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The International Journal of Biochemistry & Cell Biology 53 (2014) 218–223

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

jo u r n al homep ag e: www.elsev ier .com/ locate /b ioce l

olecules in focus

hosphorylation mediated structural and functional changes inentameric ligand-gated ion channels: Implications for drug discovery

ahil Talwara, Joseph W. Lyncha,b,∗

Queensland Brain Institute, The University of Queensland, St Lucia 4072, AustraliaSchool of Biomedical Sciences, The University of Queensland, St Lucia 4072, Australia

r t i c l e i n f o

rticle history:eceived 2 April 2014eceived in revised form 14 May 2014ccepted 19 May 2014vailable online 28 May 2014

eywords:

a b s t r a c t

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes, includingfast neurotransmission in the brain. They are targeted by a large number of clinically-important drugsand disruptions to their function are associated with many neurological disorders. The phosphorylationof pLGICs can result in a wide range of functional consequences. Indeed, many neurological disordersresult from pLGIC phosphorylation. For example, chronic pain is caused by the protein kinase A-mediatedphosphorylation of �3 glycine receptors and nicotine addiction is mediated by the phosphorylation of

ys-loop receptorentameric ligand-gated ion channelhosphorylationonformational changesrug discovery

�4- or �7-containing nicotinic receptors. A recent study demonstrated that phosphorylation can inducea global conformational change in a pLGIC that propagates to the neurotransmitter-binding site. Herewe present evidence that phosphorylation-induced global conformational changes may be a universalphenomenon in pLGICs. This raises the possibility of designing drugs to specifically treat disease-modifiedpLGICs. This review summarizes some of the opportunities available in this area.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

The pentameric ligand-gated ion channel (pLGIC) familyncludes nicotinic acetylcholine receptors (nAChRs), GABAA recep-ors (GABAARs), glycine receptors (GlyRs) and 5-HT3 receptors5-HT3Rs). Although these receptors are involved in many phys-ological processes, they are probably best known for mediatingast neurotransmission in the nervous system. They have beenmplicated in numerous channelopathies with neurological man-festations and are pivotal pharmacological targets. For example,AChRs play a clinical role in nicotine addiction and are thera-eutic targets for Alzheimer’s disease and schizophrenia (Pollockt al., 2009); GABAARs are important clinical targets for epilepsy

Abramian et al., 2010), anxiety, addiction and anaesthesia (Songnd Messing, 2005); GlyRs are emerging targets for chronic inflam-atory pain (Harvey et al., 2004).

Abbreviations: 5-HT3R, 5-hydroxytryptamine type-3 receptor; CamKII,a2+/calmodulin-dependent protein kinase; GABAAR, GABA type-A receptor; GlyR,lycine receptor; IPSC, inhibitory postsynaptic current; nAChR, nicotinic acetyl-holine receptor; PKA, protein kinase A; PKC, protein kinase C; pLGIC, pentamericigand-gated ion channel; PTK, protein tyrosine kinase; PGE2, prostaglandin type-E2;CF, voltage clamp fluorometry.∗ Corresponding author at: Queensland Brain Institute, The University of Queens-

and, Brisbane, QLD 4072, Australia. Tel.: +61 7 3346 6375; fax: +61 7 3346 6301.E-mail address: j.lynch@uq.edu.au (J.W. Lynch).

ttp://dx.doi.org/10.1016/j.biocel.2014.05.028357-2725/© 2014 Elsevier Ltd. All rights reserved.

Phosphorylation is well known to influence synaptic func-tion by directly modulating pLGICs (Swope et al., 1999). Indeed,pLGIC phosphorylation has been implicated in various disorderssuch as nicotine addiction (Wecker et al., 2001), status epilepti-cus (Terunuma et al., 2008) and chronic pain (Harvey et al., 2004).Phosphorylation of pLGICs can elicit a wide variety of effects,ranging from alterations in the level of surface expression, synap-tic targeting and receptor desensitization. Many of these effectsare summarized in Table 1. Until recently, there have been fewattempts to resolve the structural basis of these effects. By doing so,it may be possible to design drugs to specifically treat the conse-quences of pathological receptor modifications that lead to disease.This review summarizes some of the opportunities available in thisarea.

2. Structure

2.1. pLGIC architecture

Functional pLGICs comprise pentameric assemblies of identicalor different subunits (Fig. 1A). The five subunits together form a

central water-filled pore that facilitates transmembrane ion flux(Fig. 1B). Each subunit can be divided into three domains, anextracellular ligand-binding domain, four transmembrane helices(termed M1–M4) and a large intracellular M3–M4 domain which

S. Talwar, J.W. Lynch / The International Journal of Biochemistry & Cell Biology 53 (2014) 218–223 219

Table 1Effect of phosphorylation on pLGICs and their relevance to diseases.

Receptor Subtype Phosphorylationsite

Proteinkinase

Effect on receptor function Disease relevance References

GlyR �3 S346 PKA Inhibition of glycinergiccurrents

Chronic inflammatory pain Harvey et al. (2004)

�1 S409 PKA, PKC Enhanced desensitization andprolonged deactivation

Epilepsy Hinkle and Macdonald(2003)

�3 S408, S409 PKC Alterations in cell surfaceexpression.

Status epilepticus Terunuma et al. (2008)

PKC BDNF-mediated transientincrease in receptor functionfollowed by down regulation

Neuronal Excitotoxicity Jovanovic et al. (2004)

S383 CamKII Rapid insertion ofextrasynaptic receptors at thecell surface and enhanced toniccurrents in hippocampus

Neuropsychiatric disorders Saliba et al. (2012)

GABAAR �2 S327 PKC Alterations in ethanol andbenzodiazepine sensitivity

Alcoholism Qi et al. (2007)

S343 PKC Increased amplitude of mIPSCs Alcoholism Song and Messing(2005)

Y365, Y367 Src Increased postsynapticreceptor expression andenhanced frequency ofhippocampal mIPSCs

Spatial memory deficits Tretter et al. (2009),Luscher et al. (2011)

�4 S443 PKC Increased cell surface stabilityand activity

Temporal lobe epilepsy Abramian et al. (2010)

�7 Y386, Y442 Src Increased current amplitudeswith no change in surfacereceptor number

Cognition and addiction Charpantier et al.(2005)

Y386, Y442,Y317

Src Increased surface receptornumbers with no change inopen probability

Cho et al. (2005)

nAChR (neuronal) �4 S368 PKC Prolonged nicotine-induceddesensitization

Nicotine addiction Wecker et al. (2001)

S365, S472,S491

PKA Increased surface expression Guo and Wecker(2002)

S467 PKA Favoured expression oflow-affinity receptors

Bermudez and Moroni(2006), Pollock et al.(2009)

S550 PKC Dual action affecting maturereceptors to stabilize thereceptors at cell surface andincrease transport of immaturereceptors from endoplasmicreticulum to membrane

Pollock et al. (2009)

� Y390 Src Decreased receptor turnoverand metabolic stabilisation

Myasthenia gravis Rudell and Ferns(2013)

Y355 Src Enhanced desensitization andimmobilization of the receptor

Tzartos et al. (1993)

nAChR (muscle) � S361, S362 PKA Enhanced desensitization Myasthenia gravis Hoffman et al. (1994)localisr dured de

hpcepmti

2

Mavnnst

Y372 Src Stable

recepto� S353, S354 PKA Enhanc

as a length and primary structure that varies enormously amongLGIC members (Fig. 1A). The extracellular domain predominantlyomprises a twisted �-sheet sandwich with ligand-binding pock-ts located at the subunit interfaces. The principal (+) side of theocket is lined by binding domain loops A, B and C and the comple-entary (−) side is lined by binding loops D, E and F (Fig. 1A). The

ransmembrane �-helices form concentric rings around a centralon pore, which is directly lined by five M2 helices (Fig. 1A).

.2. Structure and importance of the M3–M4 cytoplasmic domain

Unlike the extracellular and transmembrane domains, the3–M4 domain is poorly conserved both in terms of length and

mino acid sequence. Therefore, it is likely to exhibit structuralariation. Low-resolution structural data suggest that the Torpedo

AChR M3–M4 domain forms a ‘hanging basket’ type structure con-ecting the pore with the cytoplasm (Miyazawa et al., 1999). Thistructure incorporates a lateral ion permeation pathway (or por-al) linking the cytoplasm with the inner vestibule at the base of

ation of theing synaptogenesis

Wagner et al. (1991)

sensitization Hoffman et al. (1994)

the pore. Charged residues lining these portals influence the singlechannel conductance of nAChRs, GlyRs and 5HT3Rs (Peters et al.,2010). Interactions between the M3–M4 loop and other proteinsor ions are well known to modulate pLGIC activity, assembly andtrafficking (e.g., Luscher et al., 2011). These interactions are highlyspecific to different pLGIC subunits. The M3–M4 domain is also theonly pLGIC region known to house phosphorylation sites.

2.3. Receptor phosphorylation: structural changes induced bykinases?

Phosphorylation results from the kinase-mediated covalentattachment of the �-phosphate group of ATP to the hydroxyl groupof serine, threonine or tyrosine (Fig. 1D). The best-characterizedprotein kinases include cAMP dependent protein kinase A (PKA),

protein kinase C (PKC) and protein tyrosine kinase (PTK). Ofthese, PKA and PKC phosphorylate both Ser and Thr residues andPTK (including Src family kinase) phosphorylates Tyr residues.Many other kinases also exist such as Ca2+/calmodulin-dependent

220 S. Talwar, J.W. Lynch / The International Journal of Biochemistry & Cell Biology 53 (2014) 218–223

Fig. 1. Structure and phosphorylation of pLGICs. (A) Side view of pentameric �3 GlyR homology model viewed parallel to the membrane showing five identical subunits,each colored differently. The large M3–M4 cytoplasmic loop is shown as a dashed line as its structure is not known. (B) Top down view of the same pentameric assemblyshowing the central pore and the (+) and (−) subunit interfaces. (C) Close-up side view of an extracellular domain subunit interface showing ligand-binding loops A, B andC on the (+) side of the subunit interface and loops D, E and F on the (−) side of the interface. (D) Ion channel phosphorylation pathway. (i) Phosphorylation starts with thebinding of ATP to the catalytic site of the protein kinase. (ii) Binding of ATP is followed by protein kinase binding to the substrate receptor. (iii) Followed by �-phosphatetransfer from ATP to Ser/Thr or Tyr of the receptor phosphorylation site. (iv) Following phosphorylation, kinase leaves the receptor with ADP bound, following which ADP isr hich

pacdapras2is(

eleased. (E) Phosphorylation of pLGICs leads to many possible outcomes, some of w

rotein kinases (CaMKII) which also phosphorylate both Sernd Thr residues (Swope et al., 1999; Davis et al., 2001). Theonsensus phosphorylation site for Ser/Thr or Tyr specificity isetermined by the structure of the catalytic cleft of the kinasend the local interactions between the kinase cleft and proteinhosphorylation site. This binding between the kinase and theeceptor protein often provides additional binding interactions,nd occasionally, provides allosteric regulation and localization topecific cellular compartments or structures (Ubersax and Ferrell,007). Phosphorylation can have many effects on pLGIC function,

ncluding alterations in open probability and desensitization,urface expression levels, turnover rate and synaptic targetingSwope et al., 1999; Davis et al., 2001) (Fig. 1E).

are indicated.

3. Biological function

3.1. Chronic inflammatory pain is mediated by phosphorylationof ˛3 GlyRs

Perhaps the most direct evidence for phosphorylation-inducedstructural changes in a pLGIC, and its relationship with a dis-ease, has emerged from studies on the �3 GlyR. This will nowbe described in detail. Inflammatory pain is mediated by the pro-duction of prostaglandins in the spinal cord. Electrophysiological

studies have identified prostaglandin E2 (PGE2) as the most activeprostaglandin in the spinal cord, where it specifically inhibitsglycinergic inhibitory postsynaptic currents (IPSCs) in lamina I and

S. Talwar, J.W. Lynch / The International Journal of Biochemistry & Cell Biology 53 (2014) 218–223 221

Fig. 2. Phosphorylation-mediated conformational changes in the �3 GlyR expressed in Xenopus laevis oocytes. (A) Cartoon depicting location of labelled residues for VCFin the ligand-binding site (N203C; green), M2–M3 linker (R19′C; blue) and the phosphorylation site (S346G, magenta). (B) Example of EC50 glycine-induced current (�I)and fluorescence (�F) responses in fluorescently-labelled �3-R19′C and �3-R19′C-S346G GlyRs before and after a 15 min forskolin (FSK) treatment and after a 15 min wash.Averaged results (right panel) reveal that forskolin inhibits glycine-mediated fluorescence responses only in �3 GlyRs containing a functional S346 phosphorylation site. (C)Example and averaged data of saturating glycine- and strychnine-induced �I and �F responses in fluorescently-labelled �3-N203C and �3-N203C-S346G GlyRs before andafter a 15 min forskolin treatment and after 15 min wash. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisa

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igure modified from Han et al. (2013).

I nociceptive neurons (Ahmadi et al., 2002). This effect is mediatedia the activation of the EP2 receptor and involves the activation ofKA. Although �3 GlyRs are generally sparsely expressed, they areighly expressed along with �1 subunits at glycinergic synapses in

amina I and II neurons (Harvey et al., 2004). As only �3 GlyRs arenhibited by PGE2 via PKA-dependent phosphorylation at S346 inhe M3–M4 domain, it was hypothesised that the �3 subunit was

odulated by PGE2 (Harvey et al., 2004). Although GlyR �3−/− miceisplayed no overt behavioural phenotype, normal and knockoutice responded similarly to non-painful tactile stimuli and acute

nflammatory pain stimuli. However, chronic inflammation pro-uced pain sensitisation in normal animals but not in the GlyR3−/− animals (Harvey et al., 2004). Thus, PGE2 induces chronicain by phosphorylating, and inhibiting, �3 GlyRs. This reduceshe magnitude of glycinergic IPSCs in spinal pain sensory neurons,hereby ‘disinhibits’ them and increasing the likelihood of painmpulses being transmitted to the brain. Thus, drugs that potentiatei.e., restore) �3 GlyR IPSCs should be analgesic. Indeed, �3 GlyR-otentiating agents do exert potent analgesic effects in animal painodels. For example, synthetic cannabinoid derivatives modified

o prevent their binding to CB1 or CB2 cannabinoid receptors exertotent analgesia via direct effects on �1GlyRs (Xiong et al., 2012).

Phosphorylation-induced conformational changes in the �3lyR have recently been probed using voltage clamp fluorome-

ry (VCF) (Han et al., 2013). VCF takes advantage of the fact thathanges in the quantum efficiency of small molecule fluorophoresccur in response to changes in their chemical microenvironment.

t involves introducing a cysteine into the domain of interest andovalently tagging it with a sulfhydryl-labeled fluorophore, often ahodamine derivative. This technique enables one to probe confor-ational changes in protein domains of interest in real time. Using

this approach, forskolin-induced PKA phosphorylation of Ser346 inthe �3 GlyR cytoplasmic domain elicited a change in the glycine-induced fluorescence response of a label attached to R19′C in theM2–M3 linker or to N203C in loop C of the glycine-binding site(Fig. 2A). This effect was not observed if Ser346 was mutated toprevent its phosphorylation (Fig. 2B and C). Thus, phosphoryla-tion exerts a global conformational change that propagates fromS346 in the internal M3–M4 domain to the �1 glycine-binding site,a distance of >80 A. The experiments shown in Fig. 2 were per-formed in Xenopus oocytes where phosphorylation does not reduce�3 GlyR current. However, when the same receptors are expressedin HEK293 cells, phosphorylation reduced current magnitude andproduced a conformational change in the glycine-binding site (Hanet al., 2013). This conformational change appears to be uniquebecause it is accompanied by an increase in both glycine sensi-tivity and the potency of the competitive antagonist, tropisetron(Han et al., 2013). These results raise the possibility of developinganalgesic drugs to specifically target disease-affected receptors.

3.2. Phosphorylation-mediated conformational and functionalchanges in other pLGICs

Phosphorylation also appears to induce conformational changesin GABAARs. For example, PKC activators altered the efficacyof benzodiazepines and barbiturates in neuronal GABAARs (Gaoand Greenfield, 2005). Another study reported that PKC activa-tion reduced the amplitude of GABAergic IPSCs in a manner that

was completely inhibited by pre-treatment with neurosteroids(Brussaard et al., 2000). Other studies suggest different PKC iso-forms differentially affect GABAAR alcohol sensitivity (Song andMessing, 2005; Qi et al., 2007). Importantly, these later effects have

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22 S. Talwar, J.W. Lynch / The International Journ

een implicated in alcoholism. Several other examples of GABAARubunit phosphorylation sites that have been linked to receptorunctional changes and neurological disease are summarized inable 1.

Many neuronal nAChRs are also regulated by phosphorylation.or example, dephosphorylation of �7 nAChRs caused a rapidncrease in acetylcholine-evoked current and alterations in �-ungarotoxin sites with no change in receptor surface expressionWiesner and Fuhrer, 2006).

Widely expressed in the brain, the �4�2 nAChRs are proposedo play a major role in the mechanisms that cause nicotine addic-ion. Another study reported that PKA phosphorylation at Ser467 inhe M3–M4 domain increases the relative abundance of �4 avail-ble for assembly, which favoured the functional expression of lowffinity �4�2 receptors (Bermudez and Moroni, 2006). In addition,revious studies had shown the possible functional significancef phosphorylation/dephosphorylation on �4�2 receptor desen-itization at the Ser368 residue. Phosphorylation-mediated timeourse recovery from desensitization implicated different recep-or conformations with continuous nicotine exposure leading ton irreversible desensitized conformation (reviewed in Quick andester, 2002). Recently, a study by Pollock and colleagues reported

dual effect of PKC at multiple sites of �4 subunits associatedith mature and immature receptors. Nicotine has been impli-

ated as a chaperone to promote subunit assembly and stabilizehe �4�2 receptors at the cell surface (Pollock et al., 2009). Otherxamples of neuronal nAChR subunit phosphorylation sites thatave been linked to receptor functional changes and neurologicalisease (notably nicotine addiction and cognitive impairment) areummarized in Table 1.

Muscle nAChRs have also been demonstrated as phosphor-lation targets. PKA phosphorylates � and � subunits, PKChosphorylates � and � subunits and PTK phosphorylates �, � and

subunits (Swope et al., 1999). Phosphorylation of muscle nAChRss related mainly to myasthenia gravis (Table 1).

. Possible medical applications

Here we demonstrate that neurological disorders can resultrom conformational changes induced by the ‘inappropriate’hosphorylation of pLGICs. For example, PKA-mediated phosphor-lation �3 GlyRs (which causes chronic pain) inhibits current andauses a global conformational change that changes the structuref the glycine-binding site (Han et al., 2013), and alcoholism canesult from a phosphorylation-induced increase in ethanol sensi-ivity of �2-containing GABAARs (Qi et al., 2007). We propose thatn these and other neurological disorders, phosphorylation induces

global allosteric conformational change that alters the structuref orthosteric or allosteric binding sites.

In classical allosteric theory, receptors are considered to existn either inactive or active states and drugs that modify their func-ion do so by stabilising one of these states (Nussinov and Tsai,014). In reality the situation is much more complex with numer-us states existing in unmodified receptors, and even more that cane induced via covalent modifications (such as phosphorylation) orrotein–protein interactions. We suggest that an ideal therapeuticrug would bind specifically to a phosphorylated (disease-affected)eceptor and promote a conformational change that specificallyompensates for the deleterious effect of phosphorylation. As far ase are aware, no such drug has yet been developed for any receptor

ype. However, there is no reason why this should not be feasible

iven that phosphorylation is well known to modulate the abilityf enzymes to bind specifically to their substrates.

The aim of this brief commentary is to raise awareness of the facthat phosphorylation can cause global allosteric conformational

ochemistry & Cell Biology 53 (2014) 218–223

changes in pLGICs and to suggest that these conformations couldin principle be targeted by drugs with therapeutic potential.

Acknowledgements

Research in the author’s laboratory is supported by the NationalHealth and Medical Research Council of Australia (grant numbers:569570 and APP1060707) and the Australian Research Council(grant number: DP120104373). S.T. is thankful to QueenslandEducation and Training International and to The University ofQueensland for PhD scholarship support.

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