experimental data. It was also concluded that
models with the same characteristics of the first
model studied can only barely simulate the
functioning of CFTR a model able to do so is
expected to resemble more the third model
analysed in this study.
COULD PKA BE LINKED TO CFTR IN ORDER TO ACTIVATE
IT EFFICCIENTLY ?
The activation mechanism of CFTR by
phosphorylation of the R domain was also studied.
It is well known that PKA is the main kinase
responsible for this activation. Yet, it is not
completely clear if in vivo the PKA enzymes that
activate CFTR are soluble or anchored to the cell
membrane (4). In excised inside out patch clamp
experiments, with multiple channels in each patch
of CACO2 cell membrane, it was possible to
observe a chloride conductance activated by cAMP
in the presence of ATP. Since the membrane patch
was nominally cytoplasm free, the results
obtained may suggest that PKA anchored to the
cell membrane is responsible for the activation
of CFTR.
1.
3.
a)
b)
ATP cAMP
cAMP (ATP already present)

Excised inside-out patch experiments with
CaCo2 cells. The membrane patch area is large
(pipette resistance 4-5 M?), and consequently,
there are too many channels to allow single
channel analysis. The currents were recorded at
45 mV applied potencial, so downward deflections
of the current are due to decreased seal
resistance (that may correspond to channel
activation). In a) this decrease was observed
after ATP and cAMP addition (at about 20 seconds
after the start of the record) to the
intracelular side of the membrane. To check if
cAMP was responsible for some of this decrease,
in a diferent experiment, b), the intracelular
side already had ATP, and cAMP was added, at
about 20 seconds after the start of the record.
It was already known that cAMP alone (without
ATP) was not able to activate any channels in
these cells. The currents shown above were
identified as chloride currents based on the
reversal potential observed in IV curves.
Conclusions
2.
References 1. Carson, M.R., Travis, S.M., Welsh,
M.J. (1995). J. Biol. Chem. 270 (4),1711-1717 2.
Venglarik, C.J., Schultz, B.D., Frizzell, R.A.,
Bridges, R.J. (1994). J. Gen. Physiol.
104,123-146. 3. Ikuma, M., Welhs, M.J. (2000)
PNAS 97 (15), 8675-8680 4. Kirk, K.L. (2000).
Cell. Mol. Lifr. Sci. 57,623-634
Whole Cell experiments in CaCo2 cells. Currents
stimulated by cAMP- IBMX. Almost totally
inhibited by DPC. This results suggest that the
currents observed are due to the presence of
endogenous CFTR in this cell line.
This group of results may present a new kind of
evidence that PKA may be anchored to the cell
membrane and maintained near CFTR molecules by
some unknown complexes. There is no previous
report of CFTR activation by cAMP in the presence
of ATP in excised patch experiments. One possible
reason for that is the general use of cells
expressing exogenous CFTR for this kind of
studies. In these cells, other components of this
membrane complex may not be correctly expressed.