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Biophys J, July 2002, p. 1-2, Vol. 83, No. 1
Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand
Ion channels within membranes catalyze the
transition of ionic species across the hydrophobic cell membrane and by
opening and closing (gating) allow this transition to be controlled
(Hille, 1992 Spontaneous calcium release events (calcium sparks; Cheng et al., 1993 While it is possible that some unknown accessory protein for
electrochemical gradient coupling could be lost during RyR isolation, is such extra molecular complexity really required? Wang et al. (2002) The paper of Wang et al. is "new and notable" for the elegance with
which quite different state of the art methods are all brought to bear
on a very difficult biophysical problem as well as showing how
reintegration with computer models allows new plausible hypotheses to
be created. It is likely that future progress in E-C coupling will also
require similarly diverse techniques and the modern biophysicist seems
to need much more than a few simple tools to open Nature's
secrets
ARTICLE
). The control of ion flow can be used to regulate
information flow (e.g., the development of inhibitory or excitatory
potentials in neurons) as well as a variety of intracellular chemical
reactions to achieve cell functions, such as muscle contraction. Muscle excitation-contraction (E-C) coupling hinges on the processes that
permit calcium ions to move down their electrochemical gradient from
the lumen of the sarcoplasmic reticulum (SR) to the cytosol (where they regulate contractile protein interaction and metabolism). The ion channels regulating this process are ryanodine receptors (RyRs)
and are one member of a super family of channels involved in calcium
homeostasis (Sorrentino and Volpe, 1993; Williams et al., 2001). The
intracellular location of RyRs essentially prevents analysis of their
gating with in situ patch clamp techniques and, until now,
biophysicists have generally relied on reconstitution experiments with
artificial lipid bilayers or extracted SR vesicles to gain
insight into their gating behavior. By combining a number of
technologies Wang et al. (2002) have now gained new insight into the
gating behavior of RyRs in their native environment.
)
can be detected in isolated cells by applying fluorescent calcium
indicators and confocal microscopy. By incorporating EGTA in
the dialysis solution of the whole cell patch clamp pipette (to limit the spread of the dye-indicator complex) the time course of
calcium release may then be measured with some fidelity. With this
array of state of the art techniques, Wang et al. (2002) were in a
position to examine the duration of spontaneous calcium release events
at selected sites. The duration of repeated spontaneous SR calcium
release events exhibited a clear mode and, since the duration of SR
release should reflect the open time of the RyRs within the cell, Wang
et al. (2002) concluded that the RyR open time distribution must also
be modal. This result was in agreement with an earlier cardiac muscle
study which showed that the amplitude of calcium sparks at given sites
are modal (Bridge et al., 1999). For those interested in muscle, this
observation would seem reasonable; the cell might well be expected to
release calcium in quanta (sparks) that are just sufficient to activate
contraction and excess calcium release would require more ATP to pump
calcium back into the SR, for no obvious benefit. However, for the
biophysicist this observation is puzzling. Since the spontaneous
calcium release events repeated, the RyRs must have been cycling though
their open states and, for a reversible reaction the open time
distribution should be exponentially distributed, not modal. In fact,
exponentially distributed lifetimes for ion channel states are
generally observed with few exceptions. To allow steady-state behavior
without micro-reversibility requires an energy source (Steinberg, 1987;
Lauger, 1983). Since potential energy is stored in the calcium
electrochemical gradient across the SR membrane and RyR gating is
controlled by calcium ions, the energy source for such non-reversible
cyclical gating would seem to be readily available. To examine this
possibility Wang et al. (2002) carried out experiments on isolated RyRs
in planar lipid bilayers with realistic transmembrane calcium
gradients. In these conditions, only exponentially distributed open
times were observed showing that the energy gradient in the SR is not simply powering the modal RyR behavior observed in situ. In connection with this observation, it is notable that the closed time
distribution (i.e., the time between spontaneous spark events) was also
modal, another result which is not simply reconciled with energy being derived from the dissipation of the SR electrochemical gradient. (Modal
RyR gating behavior has also been observed in skeletal muscle (Klein et
al., 1999; González et al., 2000) although the possible role of
the sarcolemmal voltage sensor in such behavior remains unclear.)
then turn to computer modeling and show that modal behavior could still
be observed in a cluster of RyRs even when isolated RyRs gate with
exponentially distributed open times. The open times in this model are
not calcium dependent and so are insensitive to any possible coupling
via calcium. In contrast, when an array of RyRs is considered the
ensemble open states contain transitions which are calcium sensitive
(as one or more RyRs open to join the ensemble open state). Put another
way, the energy in the SR electrochemical gradient could be coupled
into the RyR gating scheme by the cytosolic calcium which results from
the flux of calcium via a more distant RyR. This idea is made even more
attractive by the fact that E-C coupling occurs in a narrow region
between the SR and surface membranes which restricts diffusion and
thereby increases the magnitude and lengthens the time course of
gradients (Soeller and Cannell, 1997). Since the purpose of ion channel gating is to control the flow of information (implying a change in
energy content of the system) perhaps we should be more careful in our
interpretation of equilibrium (reversible) gating in isolated channel
experiments as energy sources are often modified (or removed) by the
experimenter? Of course at this point we are moving into speculation,
but the mathematical modeling of Wang et al. (2002) follows the best
biophysical traditions. Rather than `hand wave' the mathematical
models rigorously test the validity of the hypothesis and even give
insight that may be far from intuitive.
indeed, a whole box of tools would seem to be required!
Finally, the paper also reminds us that insight gained from dissection
of cell systems may be limited (if not completely flawed), since the
macroscopic behavior of protein/chemical complexes may be quite
different from their behavior in broken cell systems. After all, the
cell is alive but the molecular components of the cell are not (with
apologies to Albert Szent-Gyorgi).
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FOOTNOTES |
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Address reprint requests to Mark B. Cannell, FMHS, Park Rd., Grafton, Private bag 92019, Auckland, New Zealand. Tel.: 64-9-3737599 ext. 6201; Fax: 64-9-3737499; E-mail: m.cannell{at}auckland.ac.nz.
Submitted February 3, 2002, and accepted for publication March 12, 2002.
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REFERENCES |
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Biophys J, July 2002, p. 1-2, Vol. 83, No. 1
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