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New insights into the therapeuticpotential of Girk channelsRafael Lujan1, Ezequiel Marron Fernandez de Velasco2, Carolina Aguado1, andKevin Wickman2

1 Instituto de Investigacio n en Discapacidades Neurolo gicas (IDINE), Departamento de Ciencias Me dicas, Facultad de Medicina,

Universidad Castilla-La Mancha, Campus Biosanitario, C/Almansa 14, 02008 Albacete, Spain2 Department of Pharmacology, University of Minnesota, 321 Church Street South East, Minneapolis, MN 55455, USA

Review

G protein-dependent signaling pathways control theactivity of excitable cells of the nervous system andheart, and are the targets of neurotransmitters, clinicallyrelevant drugs, and drugs of abuse. G protein-gatedinwardly rectifying potassium (K+) (Girk/Kir3) channelsare a key effector in inhibitory signaling pathways. Girk-dependent signaling contributes to nociception and an-algesia, reward-related behavior, mood, cognition, andheart-rate regulation, and has been linked to epilepsy,Down syndrome, addiction, and arrhythmias. We dis-cuss recent advances in our understanding of Girk chan-nel structure, organization in signaling complexes, andplasticity, as well as progress on the development ofsubunit-selective Girk modulators. These findings offernew hope for the selective manipulation of Girk channelsto treat a variety of debilitating afflictions.

Introduction to Girk signalingSignal transduction involving inhibitory (Gi/o) G proteinstitrates the excitability of neurons, cardiac myocytes, andendocrine cells, actions crucial for regulating mood andcognition, nociception and antinociception, reward, energyhomeostasis, motor activity and coordination, hormonesecretion, and cardiac output. Not surprisingly, dysregula-tion of Gi/o-dependent signaling has been linked to severalneurological and cardiac disorders. Given this, and becausethe efficacy of many clinically relevant and abused drugsrelates to their actions on Gi/o-dependent signaling, it isimportant that we understand the cellular, subcellular,and molecular details of how such signaling is organized,how it is regulated, and how and when it goes awry.

Girk/Kir3 channels are a common effector for Gi/o-de-pendent signaling pathways in the heart and nervoussystem [1,2]. Studies of mutant mice and a more limitedset of linkage analyses have suggested that dysregulationof Girk signaling may contribute to particular humandiseases and disorders (Table 1). Although this work sug-gests that therapeutic approaches targeting Girk channels

0166-2236/$ – see front matter

� 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2013.10.006

Corresponding authors: Lujan, R. (Rafael.Lujan@uclm.es);Wickman, K. (wickm002@umn.edu).

may prove beneficial in some settings, there is legitimateconcern that manipulation of Girk signaling would triggerprofound and widespread off-target effects. The goal of thisreview is to highlight recent developments related to ourunderstanding of Girk channel diversity, compartmentali-zation, and plasticity. These studies suggest new opportu-nities for selective manipulation of Girk signaling, effortsthat could eventually lead to novel treatments for debili-tating human afflictions.

Girk channel structureGirk channels are tetramers formed by differential multi-merization among the products of four genes: Girk1/Kir3.1/Kcnj3, Girk2/Kir3.2/Kcnj6, Girk3/Kir3.3/Kcnj9, and Girk4/Kir3.4/Kcnj5 [1,2] (Figure 1A). Each Girk subunit pos-sesses intracellular N- and C-terminal domains, and twotransmembrane segments that flank a hydrophobic poredomain. Random assembly theoretically allows the forma-tion of many distinct Girk channel subtypes, and alterna-tive splicing of Girk1 and Girk2 gene mRNAs potentiallyadds an additional spice of diversity (e.g., [3,4]). However,two observations suggest that a more limited number ofGirk channel subtypes exist in vivo: (i) Girk1 does not formfunctional homomultimeric channels [5,6], an observationattributable at least in part to the absence of an endoplas-mic reticulum (ER) export signal that is found in Girk2 andGirk4 [7]. Surprisingly, however, introduction of a singlepoint mutation into the pore domain of Girk1 (F137S) issufficient to yield functional homomeric channels [8]. Col-lectively, these findings suggest that, although ER exportsignals are important, other factors such as protein foldingand stability may also influence the trafficking of Girkchannels. (ii) The four Girk subunits exhibit overlappingbut distinct expression patterns, and this is particularlyevident in the nervous system [9,10]. Indeed, work in thepast two decades has clarified the cell type-specific expres-sion patterns of Girk subunits (Box 1). Moreover, recentcrystallography studies have begun to provide insight intothe 3D structure of Girk channels, with resolution of themembrane-spanning and large portions of the intracellulardomains of homomeric channels [11–16] (Figure 1B).

Functional implications of Girk channel diversity

In expression systems, Girk channels of various composi-tion – including heteromers (Girk1/2, Girk1/3, Girk1/4,

Trends in Neurosciences xx (2013) 1–10 1

Table 1. Physiological and pathophysiological relevance of Girk signalinga

Girk gene(s) Mutation Phenotype Refs

Cardiovascular physiology

Heart rate Girk1, Girk4 Null Resting tachycardia [86,87]

Parasympathetic regulation Girk1, Girk4 Null Decreased chronotropic response [86,87]

Heart rate variability Girk4 Null Decreased variability [86]

Arrhythmia GIRK1, GIRK4 Multiple Long QT, atrial fibrillation [88–90]

Hypertension GIRK4 Multiple Associated with aldosteronism [91,92]

Nociception

Thermal Girk1, Girk2 Null Hyperalgesia [93,94]

Mechanical Girk2 Null Hyperalgesia [93]

Chemical Girk2 Null Hyperalgesia [93]

Antinociception

Opioid Girk1, Girk2 Null Decreased analgesia [93–97]

Girk3 Null Decreased sensitivity [98]

GIRK2 Polymorphism Increased dosing requirement [99,100]

a2 Adrenergic Girk2,Girk3 Null Decreased analgesia [93,95,98]

GABAB Girk2 Null Decreased analgesia [95]

Cholinergic Girk2 Null Decreased analgesia [95]

Cannabinoid Girk2, Girk3 Null Decreased sensitivity [95,98]

Reward

Motor activity Girk1, Girk2 Null Enhanced basal and cocaine-induced [101–103]

Natural rewards Girk2, Girk4 Null Elevated responding for food [104,105]

Girk2 Triploid Enhanced sucrose intake [58]

Self-administration Girk2, Girk3 Null Decreased (cocaine) [102]

Girk2 Null Enhanced consumption (ethanol) [106]

Dependence/withdrawal Girk2 & Girk3 Null Decreased opioid withdrawal [97]

Girk3 Null Decreased sedative-hypnotic withdrawal [107]

GIRK2 Polymorphism Association with ethanol intake and stress [108]

Learning/memory

Spatial learning/memory Girk4 Null Decreased recall [109]

Fear conditioning Girk2 Triploid Decreased contextual recall [58]

Anxiety Girk2 Null Anxiolysis [104,110]

Schizophrenia GIRK1 Polymorphism Genetic association [111]

Seizure/epilepsy Girk2 Null Increased spontaneous and PTZ-induced [74]

Energy homeostasis Girk4 Null Late-onset obesity [105]

Thermoregulation Girk2 Null Decreased drug-induced hypothermia [112]

Neurodevelopment Girk2 weaver Loss of granule and dopamine neurons [113]

aThe table lists outcomes from behavioral studies involving mice harboring mutant Girk subunits (null/knockout, triploid, or weaver) or from human linkage studies (GIRK

genes in uppercase) that identified polymorphisms or mutations in GIRK genes.

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Girk2/3, Girk2/4) and homomers (Girk2 and Girk4) – dis-play K+ selectivity, inward rectification, and G protein-dependent gating [1,2]. Although it cannot form a func-tional homomer, Girk1 is an integral subunit of mostneuronal Girk channels and the cardiac Girk channel IKACh

[5,17]. Girk1 confers robust receptor-dependent activity toGirk heteromers, attributable in part to unique residuesfound in the pore and second transmembrane helix thatenhance single-channel conductance and opening proba-bility [8,18–20]. The intracellular C-terminal domain alsocontributes to the potentiating influence of Girk1 onGPCR-dependent heteromeric channel activity, likelydue to the presence of unique Gbg, Ga, and phosphatidy-linositol 4,5-bisphosphate (PIP2) interaction domains andphosphorylation sites [20–26].

The functional relevance of Girk channel subunit com-position is nicely illustrated in the ventral tegmental area(VTA). VTA dopamine neurons express Girk2 and Girk3,whereas VTA GABA neurons express Girk1, Girk2, andGirk3 [27,28]. VTA dopamine neurons are significantlyless sensitive than VTA GABA neurons to direct GABAB

2

receptor-dependent inhibition [27,28], consistent with therelatively low sensitivity of recombinant Girk2/3 hetero-mers to Gbg-dependent activation [29]. Interestingly, ec-topic expression of Girk1 or genetic ablation of Girk3enhanced the sensitivity of the Girk channel in VTA dopa-mine neurons to GABAB receptor-dependent inhibition[28]. The negative influence of Girk3 on the sensitivityof Girk2/3 heteromers to Gbg- and GABAB receptor-depen-dent activation may be linked to intrinsic structural ele-ments that weaken its interaction with Gbg or the couplingbetween Gbg binding and channel gating, an explanationsupported by the behavior of recombinant Girk2/3 hetero-mers [29]. Alternatively, Girk3-specific interactions withnegative regulatory proteins expressed in VTA dopamineneurons (Rgs2 and/or sorting nexin 27 [28,30], discussedbelow), interactions that are presumably precluded ormitigated by the presence of Girk1, may explain the dif-ferential sensitivity of Girk channels in VTA dopamine andGABA neurons to GABAB receptor activation. Regardlessof the mechanism, the molecular and cellular diversity ofGirk channels shapes the sensitivity of VTA dopamine

Box 1. Cellular and subcellular diversity of Girk channels

Although Girk channel expression has been reported in neuroendo-

crine cells and more recently in some cancer cells (e.g., [114–117]), the

expression and relevance of Girk channels is far better understood in

the heart and nervous system. Girk1/4 heteromers comprise the

muscarinic-gated atrial K+ channel IKACh, a crucial mediator of the

parasympathetic regulation of heart rate [5,86]. Although the proto-

typical neuronal Girk channel is thought to be the Girk1/2 heteromer,

multiple Girk channel subtypes are present in the rodent nervous

system. Indeed, Girk3 is expressed throughout the central nervous

system [9] and, although Girk4 expression is not prominent in the

brain, it is found in a few regions including the hypothalamus and

cerebellum [105,118]. Girk1, Girk2, and Girk3 are coexpressed in many

neuron populations, including hippocampal pyramidal neurons

[9,10]. By contrast, dopamine neurons of the VTA and substantia

nigra pars compacta (SNc) display Girk2/3 and Girk2a/c heteromers,

respectively [27,119]. The cerebellum exemplifies the molecular

diversity that can be achieved via differential subunit expression:

seven distinct Girk expression patterns were discerned within the

various neuronal subtypes in this brain region [118].

Girk channels are distributed mainly in the somato-dendritic

compartment of neurons (e.g., [10,96,118,120]). This distribution is

consistent with most studies showing that Girk channels selectively

mediate the postsynaptic inhibitory effects of neurotransmitters and

related drugs, while making little or no contribution to their

presynaptic inhibitory effects [121]. Some ultrastructural and

functional data, however, support the contention that Girk channels

contribute to presynaptic inhibition in some neurons [122–124]. Girk

channels of distinct subunit composition can also exist within

specific subcellular compartments of the same neuron. For exam-

ple, Girk1 and Girk2 show extensive colocalization across the

different dendritic layers of the hippocampus [120]. Their expression

levels vary among dendritic regions innervated by distinct synaptic

inputs in CA1 pyramidal cells, showing a significant increase from

proximal to distal dendrites. By contrast, Girk3 is more uniformly

distributed along the cell surface of pyramidal cells, including the

presynaptic terminal [10]. In addition, Girk2 and Girk3 are evenly

distributed along the postsynaptic density (PSD) of pyramidal cells

and Purkinje cells, whereas Girk1 is absent from excitatory synapses

and only observed at perisynaptic sites [10,96,120,123]. Finally, in

the subpopulation of parvalbumin-expressing interneurons consist-

ing mainly of basket and chandelier cells, Girk1, Girk2, and Girk3

share the same localization at very similar densities [125]. Collec-

tively, these data argue that distinct Girk channel subtypes exist in a

tissue/cell type- and subcellular compartment-dependent manner, a

scenario that enhances the prospects for selective and efficacious

therapeutic manipulation of Girk signaling.

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output to GABAB receptor activation, and helps to explainthe intriguing pharmacodynamic differences between theGABAB receptor agonists baclofen (an anti-craving com-pound) and g-hydroxybutyric acid (GHB, a drug of abuse)[28].

Macromolecular organization of Girk signalingGirk channels are thought to exist in multiprotein com-plexes that include GPCRs, G proteins, and regulatoryproteins [1,2,31], a consensus that has emerged despitethe fact that relatively few protein–protein interactionsinvolving Girk channels have been demonstrated usingclassical biochemical approaches or native systems. In-deed, most reported interactions have involved overexpres-sion of Girk subunits and putative binding partners in celltypes that do not normally express Girk channels. Never-theless, data obtained using these approaches have beenvaluable in supporting a conceptual model that aligns withkey functional properties of GPCR–Girk signaling (e.g., Gi/o

coupling specificity, signaling kinetics).

A core signaling complex: GPCR–Gabg–Girk

The interaction with Gbg represents, from a structural andfunctional perspective, the best understood of the protein–protein interactions involving Girk channels. Gbg binds toGirk channels, strengthening the interaction between thechannel and PIP2, a required cofactor for channel gating[32,33]. Although biochemical and structure–functionapproaches have suggested multiple interaction domainsfor Gbg in all four Girk subunits [1,34], clear resolution ofthis crucial interaction was obtained recently with thecrystallization of a complex formed by Gbg and the Girk2homomer [16] (Figure 1B). Girk2 homomers possess fourbinding sites for Gbg found at the well-conserved cyto-plasmic interfaces between adjacent subunits that contrib-ute to formation of the extended cytoplasmic pore; Gbg

promotes a ‘pre-open’ state of the channel that is interme-diate between the closed state of the channel and the openconformation of a constitutively-active Girk2 homomer[15,16].

Girk channels also interact with Gai/o. The inactiveheterotrimeric G protein (Gai/o-GDP/Gbg), as well asGai/o-GDP and Gai/o-GTP, can bind to intracellulardomains of Girk channels [25,26,35–38]. Interactions be-tween Girk channels and Gai/o-GDP (either alone or in thecontext of the inactive heterotrimeric G protein) suppressbasal activity of Girk channels, while enhancing their Gprotein-dependent activation [25,39,40]. Moreover, the se-lective association between Girk channels and Gai/o likelyexplains in part the strict coupling specificity between Girkchannels and Gi/o-dependent signaling pathways in vivo[36,41]. This specificity may be reinforced further by selec-tive pre-coupling between certain GPCRs and Gai/o-GDP/Gbg [42], and/or by direct GPCR–Girk interactions [31].Indeed, several GPCRs that couple to Gi/o G proteins,including D2 and D4 dopamine receptors, and GABAB

receptors, have been shown to interact directly with Girkchannels [31,43,44].

Collectively, these studies support the vision of a com-pact core Girk signaling complex, wherein minor confor-mational changes triggered by agonist binding to GPCRunveils key protein–protein interaction interfaces [45].Organization of signaling elements within a multiproteincomplex affords several advantages over random, collision-based designs. The close spatial proximity of the relevantmolecules allows fast and efficient signaling, and the noiseor ‘crosstalk’ that might otherwise occur with nonspecificcollision events is minimized, creating a tailored intracel-lular response to an external stimulus. Moreover, thestrength or sensitivity of the signaling pathway can theo-retically be titrated, by altering macrocomplex composi-tion, to suit the needs of the cell under specificcircumstances. With respect to this latter point, twoGirk-interacting proteins warrant further discussion.

Sorting nexin 27 (SNX27)

Girk2c and Girk3 possess identical C-terminal class 1interaction motifs for PDZ domain-containing proteins (–ESKV) (Figure 1A). Using the distal C-terminal domain ofGirk3 as bait in an unbiased proteomic screen, sortingnexin 27 (SNX27) was identified as a Girk-interactingprotein [30]. SNX27 regulates the trafficking between cell

3

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N-terminus(A) (B)

0 100 200 300 400 500

Girk2A

Girk2C

Girk3

Girk4

ELETEEEE

EAEKAEAEDHEEEE

ESKV

Ser-9 VL

Amino acid sequenceC-terminus

PIP2

Turret

TM

CTD

Out

GγN-term Gβ

N-term

gg

Na+In

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Figure 1. Structure of G protein-gated inwardly rectifying potassium (K+) Girk channels. (A) Linear depiction of Girk channel subunits, including two prevalent Girk2 splice

variants expressed in the mouse nervous system. The four Girk subunits exhibit a high degree of homology in the membrane-spanning (M1, M2), extracellular (1, 2), and

pore (P) domains, with most inter-subunit differences observed in the distal N- and C-terminal domains. The Girk2 splice variants and Girk4 contain endoplasmic reticulum

(ER) export signals (ELETEEEE and EAEKAEAEDHEEEE, respectively) not found in the other subunits [7]. Girk2 also exhibits an N-terminal internalization motif (VL), whose

influence on channel trafficking is precluded by phosphorylation of Girk2(Ser9) [71]. Girk2C and Girk3 possess identical C-terminal PDZ interaction motifs (–ESKV). (B)

Structure of the Girk–Gbg complex from a side-view, with colors highlighting Girk2 (blue), Gb (red), Gg [green, with associated geranyl-geranyl (gg) lipid modification], PIP2

(yellow/orange sticks), Na+ ions (purple spheres), K+ ions (green spheres within the pore). Note that there are four Gbg binding sites per channel, and the Gbg facing the

viewer has been removed for clarity [16]. Other abbreviations: CTD, C-terminal domain; N-term, N-terminal domain; PIP2, phosphatidylinositol 4,5-bisphosphate; TM,

transmembrane domains.

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surface and endosome of an array of neuronal signalingproteins [46], and has been implicated in Down syndromeand addiction [47,48]. SNX27 is the only member of thesorting nexin family that has a PDZ domain and it recog-nizes class I PDZ-binding motifs [49].

Amino acids immediately upstream from the ESKVmotif in Girk3 are crucial for promoting the Girk3–SNX27 interaction, and preclude its interaction withpost-synaptic density protein PSD95 [30,50]. SNX27 alsocontains a Ras association (RA) and a lipid-binding phox-homology (PX) domain. All three functional domains arecrucial for the proper function of SNX27 – which in thecontext of Girk signaling involves targeting Girk3-contain-ing channels to early endosomes, effectively reducing thesurface expression of Girk channels, and enhancing theexcitability of host neurons [30,50,51] (Figure 2). Interest-ingly, although SNX27 can bind to Girk2c, the surfacedistribution of homomeric Girk2c channels is unaffectedby SNX27 expression, suggesting that another factor(s)may influence the trafficking fates of Girk channels [52].

The upregulation of b isoform of SNX27 (SNX27b) inresponse to in vivo exposure to the psychostimulants co-caine and amphetamine is particularly intriguing in lightof recent observations that GABAB–Girk signaling is weak-ened by acute psychostimulant exposure in VTA dopamineand GABA neurons, and following chronic cocaine treat-ment in layer 5/6 pyramidal neurons of the medial prefron-tal cortex (mPFC) [47,53–55] (discussed below). Similarly,the association between loss of SNX27 and Down syndrome[48] is interesting given that Girk signaling is enhanced inthe hippocampus and cortex of a mouse model of Downsyndrome [56,57], and because Girk2 triploid mice recapit-ulated many of the neurological phenotypes associatedwith this syndrome [58]. Although altered SNX27 expres-sion and/or function will certainly impact upon a widearray of cell signaling pathways, these studies arguethat SNX27-dependent alterations in Girk signaling may

4

contribute to some of the cellular and behavioral deficitslinked to psychostimulant addiction and Down syndrome.

R7 RGS proteins

GPCR–Girk signaling is negatively modulated by RGS(regulator of G protein signaling) proteins [1,2]. RGS pro-teins enhance the GTPase activity of Ga subunits, acceler-ating the deactivation of G protein signaling followingagonist removal [59]. Accordingly, RGS proteins acceleratereceptor-induced Girk current deactivation kinetics,among other influences [60]. Discrete protein modulesconfer unique functionality to specific RGS proteins [59],and these – together with their unique cell/tissue expres-sion patterns – appear to promote interactions with Girkchannels. For example, the selective expression of Rgs2 inVTA dopamine neurons, and its preferential associationwith Girk3, weakens the coupling between GABAB recep-tors and the Girk2/3 heteromer [28]. Interestingly, changesin the expression of Rgs2 in response to chronic GHB (andmorphine) treatment correlated with altered GABAB–Girkcoupling in VTA dopamine neurons, and this neuroadapta-tion may contribute to the development of tolerance toGHB and other drugs of abuse.

Recent data show that Girk channels are modulated byRGS proteins in the R7 subfamily, which includes Rgs6,Rgs7, Rgs9, and Rgs11. The R7 RGS proteins possess adomain resembling the G protein Gg subunit (Gg-likedomain, or GGL) that promotes association with the atypi-cal 5th member of the G protein Gb family, Gb5 [61]. Thecrystal structure of the Rgs9–Gb5 complex reveals that theinteraction between GGL and Gb5 resembles that ob-served in conventional Gbg dimers [62]. Not surprisingly,therefore, complexes formed between R7 RGS proteins andGb5 (Rgs/Gb5) bind to Girk channels and modulate thekinetics of M2 muscarinic receptor/Girk signaling in atrialcardiomyocytes (Rgs6/Gb5) [63,64] and GABAB receptor/Girk signaling in hippocampal CA1 pyramidal neurons

EE

Girk2Key:Girk2

Girk2 Girk2Girk3

Girk1Girk3 SNX27Girk1

EE

RE RE

LE-LyLE-Ly

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Figure 2. Subunit-dependent regulation of Girk channel trafficking by sorting nexin 27 (SNX27). Neuronal Girk channels are thought to internalize into early endosomes

(EE), at which point they can be recycled back to the cell surface via recycling endosomes (RE) or diverted to late endosomes/lysosomes (LE-Ly) for degradation. Via a

selective physical and functional association with Girk3, SNX27 enhances the trafficking of Girk3-containing channels to early endosomes, leading ultimately to increased

lysosomal degradation and consequently, reduced numbers of Girk channels on the cell surface (right side of schematic). By contrast, SNX27 does not influence the

trafficking of Girk2 homomers or Girk1/2 heteromers (left side of schematic).

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(Rgs7/Gb5) [44,65]. Moreover, Gb5 ablation enhanced thesensitivity of Girk channels to GABAB receptor stimulationin hippocampal neurons, indicating that Rgs/Gb5 com-plexes can also negatively influence GPCR–Girk couplingefficiency [65]. Interestingly, Rgs6/Gb5 complexes appearto modulate GABAB–Girk signaling in cerebellar granulecells [66], showing that complexes containing either Rgs6or Rgs7 are relevant to neuronal Girk signaling. In addi-tion, the Rgs/Gb5–Girk channel interaction may be con-trolled by the R7 RGS-binding protein R7BP [67],providing another layer of fine regulation of GPCR–Girksignaling and another potential therapeutic target.

Loss of the Rgs6/Gb5-dependent modulation of Girksignaling in the heart correlates with bradycardia andenhanced parasympathetic influence [63,64]. Because dys-regulation of the parasympathetic control of cardiac outputhas been linked to sick sinus syndrome, heart failure, andarrhythmia [68], selective targeting of the Rgs6/Gb5–Girkaxis may prove beneficial in clinical settings involvingcardiac disorders. Although the relevance of the Rgs7/Gb5-dependent modulation of GABAB–Girk signaling inthe hippocampus is not understood, mice lacking Gb5 weremore sensitive to the sedative effect of the GABAB agonistbaclofen [65]. Moreover, mice lacking R7BP exhibited en-hanced thermal nociceptive thresholds and augmentedanalgesic effects of opioids and baclofen, consistent withits proposed role as a facilitator of the Rgs/Gb5-dependentregulation of GPCR–Girk signaling [67]. Because en-hanced Girk signaling has been linked to depotentiation[69], a particular form of excitatory synaptic plasticity, andwith cognitive deficits associated with Down syndrome[58], it will be particularly important to explore the rela-tionship between the Rgs7/Gb5 regulation of Girk signal-ing and of hippocampal-dependent learning and memory.Moreover, it will be interesting to probe the involvement ofRgs/Gb5 complexes in other neuronal GPCR–Girk signal-ing pathways.

Plasticity of Girk signalingRecent work has shown that the strength and sensitivity ofneuronal Girk signaling is titratable and subject to regu-lation by multiple stimuli. The first clear example of Girksignaling plasticity came with the demonstration thatstimulation protocols that evoked NMDA receptor-depen-dent long-term potentiation (LTP) of glutamatergic neuro-transmission in hippocampal CA1 neurons alsostrengthened synaptic GABAB–Girk signaling [70]. Subse-quent work in cultured hippocampal pyramidal neuronsrevealed that neuronal activity triggered by NMDA recep-tor activation leads to a rapid increase in the levels of Girkchannels (Girk1/2) on the somatodendritic membrane, andenhanced Girk signaling via the A1 adenosine receptor[71]. Pharmacologic or genetic ablation of Girk signalingsuggested that this neuroadaptation is crucial for thedepotentiation of excitatory LTP [69].

Drugs of abuse

As documented in Table 1, Girk signaling shapes many ofthe behavioral effects of drugs of abuse, including opioids,psychostimulants (cocaine), and ethanol. Exposure todrugs of abuse can alter neuronal Girk signaling in durablefashion in the reward circuitry, the core of which consists ofinterconnected neurons in the VTA, mPFC, and nucleusaccumbens (NAc) [72]. For example, a single non-contin-gent exposure to cocaine suppressed GABAB–Girk signal-ing by 50% in VTA dopamine (but not SNc dopamine)neurons for several days [53], paralleling the better-under-stood enhancement of glutamatergic neurotransmissionoccurring in the same neurons [72]. Acute psychostimulantexposure also persistently suppressed GABAB–Girk sig-naling in VTA GABA neurons [54]. Finally, repeated co-caine exposure suppressed GABAB–Girk signaling in layer5/6 glutamatergic pyramidal neurons of the mPFC [55], themain source of glutamatergic input to the VTA and NAc.This neuroadaptation was specific for pyramidal neurons

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in the prelimbic cortex (as compared to pyramidal neuronsin the infralimbic and motor cortices), and persisted formore than 1 month after the final cocaine injection. More-over, persistent suppression of Girk signaling in layer 5/6of the mPFC presensitized mice to the motor-stimulatoryeffects of acute cocaine. Although more work is necessary tounderstand the behavioral relevance of drug-inducedadaptations in Girk signaling in the reward circuitry, theseearly insights, together with the anatomic and cellularspecificity of the neuroadaptations, and their durability,suggest that they drive and/or contribute to the persistentexpression of addictive behaviors including drug-seeking,craving, and relapse.

Mechanism(s) of plasticity

Plasticity of Girk signaling triggered by neuronal activityand drugs of abuse involves the redistribution of Girk2-containing channels to and from the surface membrane(Figure 3). Enhanced Girk signaling in hippocampal

Girk

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Figure 3. Plasticity of neuronal Girk signaling. Cocaine-induced suppression of Girk sign

(D–F). (A) A single cocaine injection reduced baclofen-induced (GABAB receptor-depende

days, required activation of D2 dopamine receptors, and correlated with a redistribution

intracellular sites (B,C) [53]. (D) Repeated cocaine administration suppressed baclofen-in

more than 1 month, required activation of D1 dopamine receptors, and correlated with a

receptors from the cell surface to intracellular sites (D,F) [55]. The blue line in the current

antagonist.

6

pyramidal neurons triggered by NMDA receptor activationwas linked to enhanced trafficking of Girk2-containingchannels from recycling endosomes to the cell surface[71]. By contrast, increased intracellular labeling at theexpense of surface labeling was seen for Girk2 in VTAdopamine and GABA neurons following acute cocaine andmethamphetamine treatment [53,54], respectively, and inlayer 5/6 pyramidal neurons following repeated cocaine[55]. Interestingly, a similar redistribution of the GABAB

receptor from the surface to inside the cell was observed inVTA GABA and layer 5/6 mPFC pyramidal neurons, butnot in VTA dopamine neurons, suggesting that differentmechanisms mediate Girk signaling plasticity in differentcell types. Consistent with this premise, the methamphet-amine-induced adaptation in VTA GABA neurons andcocaine-induced adaptation in layer 5/6 mPFC pyramidalneurons were both blocked by pretreatment with a D1

dopamine receptor antagonist, whereas the cocaine-induced adaptation in VTA dopamine neurons was D2

(C)

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compartment

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TRENDS in Neurosciences

aling in the ventral tegmental area (VTA) (A–C) and medial prefrontal cortex (mPFC)

nt) Girk currents in VTA dopamine neurons, an adaptation that persisted for up to 5

of Girk2-containing channels (but not of GABAB receptors) from the cell surface to

duced Girk currents in layer 5/6 pyramidal neurons, an adaptation that persisted for

phosphorylation-dependent redistribution of Girk2-containing channels and GABAB

traces shows that the baclofen-induced current was reversed by a GABAB receptor

Box 2. Outstanding questions

Pharmacology. The recent identification of the subunit-selective,

efficacious, and potent direct modulators of Girk signaling repre-

sents an important step forward in this field, and should provide a

foundation upon which efforts in synthetic chemistry, molecular

modeling/simulations, and crystallography can synergize to yield

new compounds with optimized pharmacodynamic and pharmaco-

kinetic properties. When available, these compounds will greatly

facilitate efforts directed at understanding the physiological rele-

vance of Girk channels, permitting us to move beyond studies in

mutant mice.

Macrocomplex formation. Although a large body of evidence

supports the existence of discrete signaling complexes containing

Girk channels in vivo, gaps in our understanding of Girk channel

trafficking and regulation argue that additional proteins that

influence Girk function – either via direct or indirect physical

interactions – remain to be discovered. Moreover, understanding

the molecular determinants of macrocomplex formation will be

helpful because it can potentially provide the means to manipulate

Girk signaling selectively in a subtle manner.

Physiological relevance. Further investigation into the physiolo-

gical relevance of Girk channels is necessary to understand better

the opportunities for beneficial therapeutic manipulation. The

development of novel pharmacological tools that can discriminate

among the various Girk channel subtypes existing in vivo will

facilitate this process, and will complement next-generation genetic

approaches that can give region- and cell type-dependent insights

into the function of Girk channels formed by distinct combinations

of subunits (including specific splice isoforms). In addition, efforts

that build on the growing evidence linking mutations or polymorph-

isms in GIRK genes to human disease will be invaluable.

Plasticity. We are only now beginning to understand the triggers

and mechanisms underlying the plasticity in Girk signaling. Going

forward, it will be important to understand more about the

mechanisms involved in the dynamic modulation of Girk channel

trafficking, including differences that may exist between distinct

drugs of abuse, neuron populations, Girk channel subtypes, and

GPCR–Girk combinations. It will be particularly interesting to

explore the potential relationship between SNX27, the phosphor-

egulation of Girk trafficking, and the drug-induced adaptations in

Girk signaling seen in the reward circuitry. Of course, understanding

the physiological (and perhaps pathophysiological) relevance of the

adaptations is the ultimate goal.

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dopamine receptor-dependent. Moreover, GABAB–Girksignaling (but not somatodendritic inhibitory signalingvia D2 dopamine or a2 adrenergic receptors, or somatoden-dritic GABAB-dependent signaling that did not involveGirk channels) was suppressed by repeated cocaine inlayer 5/6 mPFC pyramidal neurons, whereas Girk signal-ing via both the GABAB and D2 dopamine receptor wassuppressed by acute cocaine in VTA dopamine neurons.These observations are reminiscent of the selective en-hancement by neuronal activity of Girk signaling via theA1 adenosine (but not GABAB) receptor [69], and suggestthat some forms of Girk signaling plasticity are compart-mentalized within neurons, and are presumably driven bythe GPCR or other proteins within the signaling macro-complex.

The trafficking of Girk channels and GPCR–Girk com-plexes to and from the cell surface that underlies the formsof plasticity described above appears to be regulated byphosphorylation. For example, the potentiation of synapticGABAB–Girk signaling triggered in parallel with LTP ofglutamatergic neurotransmission was dependent on Ca2+/calmodulin-dependent protein kinase (CaMKII) activity[70]. In addition, in cultured hippocampal neurons, CaM-KII activation (via prolonged morphine treatment, activa-tion of metabotropic glutamate receptors, or aconstitutively active CaMKII mutant) shifted the distribu-tion of Girk2 from dendritic shafts to spines, leading toenhanced Girk signaling via 5-HT receptors and decreasedGABAB–Girk signaling [73]. Although the direct moleculartarget of CaMKII was not determined in these studies,Girk2(Ser9) is a reasonable candidate. Phosphorylation ofGirk2(Ser9), which sits upstream of a unique internaliza-tion motif (VL) [7], suppresses surface trafficking of Girk2-containing channels [71]. Conversely, dephosphorylationof Girk2(Ser9) via protein phosphatase 1 (PP1) promotessurface trafficking of Girk2-containing channels from recy-cling endosomes, explaining the activity-dependent poten-tiation of Girk signaling linked to the depotentiation ofLTP [69,71]. Although dephosphorylation of Girk2(Ser9)promotes enhanced surface distribution of Girk channels,psychostimulant-induced suppression of GABAB–Girk sig-naling in VTA GABA and layer 5/6 mPFC neurons is morelikely related to the phosphorylation status of the GABAB

receptor [54,55]. Surprisingly, despite the durable natureof the drug-induced suppression of GABAB–Girk signalingin these pyramidal neurons, acute intracellular treatmentwith okadaic acid, an inhibitor of protein phosphatasesPP1/PP2a, restored normal Girk signaling.

Pharmacologic manipulation of Girk channelsGiven the broad distributions and roles of Girk channels inthe nervous system, and their contributions to cardiac andendocrine physiology, global and direct pharmacologicmanipulation of Girk signaling should evoke an array ofconsequences, many undesirable (Table 1). Indeed, globalconstitutive ablation of Girk2 triggers an array of pheno-types, most notably a shortened lifespan due to spontane-ous lethal seizures [74]. Although constitutive ablation ofother Girk subunits correlates with less-severe pheno-types, the full therapeutic potential associated with inhi-biting or enhancing Girk signaling will likely not be

achieved without regional and/or Girk subunit-selectivemanipulation. For example, an agonist with selectivity forGirk2/3 heteromers, the Girk channel subtype thatappears to be uniquely expressed in VTA dopamine neu-rons [28], could be useful as an anti-craving compound. Inaddition, drugs that selectively activate or inhibit Girk1/4heteromers, and which cannot pass the blood–brain barri-er, could represent efficacious therapies for particulartypes of arrhythmias. Indeed, two drugs that can inhibitGirk1/4 heteromers (NTC-801 and NIP-151) showed prom-ise in preclinical studies in the treatment of atrial fibrilla-tion [75,76].

Although Girk channels are activated in a G protein-independent manner by ethanol [77,78], volatile anes-thetics [79,80], and the flavonoid naringin [81], and areblocked by an array of psychoactive compounds (many ofwhich are clinically-relevant) (e.g., [82]), most of the com-pounds have other primary molecular targets and/or thereis little evidence for Girk subtype-specificity or pharmaco-kinetic advantages. Recently, however, a new class ofsubunit-selective, efficacious, potent, and direct-actingGirk channel agonists and antagonists was identified[83–85]. The prototype (ML297) is strikingly selective for

7

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Girk1-containing heteromers, and was efficacious in micein delaying seizure onset in a maximal electroshock modelof epilepsy, and was able to prevent convulsions andlethality in a chemically induced epilepsy model [84].Accordingly, this family of compounds and derivativesshould afford an excellent opportunity to investigate thepotential therapeutic benefits associated with direct acti-vation or inhibition of Girk1-containing channels, and mayserve as a platform for the identification of compounds thatcan discriminate between a wide range of channel sub-types.

Concluding remarksAlthough several outstanding questions remain (Box 2),efforts by many research groups over the last two decadeshave revealed key functional, structural, and regulatoryfeatures of Girk channels. This body of evidence, combinedwith our evolving understanding of Girk channel contribu-tions to physiology and disease, and a continually improv-ing capacity for pharmacologic manipulation, set the stagefor an exciting future of investigations into the therapeuticpotential of this interesting and important channel class.Such efforts hold the promise of yielding novel therapeuticapproaches to the treatment of many forms of neurological,cardiovascular, and neuroendocrine disorders.

AcknowledgmentsThis work was supported by National Institutes of Health (NIH) grants toK.W. (MH061933, HL105550, and DA034696) and by the SpanishMinistry of Science and Innovation BFU2012-38348 and CONSOLI-DER-Ingenio CSD2008-0000 (to R.L.). The authors thank members of theWickman and Lujan laboratories for their suggestions for the manuscript.

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