From the Institut für Biologische Informationsverarbeitung, Forschungzentrum Jülich, 52425 Jülich, Germany
Most ion channels consist of several different subunits,
and figuring out the exact role that each subunit
polypeptide plays in channel regulation is a daunting
and rewarding task. The challenge is to find out (a)
which subunits coassemble to form the native channel
protein, (b) how many copies of each individual polypeptide are contained in the channel, (c) which subunits contribute to the lining of the conducting pore
and which just stick to the internal or external face of
the channel, and (d) which of the subunits are neighbors in the native channel. The reward has two equally
exciting aspects: the studies can yield glimpses into the
mechanisms for fine tuning nearly all functional parameters, including ion selectivity, regulation of open
probability (gating), and channel expression. In addition, a modular system of channel assembly may become apparent in which a cell chooses from a repertoire of subunits to build the channel it needs. The report by
Hackos and Korenbrot (1999) CNG channels work as transducer channels in photoreceptors of the vertebrate retina and in olfactory sensory neurons (OSNs) of the nose. In the light-sensitive
outer segment of rod and cone photoreceptors, CNG
channels conduct a steady inward current in the dark,
the "dark current." Activated by cGMP, the second messenger of visual transduction, the channels have an open
probability in the dark of just 1-5%, and they close when
cGMP is hydrolyzed upon illumination. Thus, photoreceptor channels always work at very low activation levels, prompting Hackos and Korenbrot to study their
conducting properties during low activation. The closing of CNG channels in light not only hyperpolarizes
the membrane, it also induces a Ca2+ signal that plays a
pivotal role in phototransduction. The dark current is a
mixed cation current with a Ca2+ fraction between 12 and 21% (Nakatani and Yau, 1988 In OSNs, CNG channels are activated by cAMP,
which acts as second messenger during odor stimulation in the sensory cilia. Although our understanding
of olfactory signal transduction is by far not as detailed
as our concept of phototransduction, it is becoming
clear that the ability of CNG channels to conduct Ca2+
determines both the rise time and the amplitude of the
olfactory receptor current, as well as its termination after the stimulus. Ca2+-gated Cl How the channels interact with Ca2+ depends on the
set of subunits that coassemble to form the channel
protein. CNG channels can form heteromeric proteins
containing at least two types of subunits: principal All known subunits of CNG channels are integral
membrane proteins and appear to contribute to the
formation of the channel pore. This is particularly
important for cation permeation because the To most people, relative Ca2+ permeability is a somewhat cryptic parameter. It is usually interpreted as the
relative ease with which two ion species (here Ca2+ and
Na+) can enter a channel, but it doesn't tell you how efficiently a channel conducts Ca2+ into the cell. Recent
studies of Ca2+ interaction with CNG channels have
yielded a concept for Ca2+ permeation that may help us
appreciate the results presented by Hackos and Korenbrot (1999) Thus, Ca2+ influx is inversely related to Ca2+ affinity
in these channels. But how is the relative Ca2+ permeability (as determined by Hackos and Korenbrot from
reversal potentials with intracellular Ca2+) related to
Ca2+ affinity (as determined from the blockage of
monovalent currents by extracellular Ca2+)? Earlier
studies have shown that the higher the Ca2+ affinity of a
CNG channel, the lower is its relative Ca2+ permeability
(Frings et al., 1995 Taken together, high values of relative Ca2+ permeability suggest high levels of Ca2+ influx and low Ca2+
affinity in CNG channels. As permeability and flux
rates are not necessarily linked (one reflecting the access to the pore, the other the binding strength), this
phenomenological correlation is food for thought and
may stimulate further investigations into Ca2+ permeation in these channels. But it already gives some insight into how the dark current is shaped in such a way
that current amplitude and Ca2+ influx maintain just
the right balance necessary for phototransduction: at
the low cGMP concentrations in photoreceptors, relative Ca2+ permeability of CNG channels is low, implying
that Ca2+ affinity is high. This means that Ca2+ efficiently suppresses Na+ influx and that Ca2+ influx is retarded by strong binding. The result is a small dark current (approximately Interestingly, the authors demonstrate that both rod
and cone CNG channels show cGMP dependence of
relative Ca2+ permeability. Since this property is conferred to the rod channel by its
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in this issue is an excellent case in point: cyclic nucleotide-gated (CNG) channels display a fascinating dynamic fine tuning of Ca2+
selectivity and this phenomenon depends on the presence of a modulatory
subunit.
; Perry and McNaughton, 1991
). Steady Ca2+ influx in the dark is balanced
by Ca2+ extrusion through Na+/Ca2+,K+ exchangers,
resulting in a stable free Ca2+ concentration of ~500
nM. When CNG channels close in light, [Ca2+] drops
to ~50 nM due to continuous extrusion by the exchangers, a signal that is sensed by a set of Ca2+-regulated proteins that help the photoreceptor recover
after the stimulus (Gray-Keller and Detwiler, 1994
). Letting Ca2+ into the outer segment is thus an essential
part of CNG channel function in photoreceptors.
channels are triggered
by odor-induced Ca2+ influx through CNG channels
and cause a depolarizing Cl
efflux that amplifies the
receptor current (Lowe and Gold, 1993
). And among
the various processes that terminate the receptor current, probably the most rapid is the negative feedback
inhibition of CNG channels by Ca2+/calmodulin (Chen
and Yau, 1994
; Kurahashi and Menini, 1997
). Thus, Ca2+
signals generated by CNG channels are at the heart of
sensory transduction in vision and olfaction.
subunits and modulatory
subunits. Three homologous genes encode distinct
subunits in rods, cones,
and OSNs, and a fourth gene supplies two different
splice forms of
subunits in rods and OSNs (Chen et al.,
1993
, 1994
; Körschen et al., 1995
; Sautter et al., 1998
;
Bönigk et al., 1999
). In addition, a second type of modulatory subunit is part of the olfactory channels (Bradley et al., 1994
; Liman and Buck, 1994
; Shapiro and
Zagotta, 1998
). Consequently, three different subunits
form the transduction channels of OSNs, and the rod
photoreceptor channels have at least two different subunits. It is not clear whether
and
subunits are coassembled in the channels of cone photoreceptors.
subunits contribute negatively charged amino-acid residues (glutamate or aspartate) to an intrapore cation-binding site.
subunits, on the other hand, have an
uncharged glycine in the respective position and attenuate cation binding. The report by Hackos and Korenbrot (1999)
now reveals a link between ion selectivity and open probability conferred on the photoreceptor
channel by the
subunit. The authors show that the
relative Ca2+ permeability of heteromeric channels displays a pronounced dependence on the cGMP concentration, with unexpectedly small values at low (physiological) activation levels. This is a surprising result because selectivity and gating are traditionally thought of
as independent and associated with different parts of
the channel protein. The selectivity filter is determined
by geometry and charge density of the intrapore ion-binding site, which is regarded as a fixed feature of the
channel (with the notable exception of purinergic receptor channels, which change selectivity with time after activation; Khakh et al., 1999
). But this view, as well
as the textbook notion that sees the channel gate simply as a plug in the pore, controlled in an all-or-nothing
fashion by a voltage sensor or a ligand-binding site, is
obviously inappropriate for CNG channels. Apparently,
changes of ion selectivity with open probability reflect
the ability of photoreceptor CNG channels to adopt
more than a single conducting state: at low cGMP concentration, partially liganded channels may open into a
subconductance state with relatively low Ca2+ permeability. Fully liganded channels switch at elevated cGMP into a different state, which is characterized by higher
conductance and increased Ca2+ permeability. A similar dependence of ion selectivity on distinct conductance
states recently was demonstrated for mutant Shaker K+
channels (Zheng and Sigworth, 1997
) and for an NMDA-receptor channel mutant (Schneggenburger and Ascher,
1997
). To my knowledge, the report by Hackos and
Korenbrot (1999)
is the first evidence for such a phenomenon in a native channel, and it has immediate significance for CNG-channel research: physiologically
meaningful studies of Ca2+ permeation have to be
done at the right activation level!
. Interaction of CNG channels with extracellular Ca2+ is determined by the Ca2+ affinity of the
intrapore binding site. This site is formed by a set of
four negatively charged residues in channels consisting of only
subunits or by a combination of charged and
uncharged residues in channels containing
and
subunits. The
subunits of rods, cones, and OSNs
show marked intrinsic differences in Ca2+ affinity, and
coassembly with
subunits reduces Ca2+ affinity (Dzeja
et al., 1999
; Seifert et al., 1999
). Consequently, a variety
of CNG channels with quite diverse affinities for extracellular Ca2+ results from the combinations of the various
and
subunits. When Ca2+ enters a high-affinity
CNG channel, it is tightly bound, blocks the passage of
monovalent cations, and stays in the pore for a relatively long time. Therefore, Ca2+ blockage of monovalent currents is very effective in high-affinity channels,
but the rate of Ca2+ permeation is low. In contrast, low-affinity CNG channels show a less efficient Ca2+ block
of monovalent current but allow higher rates of Ca2+
permeation (a larger Ca2+ influx) because Ca2+ ions
move through the pore more easily.
). Consistent with this result, Hackos
and Korenbrot (1999)
show that recombinant rod
photoreceptor channels containing both
and
subunits have a higher relative Ca2+ permeability than
homomers; and the Ca2+ affinity in rod
channels is
lower than in
homomers (Körschen et al., 1995
). Furthermore, the reduced relative Ca2+ permeability at
low activation levels found by Hackos and Korenbrot (1999)
is associated with an increase of Ca2+ affinity, as
reported by Colamartino et al. (1991)
.
40 pA) with a relatively high
Ca2+ fraction (12-21%). At higher cGMP levels (which
apparently don't occur in photoreceptors), CNG channels would decrease their Ca2+ affinity. This would lead
to larger currents (exceeding the increment caused by
increased open probability) with a relatively smaller Ca2+ component, but a larger overall Ca2+ influx. These
relations between Ca2+ affinity, fractional Ca2+ current,
and Ca2+ influx govern the physiological functions of
CNG channels and should be kept in mind when predicting effects of the fine-tuning of Ca2+ permeation
described Hackos and Korenbrot (1999)
.
subunit, maybe cone
channels also possess a
subunit. This is particularly interesting because the
subunit contains a calmodulin-binding site (Weitz et al., 1998
), which may mediate regulatory effects by Ca2+/calmodulin in rods and
cones. The relative Ca2+ permeability at low activation
levels is much higher in cones than in rods. Such a pronounced difference in Ca2+ permeation is expected to
cause differences in the dynamics of Ca2+ handling between the two photoreceptor types and may be one of the reasons why cones show faster recovery after a light
stimulus. Finally, in cells where CNG channels can
reach high levels of activity, the dynamic tuning of Ca2+
permeation may constitute a regulatory mechanism
that becomes effective as Ca2+ affinity changes with
open probability.
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FOOTNOTES |
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Original version received 26 April 1999.
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