Review
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41
Functional organization of PLC
signaling microdomains in neurons
Patrick Delmas1, Marcel Crest1 and David A. Brown2
1
Intégration des Informations Sensorielles, CNRS, UMR 6150, IFR Jean Roche, Faculté de Médecine, Boulevard Pierre Dramard,
13916 Marseille, France
2
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
Our understanding of receptor transduction systems
has grown impressively in recent years as a result of
intense efforts to characterize signaling molecules and
cascades in neurons. A large body of evidence has
recently accrued regarding the fast and effective signal
transfer that occurs during phosphoinositide signaling.
In particular, dissection of the Drosophila phototransduction pathway has enabled a greater understanding
of the molecular organization of phospholipase C (PLC)
signaling. Supramolecular complexes organize the correct repertoires of receptors, enzymes and ion channels
into individual signaling pathways. Such mechanisms
involve localization of signaling molecules to sites of
action by scaffold and anchoring proteins, ensuring
speed and specificity of signal transduction events.
However, not all PLC signals nucleate around scaffold
proteins, although mechanisms for selectivity and discrimination remain. This article reviews recent
advances on the molecular organization and functional
consequences of PLC signaling domains, which link
membrane receptors to ion channels, including TRP and
KCNQ channels.
The phospholipase C (PLC) signaling system constitutes a
virtually universal signal-transduction mechanism in
both neural and non-neural cells. In the common form of
this pathway, PLCb is stimulated by the a-subunit of a
member of the Gq family of G proteins (which includes Gaq,
Ga11, Ga14, Ga15 and Ga16) following the activation of the
G protein by a G-protein-coupled heptahelical receptor.
PLCb then catalyses the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to yield
the diffusible messenger inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3] and the membrane-associated fatty acid
diacylglycerol (DAG) [1]. Ins(1,4,5)P3 releases Ca2þ from
the endoplasmic reticulum while DAG activates protein
kinase C (PKC). These two tertiary messengers – acting
independently or synergistically – can then modify the
activity of a wide variety of downstream targets including
ion channels, enzyme systems and structural proteins [1].
In the original conception of G-protein-mediated signaling pathways, it was envisaged that all of the major
components – receptors, G proteins and enzyme targets –
were free to diffuse within the membrane [2] and that
Corresponding author: Patrick Delmas (delmas.p@jean-roche.univ-mrs.fr).
information transfer occurred by ‘collision-coupling’. However, this raises two difficulties, especially with respect to
the PLC system. First, a very large number of receptors for
different neurotransmitters, hormones and sensory stimuli are coupled to this system. Nonetheless, neurons seem
to be able to distinguish one stimulus from another: given
that the downstream pathway is predetermined, how do
they do this? Second, with so many steps between stimulus
and final response, how is the message conveyed within a
reasonable timescale?
The emerging answer to both of these conundra seems
to be that various components of the signaling pathway –
receptors, G proteins, PLC and (sometimes) the final
target – tend to be aggregated within anatomically
restricted signaling ‘microdomains’ with the aid of submembrane scaffolding or anchoring proteins. Here, this is
illustrated with respect to two systems – one specialized
for speed (the Drosophila photoreceptor system) and one
that is slow but allows discrimination between different
receptors (signaling to KCNQ/M Kþ channels).
PLC-mediated signaling to TRP channels: an example of
receptor– ion-channel segregation
The sophistication of PLC signaling is best illustrated by
the phototransduction cascade in the fruitfly Drosophila
[3– 5]. This PLCb-coupled mechanism represents the
fastest Gq-protein-signaling pathway known: the absorption of a single photon by rhodopsin is translated into a
physiological response (a depolarization) in just 20 ms [6].
Nonetheless, phototransduction in flies is a complex,
multiprotein cascade involving many enzymatic processes.
First, light signal is transmitted from the Gaq-coupled
rhodopsin photoreceptor to PLCb4 (which is encoded by
the norpA gene), activation of which leads to the
production of Ins(1,4,5)P3 and DAG. This causes photoreceptor to depolarize through the opening of Ca2þpermeant cation channels TRP, TRPL and possibly
TRPg, all of which belong to the transient receptor
potential channel (TRP) superfamily [7 – 9]. Gating of
TRP channels by PLCb is still controversial [10– 12] but
clearly appears to be independent of Ins(1,4,5)P3 and
ryanodine receptors [13 – 15]. The favored models instead
suggest that TRP channels are gated by DAG or its
metabolites (polyunsaturated fatty acids) [16,17] or via
membrane depletion of PtdIns(4,5)P2 [18,19]. Finally, the
photoresponse is terminated via a multifaceted regulatory
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42
Review
TRENDS in Neurosciences Vol.27 No.1 January 2004
mechanism that involves many different players, including intracellular Ca2þ [5], the eye-specific kinase C (INAC)
[20,21], arrestin [22,23], the unconventional myosin III
(NINAC) [24] and various phosphatases and kinases
(Figure 1). This discussion is by no means exhaustive
and readers are referred to several more detailed reviews
on phototransduction [3 – 5].
How are speed and specificity achieved in Drosophila
phototransduction? Importantly, proteins crucial for
phototransduction are localized in the rhabdomere, a
specialized photoreceptor membrane compartment that
concentrates the key players and limits potential interactions with superfluous signaling molecules. Phototransduction signaling proteins then join this specialized
cellular architecture as a supramolecular complex often
referred to as the signalplex or transducisome, which is
nucleated around the scaffolding protein inactivation-noafterpotential D (InaD) [25] (Figure 1). InaD shares
homology with the PDZ domain proteins postsynapticdensity 95-kDa protein (PSD95), discs large and zona
occludens 1, and encompasses five PDZ motifs [26]. PDZ
domains are typically implicated in the correct localization
and clustering of multiple proteins through interaction
with their C-terminal domains [27 –29]. The InaD core
complex consists of InaD, TRP, PLCb and PKC, which are
present in the complex at an approximately equimolar
ratio. TRP, PLC and PKC are constitutively bound to InaD
and degraded in various InaD mutants (e.g. inaD1 and
inaDP125) [30,31], demonstrating that InaD is crucial for
the stability and proper localization of the core components
in the rhabdomere. The reported binding interactions,
summarized in Figure 2, show little specificity in the
different partners for the individual PDZ domains,
suggesting that ligands bound to individual InaD molecules might vary from molecule to molecule in vivo [32].
Importantly, InaD can self-multimerize via PDZ– PDZ
interactions, allowing the assembly of individual InaD
complexes into a larger supramolecular network. Other
proteins are also known to be associated with the core
complex but appear to bind to InaD transiently [33,34].
They include rhodopsin, TRPL, NINAC and calmodulin –
none of which depends on InaD for retention in the
rhabdomere.
The InaD transduction complex is necessary for proper
signaling in vivo and is key to understanding the rapid
kinetics and adaptational capacity of the fly photoreceptors. Mechanistically, InaD retains the signaling
molecules in a highly organized molecular complex and
TRP, TRPL
TRP, TRPL
Ca2+ Na+
Ca2+ Na+
PIP3
PIP2
Rh1
CaM
2+
Ca2+ Ca
Rh1
P
Arr2
PIP2
Gq
RK
PUFAs
Ca2+ CaM
Ca2+
P
PLCβ4
RDGC
Ca2+
CaMKII
SOC?
DAG
IP3
CaM
InaD
InaD
IP3
Ca2+
P
PKC
Ca2+
RyR
Ca2+
Ca2+
PKC
InaD
P
?
Ca2+
IP3R
NINAC
F-actin
P NINAC
NINAC
CaM
CaM
NINAC
CaM
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
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Figure 1. Phospholipase C (PLC) signaling to transient receptor potential (TRP) channels. Several components of the Drosophila melanogaster phototransduction cascade,
including the light-sensitive TRP and TRPL channels, protein kinase C (PKC), PLC, calmodulin (CaM) and the unconventional myosin III (NINAC), are coordinated into a signaling complex by the scaffolding protein inactivation-no-afterpotential D (InaD). In addition, InaD forms homomultimers that are tightly associated with the actin cytoskeleton via NINAC. When light is absorbed, rhodopsin (Rh1) is photoisomerized to metarhodopsin (not illustrated), which activates the heterotrimeric Gq G-protein, releasing
the Gaq subunit. This leads to activation of PLCb, generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3, or IP3], and subsequent opening of the
light-sensitive TRP and TRPL channels by an unknown mechanism. Potential mechanisms of TRP and TRPL channel opening could involve DAG, its metabolites (polyunsaturated fatty acids, or PUFAs) and depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, or PIP2] from the membrane. At the base of the rhabdomere, a system
of submicrovillar cisternae has been proposed to represent Ca2þ stores endowed with Ins(1,4,5)P3 receptors (IP3R) and ryanodine receptors (RyR). In heterologous systems,
TRP channels lacking interaction with InaD might be prone to store-dependent regulation when stores of Ca2þ in the endoplasmic reticulum are emptied (SOC, store-operated channels). Termination of the photoresponse is a complex multistep process. First, Gaq is inactivated by the GTPase-activating activity of PLCb. Second, rhodopsin is
inactivated by binding to arrestin 2 (Arr2), translocation of which is regulated by phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] [23]. Arrestin 2 and rhodopsin
undergo rapid light-dependent phosphorylation by Ca2þ/CaM-dependent protein kinases (CaMKII) and rhodopsin kinases (RK), respectively. Phosphorylation of arrestin 2
appears to regulate its release from rhodopsin. Rhodopsin must be subsequently dephosphorylated, possibly by a CaM-regulated rhodopsin phosphatase (RDGC), before
inactivation and recycling is complete. Third, TRP and TRPL channels are rapidly inactivated by Ca2þ, possibly via release from ryanodine-sensitive stores and TRP-attached
calmodulin. Finally, DAG-activated PKC is also required for rapid termination of the photoresponse, with potential phosphorylation targets being the TRP channel, InaD
and NINAC. The overall organization of the TRP –InaD signalplex promotes very fast and efficient kinetics of activation and deactivation. Broken arrows indicate hypothetical pathways.
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Review
Rh1?
Rh1?
PKC
InaD
InaD
PLCβ
PKC
PKC
PKC
TRP
TRP
PLCβ
PDZ2
PDZ3
PDZ4
PDZ5
NINAC
CaM
PDZ1
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245
333 362 449 485
577 580
665
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Figure 2. Inactivation-no-afterpotential D (InaD) coordinates the Drosophila melanogaster phototransduction cascade. The PDZ domains and major binding partners of InaD are indicated. InaD interacts both in vitro and in vivo with the effector
phospholipase Cb (PLCb), the light-activated transient-receptor-potential channels
TRP and TRPL, an eye protein kinase C (PKC), calmodulin (CaM) and NINAC, an
unconventional myosin III. Rhodopsin (Rh1) might also be a putative ligand. Note
that the specificity of binding of some partners (e.g. PLCb, PKC and TRP) is not
restricted to a single PDZ domain of InaD. PLCb binds to PDZ1 with the C-terminal
motif F-C-A and also to PDZ5 with an internal sequence overlapping the G-proteinbinding site. Calmodulin is the only ligand to bind InaD not at one of its PDZ
domains but instead in the area between PDZ1 and PDZ2. Importantly, InaD molecules multimerize via PDZ– PDZ interactions, allowing the assembly of transducisomes from individual InaD complexes. Numbers indicate the amino acid
positions of the PDZ domain.
maintains the different partners in the proper stoichiometry. Thus, acceleration of signaling by restricting
diffusion might be the most prevalent contribution of a
multi-PDZ scaffolding protein. Pre-coupling of receptor to
G proteins and downstream effectors is indeed important
in Gq-mediated signaling, because the Gaq subunit does
not bind to the receptor until the latter is activated [35]. In
addition, because collision coupling is relatively slow and
rate limiting, pre-coupling of receptors, G proteins and
other signaling molecules is crucial for achieving signaling
speed.
A key insight into the function of the signalplex comes
from fly mutants in which PLCb levels are reduced [36].
Analysis of quantum bumps (the unitary response to a
single photon) in these flies shows increased bump latency
but no change in the size of the quantum bump. This is
consistent with a model in which the latency of the
response is determined by events upstream of PLCb,
whereas amplification occurs only downstream of PLCb,
possibly at the level of TRP channel gating (however,
see Ref. [37]). InaD is also instrumental in mediating
Ca2þ-dependent adaptation of the photoresponse by
assembling the Ca2þ-sensitive PKC with its potential
substrates, including PLC and TRP [38,39]. This requires
the PKC to be situated near the light-dependent Ca2þ
influx mediated through TRP channels. Thus, tethering of
the signaling molecules by InaD appears not to facilitate
amplification but instead to ensure speed and efficiency of
signaling and rapid regulatory feedbacks necessary for
termination.
PLC-mediated signaling to KCNQ/M channels: an
example of receptor segregation
Neural KCNQ/M channels can be shut down by virtually
any Gaq – PLC- or Ga11 – PLC-coupled receptor [40,41].
Indeed, all of their putative subunits (KCNQ2 – 5) – and
even the cardiac homolog KCNQ1 – appear to be equally
susceptible to suppression by an appropriate Gaq- or Ga11http://tins.trends.com
43
linked G-protein-coupled receptor [42]. Nevertheless,
there seems to be some functional discrimination between
different receptors, at least mechanistically.
Thus, KCNQ/M channels in sympathetic neurons are
closed by ACh (the natural transmitter released from the
preganglionic nerve fibers) acting on M1 muscarinic ACh
receptors, or by the peptide hormone bradykinin, which
can access the neurons via the blood supply to act on B2
receptors. When activated, both of these receptors stimulate the PLC system, yet seem to close the KCNQ/M
channels through entirely different mechanisms (Figure 3).
This is related to the fact that KCNQ/M channels are gated
by at least two molecules. First, like many other channels
[43], the open state is maintained by binding of membrane
PtdIns(4,5)P2 [44]. However, KCNQ/M channels are also
exquisitely sensitive to intracellular Ca2þ and are closed
when intracellular Ca2þ concentration rises appreciably
above , 100 nM [45] – probably through interaction with
calmodulin, which is bound to the KCNQ/M channel
subunits [46– 48]. Bradykinin receptors use this latter
(Ca2þ-dependent) mechanism [49], whereas muscarinic
ACh receptors do not, but instead appear to induce closure
as a direct result of PLC-mediated PtdIns(4,5)P2 hydrolysis and consequent loss of PtdIns(4,5)P2 gating [44,49,50].
The reason for the difference is that bradykinin produces a
rise in intracellular Ca2þ levels, whereas any rise produced by ACh (or its analogs) is too small to be effective [51].
This seems odd because – as already stated – both
receptors use the same (or at least equivalent) primary
pathways. So why does one raise Ca2þ concentration and
the other not? The answer seems to be that bradykinin
receptors are organized in special ‘microdomains’ in which
they are closely associated with Gq, PLCb and Ins(1,4,5)P3
receptors, such that the local formation of Ins(1,4,5)P3 can
induce a vigorous release of Ca2þ from the endoplasmic
reticulum [52]. By contrast, muscarinic ACh receptors are
more diffusely distributed and more remote from the
Ins(1,4,5)P3 receptors. Thus, using membrane-expressed
TRPC1 and TRPC6 channels as on-line detectors of
Ins(1,4,5)P3 and DAG, respectively, Delmas et al. [52]
showed that B2 receptor stimulation activated both TRPC
channels and, hence, elevated levels of both Ins(1,4,5)P3
and DAG. By contrast, stimulation of muscarinic ACh
receptors gave an equally vigorous signal for DAG but
failed to generate enough Ins(1,4,5)P3 in the vicinity of
TRPC1 to activate the channel. In consequence, muscarinic ACh receptors failed to elevate intracellular Ca2þ
levels as measured using Indo-1 or the Ca2þ-sensing
domain of PKC as a sensor for submembrane Ca2þ. That
the stronger Ins(1,4,5)P3 response to bradykinin resulted
from a closer association of the receptor with the
Ins(1,4,5)P3 receptor was shown both by coimmunoprecipitation of the B2 receptor and Ins(1,4,5)P3 and by
colocalization in the neural membrane. The Ins(1,4,5)P3
signal in response to B2 receptor stimulation was lost on
pretreatment with cytochalasin D, suggesting that the
submembrane actin cytoskeleton played a key role in the
organization of the B2 – Ins(1,4,5)P3 receptor microdomain.
Conversely, downtuning of Ins(1,4,5)P3 receptor sensitivity to Ins(1,4,5)P3 by calmodulin appeared to act as a
shield against stimulation of the Ins(1,4,5)P3 receptor by
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KCNQ2–5
PIP2
PIP2
BK
PIP2
ACh
PIP2
B2R
M1
Gq/11
PLCβ
PLCβ
DAG
IP3
IP3
CaM
Ca2+
Ca2+
K+
Gq/11
P
PKC
AKAP
IP3
DAG
IP3R
Ca2+
Ca2+
Ca2+
Ca2+
IP3R
F-actin
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
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Figure 3. Signaling of phospholipase C (PLC) to KCNQ/M Kþ channels. KCNQ/M channels (of subtypes KCNQ2– KCNQ5) bind phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2, or PIP2], calmodulin (CaM) and A-kinase-anchoring protein 150 (AKAP). PtdIns(4,5)P2 and calmodulin are required for KCNQ/M channel opening and AKAP
facilitates phosphorylation of KCNQ/M serine residues by protein kinase C (PKC). M1 muscarinic ACh receptors and B2 bradykinin (BK) receptors (B2R) couple to Gaq or
Ga11 G-proteins (Gq/11) and activate PLCb (although not necessarily the same subtype). This leads to hydrolysis of PtdIns(4,5)P2 to produce inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3, or IP3] and diacylglycerol (DAG). B2 receptors are closely connected to Ins(1,4,5)P3 receptors (IP3R) on the endoplasmic reticulum (dashed line), assisted by the
F-actin cytoskeleton. Hence, the local rise in Ins(1,4,5)P3 levels produces a vigorous release of Ca2þ sufficient to bind to KCNQ/M-attached calmodulin and close KCNQ/M
channels. M1 receptors are not directly associated with Ins(1,4,5)P3 receptors, so Ins(1,4,5)P3 has to diffuse further and is ineffective in releasing Ca2þ from the endoplasmic
reticulum. Channel closure results from hydrolysis and depletion of membrane PtdIns(4,5)P2. Dissociation of PtdIns(4,5)P2 is facilitated by activation of AKAP-bound PKC
by DAG and KCNQ/M channel phosphorylation. [Note that the same process – hydrolysis and depletion of membrane PtdIns(4,5)P2 – also occurs after B2 receptor stimulation: this could maintain KCNQ/M channels in closed state after Ca2þ has declined.] Broken arrows indicate hypothetical pathways.
the lower levels of Ins(1,4,5)P3 generated by stimulating
muscarinic ACh receptors.
The KCNQ/M channels themselves might also be
organized in a channel ‘microdomain’, as the channels
coprecipitate with A-kinase-anchoring proteins (AKAPs),
tubulin and actin [53]. It was subsequently established
that AKAP150 (the rat homolog of AKAP79) binds directly
to a region encompassed by residues 321– 499 of the
C terminus of KCNQ2 [54] (Figure 4).
AKAPs are anchoring molecules that bind and direct
protein kinase A (PKA) – and some other enzymes – to
specific targets [55]. In this case, however, the primary role
of the AKAP appears to be to direct phosphorylation of the
KCNQ/M channels by PKC [54]. Thus, the binding site on
AKAP150 was mapped to the N-terminal residues 1 –143.
This region contains the ‘A-site’, which has previously
been identified as the binding site for PKC [56], and Hoshi
et al. [55] found that KCNQ2, PKCa/b and AKAP150
coprecipitated as a trimeric complex.
It has long been known that activation of PKC with
phorbol esters shuts down KCNQ/M channels [41] and it is
also clear that activation of Gq- or G11-coupled receptors
generates DAG in sympathetic neurons [52]. However,
previous pharmacological tests to assess the contribution
of PKC activation to the receptor-mediated closure of
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KCNQ/M channels have led to equivocal and sometimes
contradictory results [41]. The experiments by Hoshi et al.
[54] help to resolve this question. They showed that
stimulation of M1 muscarinic ACh receptors induced a
PKC-mediated serine phosphorylation of rat KCNQ2
(which includes serine residues 534 and 541, equivalent
to S523 and S530 in human KCNQ2). This phosphorylation was prevented by overexpressing mutant AKAP150
(A1– 143) lacking the PKC-binding site. Importantly, the
same mutant acted as a functional dominant negative and
also reduced the M1-induced inhibition of both expressed
KCNQ2 channel currents and native KCNQ/M channel
currents in sympathetic neurons. Furthermore, both
phosphorylation and inhibition of the KCNQ/M current
were reduced by PKC inhibitors that act at the DAG site
(e.g. calphostin-C or salfingol) but not by those that act at
the catalytic domain (e.g. staurosporin or bisindolylmaleimide). This suggests that PKC is anchored to AKAP150 in
such a way as to shield the catalytic site – perhaps by pretethering in close proximity to the target site on KCNQ/M
channels. The effects of both the dominant-negative
mutant AKAP and the DAG-site inhibitors were strikingly
similar, in that both produced about a threefold shift of
the M1-agonist-concentration – KCNQ/M-current inhibition curve, without affecting the maximal inhibition.
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K+
(a)
KCNQ2
1
2
3
4
5
P
6
PIP2
523 530
310 321 328
NH2
G
341
372
499 501
IQ R
H
T
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847
S ES
COOH
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P
P
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AKAP
(c)
300 BK
[Ca2+]i(nM)
[Ca2+]i(nM)
(b)
529
10 s
100
–20mV
–50mV
Ctr
300
Oxo-M
10 s
100
–20mV
–50mV
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BK
Oxo-M
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Figure 4. Molecular partners for KCNQ/M channels: interaction sites on the C-terminal tail of KCNQ2. (a) KCNQ/M channels are voltage-gated Kþ channels. Each KCNQ/M
subunit has a conventional Shaker-like structure, with six transmembrane domains (1– 6), a pore (P) loop and a long C-terminal tail. In KCNQ2, the C-terminal tail has binding sites for several potential regulatory molecules: phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, or PIP2] [44]; calmodulin (CaMI and CaMII represent the two binding sites) [46]; and A-kinase-anchoring protein (AKAP) [54]. S523 and S530 are serine residues phosphorylated by protein kinase C (PKC) [54]. Numbering is according to
the human KCNQ2 sequence. (b,c) Inhibition of native KCNQ/M channels in rat sympathetic neurons by the peptide bradykinin (BK) (b) and by oxotremorine-M (Oxo-M, a
muscarinic-ACh-receptor stimulant) (c). The currents were pre-activated by depolarizing the neurons to 220 mV and then deactivated by stepping to 250 mV. Note that
bradykinin also raised intracellular Ca2þ concentration ([Ca2þ]i), whereas oxotremorine-M did not (upper records, on slower timescale) (from Ref. [52] and P. Delmas and
D.A. Brown, unpublished; see also Ref. [51]). This difference reflects the closer coupling of the bradykinin receptor to the Ca2þ release system [52]. The KCNQ/M current normally acts as a negative-feedback regulator of neuronal excitability. Thus, mutations of KCNQ2 or KCNQ3 give rise (in humans) to a form of juvenile epilepsy [62]. Likewise,
hormones or transmitters that reduce the current (e.g. ACh and bradykinin) enhance neuronal excitability. Upper panels of (b) and (c) reproduced, with permission, from
Ref. [52].
Thus, it seems that PKC-mediated KCNQ/M channel
phosphorylation sensitizes the channels to receptormediated inhibition. In this context it is worth noting
that AKAP also binds PtdIns(4,5)P2 and calmodulin (two
other regulators of KCNQ/M channel activity), suggesting
a central role as an integrating signal transducer.
In contrast to some other signaling systems, including
the signalplex, the receptor and effector complexes of
KCNQ/M channel regulation appear to be separate: there
is no evidence to suggest that they are associated in a
single supramolecular complex. For example, there have
been no reports to show the colocalization or association of
KCNQ/M channels and individual G-protein-coupled
receptors. Indeed, there seems to be no particular reason
why there should be, because the transduction process
from receptor to channel is relatively slow. Thus, even for
synaptically driven KCNQ/M channel closure, the minimum latency in sympathetic ganglia is , 250 ms [57],
reflecting the indirect nature of the response. This
contrasts with the almost tenfold shorter latency for
the direct Gbg-mediated gating of inward rectifier Kþ
channels or transmitter-releasing Ca2þ channels by
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receptors coupled to the G proteins Gi or Go; indeed, Go
has been reported to copurify with the Ca2þ channels
involved in transmitter release [58]. Nevertheless, because
the cytoskeletal proteins actin and tubulin are associated
with KCNQ/M channels, because actin appears to play a
role in localizing some receptors that regulate KCNQ/M
channels, and because tubulin has been reported to be
associated with Gq [59], the molecular infrastructure for
potential receptor– transducer– KCNQ/M channel complexes clearly exist: perhaps finding it is just a matter
of looking.
Concluding remarks: new principles for PLC signaling
Because the PLCb cascade is utilized by a large numbers of
transmembrane receptors and is present in virtually all
cells, a central question is how these cells generate
receptor-specific signals. From the two examples discussed
here, it appears that PLC signaling pathways are tightly
coupled, forming architecturally distinct signaling complexes or microdomains. Two strategies also seem to
emerge. In the first model, exemplified by the Drosophila
phototransduction, the PLC signaling complex allows
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TRENDS in Neurosciences Vol.27 No.1 January 2004
interaction of key signaling molecules while preventing
unwanted cross-talk with other pathways. In this prototype, the complex is formed from the assembly of four
classes of proteins – the receptor, the signaling enzyme,
the cytoskeletal protein and the ion channel – coordination of which is prearranged by the scaffold protein InaD
[4]. This highly ordered structure is relatively rigid but
ensures that the extracellular signal – a single photon – is
relayed to specific intracellular targets with extreme
fidelity, reliably resulting in the opening of a large and
predetermined number of TRP channels (the ‘quantum
bump’). In other words, PLC – TRP signaling complex in
Drosophila subserves only one purpose, phototransduction.
However, as illustrated with the KCNQ/M-current
modulation, PLC pathways can be organized within
signaling modules that promote clustering of receptors
and ion channels with enzymes but as separate entities.
This allows many protein kinases and phosphatases,
which have relatively broad substrate specificities, to
serve in varying combinations and achieve distinct
biological functions. These modules have a unique role
in the spatial organization of PLC signaling components in
sensory neurons and at brain synapses, and adds much to
the versatility of PLC and Ca2þ signals. They are likely to
represent the physical manifestation of the rich diversity
of the spatiotemporal signaling patterns observed in
various polarized cell types [60,61].
Although assemblies of PLC multiprotein complexes
have been described in an increasing number of systems,
much remains to be learned about their roles in signal
processing. For example, how does the unique structure
and composition of these multiprotein machines define
their functions? What controls their localization and
assembly – are they dynamically regulated? Is the
interaction of scaffold and anchoring proteins with the
diverse signaling proteins constitutive or regulated – do
scaffold proteins also play a regulatory role for their target
molecules? And, finally, does the prevalence of specialized
scaffolding modules in metazoans simply reflect the
increased signaling needs of multicellular organisms?
Studies over the next few years should shed light on these
and other important issues.
Acknowledgements
Our work was supported by the Centre National de la Recherche
Scientifique (CNRS) and by grants from the UK Medical Research Council
and the Wellcome Trust.
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