emerging roles for g protein-gated inwardly rectifying potassium (girk) channels in health and...

Much of the interplay between excitatory and inhibitory signals that are required for normal neuronal function occurs in the dendrites of neurons1. Detailed electron and light microscopic studies show that fast excitatory inputs are mediated by ionotropic glutamate receptors such as NMDARs (N-methyl-d-aspartate receptors) and AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors), located in the postsynaptic density at the head of the spine. By contrast, inhibi-tory synapses typically form on the soma and dendritic shafts1. The fast inhibitory signals are mediated by ionotropic GABAA (γ-aminobutyric acid type A) and glycine receptors. In addition to this fast inhibition, a slow inhibitory postsynaptic current (sIPSC) exists that is mediated by G protein-activated inwardly rectifying K+ (GIRK; also known as Kir3) channels that are located perisynaptically outside the postsynaptic density in the spine and on the shaft2. This Review discusses recent findings on the physiology, function and dysfunction of GIRK channels in the brain.

Physiology of GIRK channelsGIRK channels are members of a large family of inwardly rectifying K+ channels (Kir1–Kir7). The term ‘inward rectification’ refers to a change in slope of the current–voltage relationship of the channel at the reversal potential (that is, the zero current level, which occurs at

the equilibrium potential for K+ (EK)). At membrane potentials well above EK, the outward current is very small compared to the inward current at potentials well below EK. This rectification is due to occlusion of the cen-tral pore by intracellular Mg2+ and polyamines at poten-tials above EK (ref. 3). Under physiological conditions, the resting membrane potential of a typical neuron is positive to EK, and the small outward K+ current through GIRK channels decreases the excitability of a neuron (fIG. 1a). Various neurotransmitters, such as acetylcholine, dopamine, opioids, serotonin, somatostatin, adenosine and GABA, activate these channels by stimulating their cognate G protein-coupled receptors (GPCRs), which couple specifically to pertussis toxin (PTX)-sensitive heterotrimeric G proteins that activate GIRK channels4. Whether the activated Gα subunit or Gβγ dimer directly open GIRK channels was the focus of an animated debate in the 1980s (BOX 1). Now, a preponderance of evidence supports the conclusion that Gβγ dimers released from PTX-sensitive (Gαi and Gαo) G proteins bind directly to GIRK channels, causing them to open5–9 (BOX 1).

Building neuronal GIRK channelsMammals express four GIRK channel subunits: GIRK1 (also known as Kir3.1), GIRK2 (also known as Kir3.2), GIRK3 (also known as Kir3.3) and GIRK4 (also known as Kir3.4). In the brain, GIRK1–GIRK3 subunits are

*Department of Basic Neurosciences, Medical Faculty, University of Geneva, 1 Michel Servet, CH‑1211 Geneva, Switzerland.‡Clinic of Neurology, Department of Clinical Neurosciences, Geneva University Hospital, CH‑1211 Geneva, Switzerland.§Geneva Neuroscience Center, 1 Michel Servet,1211 Geneva, Switzerland.||Peptide Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.e‑mails: [email protected]; [email protected]:10.1038/nrn2834Published online14 April 2010

Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and diseaseChristian Lüscher*‡§ and Paul A. Slesinger||

Abstract | G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to activation of many different G protein-coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on the channel subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that GIRK activity has an important role in physiological responses, including pain perception and memory modulation. Moreover, abnormal GIRK function has been implicated in altering neuronal excitability and cell death, which may be important in the pathophysiology of diseases such as epilepsy, Down’s syndrome, Parkinson’s disease and drug addiction. GIRK channels may therefore prove to be a valuable new therapeutic target.

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Nature Reviews | Neuroscience

EKa b

c

Voltage (mV)

GIRK1–GIRK2

Low stimulation

d e

High stimulation

Anchoring

Glutamate

AMPAR orNMDAR

Action potential

GABAB –GIRK

GABAA

GABA

GIRK2–GIRK3

GIRK1–GIRK3

GIRK2–GIRK2

Relative current (%)

50

Vrest

–50–100

Basal

Agonist

GABAA

GABAB –GIRKGIRK2–/– GIRK2+/+

GA

BAB–

GIR

K (p

A)

GABAA (nS)

200 pA20 pA

200 ms

0

20

40

60

80

3 9 15 21 27 33 45 51 57 636939

20pA

200ms

fIPSC fIPSCsIPSC sIPSC

200ms200ms

25

25

50

75

100

High external[K+]

Figure 1 | Physiology of GirK channels in the mammalian brain. a | A hypothetical example of inwardly rectifying K+ currents through G protein-gated inwardly rectifying K+ (GIRK) channels. Current is plotted as a function of voltage. With physiological levels of extracellular K+, the current reverses (zero current potential) near –90 mV, which is the equilibrium potential for K+ (E

K). The basal current (shown in red) and agonist-induced current (shown in blue) display inward

rectification (large inward current and small outward current). The outward flow of current near the resting potential of the cell (shaded blue) hyperpolarizes the membrane potential of the neuron. With high extracellular K+ (for example, 20 mM KCl, a concentration commonly used to study GIRK channels), the current–voltage relationship shifts to the right to the new E

K

(dashed line), demonstrating a universal property of inwardly rectifying K+ channels. b | Three primary GIRK subunits exist in the brain and form heterotetramers (GIRK1–GIRK2, GIRK2–GIRK3 and GIRK1–GIRK3) and homotetramers (GIRK2–GIRK2)3,19–23. Less is known about GIRK4 in the brain. c | Generation of the slow inhibitory postsynaptic current (sIPSC). Two levels of synaptic activity are shown. Low stimulation of a GABA (γ-aminobutyric acid)-ergic neuron releases GABA at levels sufficient to only activate GABA

A channels (left panel). High stimulation (right panel) releases more GABA, which

accumulates in the synaptic cleft and diffuses to neighbouring GABAB–GIRK complexes, located on the shaft and dendritic

spine118–120. The resulting hypothetical fast IPSCs (fIPSCs) and sIPSCs from these two scenarios are shown below the synapse schematics. d | Electrophysiological recording of the fIPSC (GABA

A) and sIPSC (GABA

B–GIRK) from a hippocampal slice. The

relationship between the GABAA and GABA

B response is nonlinear, suggesting accumulation of GABA is needed to activate

GABAB receptors. For the scale bar, 200 pA refers to GABA

A receptors and 20 pA refers to GABA

B receptors. e | The GABA

B

receptor-dependent sIPSC is absent in the hippocampus of mice lacking GIRK2 channels. AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor; NMDAR, N-methyl-d-aspartate receptor. Part d is reproduced, with permission, from ref.119 © (2000) Cell Press. Part e is reproduced, with permission, from ref. 27 © (1997) Cell Press.

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Control ACh (bath) ACh (pipette)

a b c

GIRKGPCR

ACh

5 pA40 ms

G protein

common (fIG. 1b), whereas GIRK4 expression is low and therefore does not make a substantial contribution to cerebral GIRK currents10. The three splice variants of GIRK2 that are expressed in the brain differ in the length of the carboxy-terminal domain: GIRK2c con-tains a PDZ-binding motif that is absent in GIRK2a and GIRK2b11–14. For clarification, GIRK2a has been previously referred to as GIRK2 (ref. 14) and GIRK2-1 (ref. 15), and GIRK2c has been previously referred to as KATP-2 (ref. 16), GIRK2A-1 (refs 14,15) and BIR1 (ref. 17). GIRK1 splice variants exist in the brain but have not been investigated in detail18, and no splice variants of GIRK3 and GIRK4 have been reported. GIRK sub-units assemble into tetrameric channels in heterologous expression systems (for example, Xenopus laevis oocytes and HEK-293 cells) and native tissues3,19–23. Because GIRK1 and GIRK3 subunits are unable to independently form functional channels on the plasma membrane, they form heterotetrameric channels (GIRK1–GIRK3 and GIRK2–GIRK3)20,24,25 (fIG. 1b). By contrast, GIRK2

is unique in that it can form homotetramers11,22,23 and can assembles with other GIRK subunits to form hetero-tetramers, such as GIRK1–GIRK2 and GIRK2–GIRK3.

Biochemical and molecular genetic experiments indicate that the predominant form of GIRK chan-nels in the brain is a GIRK1–GIRK2 heterotetramer19. Whether asymmetrical arrangements of GIRK channel heterotetramers, such as a GIRK1–GIRK1–GIRK2–GIRK3, exist in neurons remains unknown. Studies with GIRK-knockout mice show that GIRK2 has a primary role in generating GIRK currents in neurons (TABLe 1); mice lacking GIRK2 channels exhibit little or no GIRK current in a number of brain regions (fIG. 1e; TABLe 1), including the hippocampus, cerebellum, sub-stantia nigra, locus coeruleus and ventral tegmental area (VTA)26–32. Interestingly, GIRK3-knockout mice are indistinguishable from wild-type controls in some behavioural tests33 (for example, open-field motor activ-ity and motor coordination) and have similar agonist-induced currents, but exhibit less severe withdrawal from

Box 1 | The great debate of the 1980s: activation of GIRK channels by Gβγ or Gα G protein subunits?

Over 20 years ago, an impassioned debate arose over the mechanism of G protein-gated inwardly rectifying K+ (GIRK) channel activation. At that time, the canonical pathway for G protein signalling involved GTP exchange for GDP on the Gα subunit, and subsequent Gα–GTP-dependent activation or inhibition of cellular enzymes, such as adenylyl cyclase or phospholipase C. By contrast, cardiac GIRK channels, referred to as I

KACh, were hypothesized to be activated directly by

G proteins through a membrane-delimited pathway4,166. One of the first suggestions of a novel signalling pathway came from studies using patch clamp recordings from rabbit atrial cells: bath application of acetylcholine (ACh) did not alter GIRK channel activity recorded in cell-attached patches (see the figure, part a versus part b), but when ACh was included in the recording pipette (see the figure, part c), GIRK channel activity greatly increased. These results suggested that activation of GIRK channels by G protein-coupled receptors (GPCRs) occurs through proteins anchored to the membrane and not through freely diffusible cytoplasmic second messengers.

Two groups examined whether GIRK channels were activated directly by G proteins. One group showed that application of purified native or recombinant Gα pre-activated with the non-hydrolysable analogue of GTP, GTPγS, led to robust GIRK channel activation167,168. Another group found that application of purified Gβγ subunits, and not Gα–GTPγS, directly activated GIRK channels5. Thus, there was agreement on the direct activation of GIRK channels by G proteins but little consensus on whether Gα or Gβγ subunits mediated receptor activation. With the cloning of GIRK subunits and improved protein expression and purification techniques, two papers published in 1994 provided new evidence for Gβγ-dependent activation. In one study, expression of recombinant Gβγ subunits led to sustained activation of GIRK channels, which was attenuated by a novel type of Gβγ binding protein, the β-adrenergic receptor kinase (βARK)7. A second study showed that different combinations of recombinant and purified Gβγ dimers directly activated GIRK channels6. Today, there is agreement that Gβγ subunits activate GIRK channels and, although there have been no further experiments showing that Gα activates GIRK channels, Gα

i and Gα

o G proteins are still considered important regulators of GIRK channels that affect

receptor specificity and basal channel activity82,84,85,92,99,100,169. Figure is reproduced, with permission, from ref. 166 © (2002) Springer.

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sedatives34 and reduced cocaine self-administration35. These findings suggest that GIRK3 may have a regula-tory role in GIRK signalling in the brain.

Single-channel recordings reveal that heterotetrameric and homotetrameric channels have different biophysical properties. Whereas GIRK2 homotetrameric channels have short open times (<0.5 ms mean open time), the heterotetrameric channels (for example, GIRK1–GIRK2, GIRK1–GIRK3 or GIRK1–GIRK4) display longer open times (~1–2 ms mean open times)3,11,23,25,36. The impor-tance of different single-channel kinetics is not clear, but the direct association of G protein Gβγ subunits with GIRK channels might alter the frequency of long open-ings, giving rise to large receptor-activated currents37,38.

Interestingly, the single-channel conductances of channels expressed in heterologous expression systems (38–40 pS) are fairly uniform compared with those observed in neurons, which show brain region-specific differences (TABLe 2). In native tissues, the subunit compo-sition is not known but single-cell reverse-transcription PCR data have recently provided information about possible types of heterotetramers (TABLe 2). Differences in conductance between native and heterologously expressed GIRK channels suggest that the subunit com-position of native channels might not be symmetrical (for example, two GIRK1 subunits, one GIRK2 subunit and one GIRK3 subunit) or that there could be channels composed of currently unknown subunits. More detailed single-channel and biochemical studies are needed to resolve these differences.

GIRK motifs: trafficking and binding partnersA number of intrinsic amino-acid sequences have been identified that control the intracellular trafficking of GIRK channels. GIRK2 contains a strong endoplasmic reticulum (ER) export signal (acidic residues) as well as

an internalization (Val-leu or ‘Vl’) motif 39, enabling this subunit to form homotetramers or heterotetramers. GIRK1, by contrast, lacks an ER export signal and must associate with another GIRK subunit (for example, GIRK2 or GIRK3) for expression on the plasma mem-brane24,39. Although GIRK3 cannot form functional homotetramers, it can co-assemble with GIRK1 or GIRK2 to form functional heterotetramers11,20,23,25. Unique to GIRK3, however, is a lysosomal targeting sequence (Tyr-Trp-Ser-Ile or ‘yWSI’) that promotes deg-radation of GIRK channels39. Both GIRK2c and GIRK3 contain a class I PDZ-binding motif and interact directly with a novel PDZ-containing trafficking protein, sorting nexin 27 (SNX27; also known as MRT1)40. Association of SNX27 with GIRK3-containing channels leads to a reduction of GIRK signalling at the plasma membrane, probably by promoting internalization of the channel40. It is unclear whether GIRK2c and GIRK3 also interact with classical PDZ-containing proteins, such as postsynaptic density protein 95 (also known as DlG4), in neurons41,42. The interplay of these trafficking motifs suggests that GIRK2 has a primary role in forming native GIRK cur-rents, whereas GIRK3 may regulate the availability of GIRK channels on the plasma membrane.

Although most neurons express GIRK1, GIRK2 and GIRK3, dopamine-containing neurons of the substantia nigra and VTA do not express GIRK1-containing channels. In substantia nigra dopaminergic neurons, for example, GIRK channels are comprised of GIRK2a–GIRK2c sub-units41. In the VTA dopaminergic neurons, some GIRK channels are heterotetramers of GIRK2c–GIRK3 subunits (ref. 43). It is not well understood why dopaminergic neu-rons express a unique combination of GIRK subunits, but GIRK2–GIRK3 channels are less sensitive to Gβγ subunits and have a higher effector concentration for half-maxi-mum response (EC50) for coupling to GABAB receptors

Table 1 | Summary of G protein-gated inwardly rectifying K+ (GIRK) channel-knockout mice

GirK knockout

VTA* sNc* cA1* Lc* spinal cord L ii*

cerebellum* Heart atrium*

Behavioural phenotype

GIRK1–/– (ref. 172)

NC26 NC28 ↓28 ND ↓31 ND ↓172 • Reduced score in anxiety behaviour testing33

GIRK2–/–

(ref. 145)↓26 ↓28 ↓27,28 ↓29,30 ↓31 ↓32 ND • Reduced anxiety with signs of hyperactivity33,173

• Abolishment of gender difference in latency for tail withdrawal to radiant heat137,138

• Reduced cocaine self-administration35

• Reduced conditioned taste aversion158

• Spontaneous convulsions and propensity for generalized seizures145,174

GIRK3–/–

(ref. 29)↓26 NC28 NC28 NC29

↓30NC31 ND ND • Decreased morphine- and clonidine-mediated

analgesia137

• Less severe withdrawal from sedatives34

• Reduced cocaine self-administration35

GIRK2–/–

GIRK3–/– (ref. 29)

↓26 ND ↓28 ↓29,30 ND ND ND • Attenuation of morphine withdrawal30

• Propensity for spontaneous and lethal seizures between 2 and 8 months of age29

• Increased cocaine self-administration35

GIRK4–/–

(ref. 175)ND ND ND ND ND ND ↓175 • Impaired performance in the Morris water maze10

GIRK, G protein-gated inwardly rectifying K+ channel; NC, no change; ND, not determined. *Data in these columns refer to agonist-evoked GIRK currents in these regions.

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than GIRK1-containing channels20,43. This difference in sensitivity has been linked to important changes in the output of the drug reward pathway (see below).

Several modulators have been described that alter GIRK channel activity, including Na+ (refs 44,45), ethanol46–48 and phosphorylation by protein kinase A (PKA)49,50 and protein kinase C (PKC)51–57. Both Na+ and ethanol seem to stimulate GIRK channels through a spe-cific binding site on the channel (see below and fIG. 2). PKC-dependent phosphorylation decreases, and PKA-dependent phosphorylation enhances, channel activ-ity49–57 (fIG. 2c). In addition to these modulators, changes in the levels of the membrane phospholipid phosphatidyl-inositol-4,5-bisphosphate (PtdIns(4,5)P2) can regulate the activity of GIRK channels58–60. GPCRs that couple to Gq G proteins stimulate phospholipase C (PlC) and deplete plasma membrane levels of PtdIns(4,5)P2, leading to desensitizing GIRK currents55,61,62 (but see also ref. 63). Activation of PlC also leads to stimulation of PKC, enabling crosstalk of these two pathways (PtdIns(4,5)P2 depletion and PKC-mediated phosphorylation) in the regulation of GIRK channels55,56.

Structural insights into GIRK functionAlthough a high-resolution three-dimensional structure of a full-length GIRK channel is not currently availa-ble, structures of the cytoplasmic domains of GIRK1, GIRK2 and a G protein-insensitive inwardly rectify-ing K+ channel (IRK1; also known as Kir2.1) have been solved64–66. Comparison of these structures highlights a conserved secondary structure for inwardly rectifying K+ channels, consisting of 14 β-strands and 2 α-helices (fIG. 2a,b). These cytoplasmic high-resolution structures have revealed a new structure, the G loop formed by the βH–βI sheet65, that is important in channel gating65,67,68, and identified several amino acids involved in regulating inward rectification and K+ binding64,65,69–71. The three-dimensional structure of a full-length chimeric channel, containing the transmembrane domains and pore of a bacterial inward rectifier channel and the cytoplasmic N-terminal and C-terminal domains of GIRK1 has been solved in two conformations72, revealing the rela-tive positions of the pore (selectivity filter), G loop and M2 gates, and the cytoplasmic interaction sites (fIG. 2a). Notably, these crystals revealed two positions of the G loop, which have been tentatively ascribed to putative open and closed states of the channel72. Although more studies are needed, a structural view of GIRK channel

gating is emerging that involves the N-terminal slide helix73, binding sites for K+ and polyamines69,70,71,74, and movement of the M2 and G loop gates65,69,72,73.

High-resolution structures of GIRK channels provide key information on the structural context of sites that were previously identified for G protein regulation. GIRK channels are predominantly closed at resting membrane potentials (agonist-independent, basal activity) and become activated upon stimulation of PTX-sensitive Gi and Go G proteins (producing a receptor-induced current). Both forms of activation involve binding of Gβγ directly to the GIRK channel8,9,36,75,76. Functional and biochemical studies have identified sequences in the N-terminal and C-terminal domains of GIRK1–GIRK4 that are involved in both agonist-independent and receptor-induced Gβγ activation9,75–82 (fIG. 2a,b). In particular, a leu residue (GIRK1-l333, GIRK2-l344 and GIRK4-l339) in the C-terminal domain of GIRK channels (βl–βM sheet) seems to have a crucial role in the Gβγ-dependent activation of GIRK channels77,80,81. Similarly, functional and biochemical studies have identified Gα-binding domains in the N-terminal and C-terminal domains of GIRK channels82–85.

Mapping these G protein interaction sites on the high-resolution structures provides clues to possible mecha-nisms of Gβγ gating (fIG. 2a,b). For example, many of the regions implicated in Gβγ-dependent activation are on the external side of the cytoplasmic domains, along an interface formed by two adjacent subunits (fIG. 2b). originating from different subunits, sequences from the N-terminal domain and the βD–βE and βl–βM sheets in the C-terminal domain contribute to this interface. An interaction between the N-terminal and C-terminal domains of two subunits has been shown to be impor-tant for assembly and gating of GIRK channels86. Recently, a hydrophobic pocket has been identified in this subunit interface and implicated in G protein-dependent activation of GIRK channels46. Fluorescence resonance energy transfer measurements (BOX 2) further reveal that Gβγ-dependent activation elicits a structural change, possibly a rotation, in the GIRK N-terminal and C-terminal domains87. Systematic mutagenesis studies have indicated that specific amino acids in the M2 transmembrane domain88–90 and G loop65,67,68 form a barrier to ion conduction in the closed state; thus, conformational changes triggered in the cytoplasmic domains must couple to the channel gates to open the channel. For example, the binding of Gβγ subunits to

Table 2 | Brain G protein-gated inwardly rectifying K+ (GIRK) channels

Brain region single-channel conductance composition

Pyramidal cells of the hippocampus111,176,177 33–38 pS GIRK1, GIRK2a, GIRK2c and GIRK3

Substantia nigra41,178 56 pS GIRK2a and GIRK2c

Locus coeruleus179–181 30 pS and 45 pS GIRK1, GIRK2, GIRK3 and GIRK4

Nucleus basalis181,182 32–39 pS GIRK1, GIRK2, GIRK3 and GIRK4

Ventral tegmental area43 Not determined GIRK1, GIRK2c and GIRK3

Medial prefrontal cortex57 30 pS Not determined

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c

Weavermutation site

G protein-andalcohol-bindingdomain

M2 gate

G loop gate

αi/o βγβγ

αq

RGS

PP1

PKA

CAMK2

OpenPTX

GPCR GPCR

SNX27

PSD95

PtdIns-(4,5)P2

GIRKK+

K+K+

PLC

PKC

TK

GIRK2

a b Gβγ-binding domain Gα-binding domain

Alcohol-binding domain

In

Out

βL-βM

L257βD-βE

βL-βM

βD-βE

Pore

M1

M2

Slide helix

N-terminus

N-terminus

N-terminus

Na+

PtdIns(4,5)P2

C-terminus

C-terminus

A323

C321G318H69

L344

L273

Mg2+

Polyamines

the channel could induce a conformational change involving the βl–βM sheet (containing the leu residue implicated in Gβγ activation), the N-terminal domain and the slide helix, which is then relayed to the M2 and G loop gates near the membrane. High-resolution

structures of Gβγ or Gαβγ complexed with full-length GIRK channels are needed to clarify our understanding of the molecular and structural mechanisms underlying Gβγ-dependent activation and to identify amino acids in the Gβγ and Gα subunits that interact with GIRKs.

Figure 2 | structural insights into gating and the formation of a macromolecular GirK signalling complex. a | A structure (ribbon model superimposed on a space-filled, electrostatic model) of a chimeric channel comprised of the bacterial inwardly rectifying K+ channel Kirbac1.3 and the G protein-gated inwardly rectifying K+ channel 1 (GIRK1)72, shown in a typical lipid bilayer. This structure contains the cytoplasmic domains (N- and C-termini) of GIRK1, and the transmembrane domains (M1 and M2) and pore region of Kirbac1.3. The structure of the GIRK1 cytoplasmic domain in this full-length chimeric protein is similar to those of other inwardly rectifying channels64–66. The K+ selectivity filter is the site of the mutation in weaver mouse models22,133,134. The general region implicated in Na+ and phosphatidylinositol-4,5-bisphos-phate (PtdIns(4,5)P

2) association is shown58–60,66,91. Two channel gates comprising the M2 transmembrane domain and

cytoplasmic G loop form a physical barrier to ion permeation65,67,68,88–90, with a constriction formed by the G loop gate65. Two opposing units are shown for clarity, as indicated by the cylinder cartoon. b | Modulation sites are shown from a side-on perspective in two adjacent subunits of GIRK2 (ref. 66). Highlighted are regions of the GIRK that are implicated in Gβγ-dependent activation (light and dark green β-strands; and residues H69, L273 and L344, shown in yellow)9,75–82, Gα

i and

Gαo association (red and pink β-strands; and residues G318, C321 and A323, shown in blue)80,84,85 and ethanol-dependent

activation (N-terminus, shown in purple; βD–βE sheet, shown in red; βL–βM sheet, shown in green; and residue L257, shown in blue)46. c | The schematic shows a macromolecular signalling complex that contains a G protein-coupled receptor (GPCR), which couples to pertussis toxin (PTX)-sensitive Gα

i and Gα

o G proteins, a GIRK channel, a regulator of G protein

signalling (RGS) protein, sorting nexin 27 (SNX27), and possibly postsynaptic density protein 95 (PSD95). Modulators of GIRK channels are also shown, including tyrosine kinase (TK), Ca2+–calmodulin-dependent protein kinase 2 (CAMK2), protein kinase A (PKA), protein kinase C (PKC) and protein phosphatase 1 (PP1)49–57,129,130,171. A second GPCR, which couples to the G

q pathway, is shown. Activation of this pathway stimulates phospholipase C (PLC), which leads to activation of PKC

and depletion of PtdIns(4,5)P2, both of which reduce GIRK channel activity55,61,62. PTX inhibits activation of GIRK channels

through Gαi and Gα

o G proteins4.

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High-resolution structures of inwardly rectifying K+ channels have also enabled visualization of the amino acids implicated in PtdIns(4,5)P2 binding, as well as pos-sible gating mechanisms for modulation by ethanol and Na+. Clusters of basic (positively charged) amino acids of the cytoplasmic domain58–60 are positioned close to the plasma membrane, where they are poised to interact directly with negatively charged membrane phospholipids and couple with the two gates (M2 and G loop) (fIG. 2a). Recently, a hydrophobic pocket formed by the N-terminal domain and the C-terminal βD–βE and βl–βM sheets has been identified as a site for ethanol-dependent activation of GIRK channels46. Bulky amino-acid substitutions of a leu residue located in the βD–βE sheet of the hydro-phobic pocket decreases ethanol-dependent activation46 (fIG. 2b). A structural analysis of GIRK2 has also provided a possible mechanism for Na+-dependent regulation of GIRK channels: Na+ may promote PtdIns(4,5)P2 binding to GIRK channels by breaking a hydrogen bond formed between an Asp residue and an Arg residue in the βC–βD sheet66,91. These high-resolution protein structures have provided important ‘snapshots’ of inwardly rectifying channels in different conformational states, as well as detailed maps for the location of functional domains. The next wave of high-resolution structural analysis should provide a more precise three-dimensional model of the conformational changes that underlie the gating mecha-nisms of inwardly rectifying channels.

A macromolecular signalling complexover the past few years, several studies have provided evidence for the existence of a macromolecular signal-ling complex on the plasma membrane. This complex is postulated to contain G proteins, GPCRs, GIRK channels and regulatory proteins92–97 (for a review, see ref. 2) (fIG. 2c). First, because GIRK channels remain open while Gβγ subunits are available, Gα subunits are expected to remain near the channel to terminate GIRK channel activation, through the formation of the inac-tive Gαβγ heterotrimer (BOX 1). Second, PTX-sensitive Gα subunits can associate directly with GIRK chan-nels and alter channel gating84,85,98,99. Gα may also be

involved in establishing receptor specificity such that stimulation of GPCRs that couple to only PTX-sensitive G proteins activates GIRK channels84,100. Third, spec-troscopic studies with fluorescently tagged GPCRs, G proteins and regulator of G protein signalling (RGS) proteins have provided details on the spatial relation-ship between proteins in this macromolecular signalling complex26,92,94,96,101,102 (BOX 2). Interestingly, spectro-scopic studies with Gαiβγ hetero trimers have suggested that G protein activation involves a conformational rearrangement of the Gαi and Gβγ subunits, in contrast to the canonical model whereby Gα and Gβγ subunits physically separate103. However, the ability to undergo rearrangement may be unique to Gαi, as other studies suggest Gαo dissociates from the Gβγ subunit104–107. Together, these studies support the view that GIRK channels reside in a macromolecular signalling complex that is governed by protein–protein interactions, limited diffusion and lipid domains2.

Recent studies have begun to investigate the regu-lation and localization of GIRK channels expressed in their native environment. In cerebellar granule cells, bio-chemically restricting the movement of μ-opioid recep-tors does not alter the rate of GIRK activation, revealing that these signalling proteins are localized in membrane compartments and cannot freely diffuse in the mem-brane97. Using neuronal PC12 cells, it was discovered that chronic stimulation of endogenous M2 muscarinic receptors (M2Rs) leads to downregulation of both the receptors and GIRK channels108, indicating M2Rs and GIRKs are co-regulated in a complex. Furthermore, GABA type B receptor sub unit 2 (GABABR2) has been shown to dimerize with the M2R and form an even larger functional M2R–GABABR2–GIRK heteromeric com-plex109. Electron microscopic immunohistochemical studies on brain tissue provide information on the locali-zation of native GIRK channels. In the hippocampus, GIRK channels are expressed on the dendritic shafts of hippocampal neurons110, which has been confirmed by cell-attached patch clamp recordings111. In addi-tion to the shaft, immuno-gold labelling shows a tight association of GIRK channels and GABAB receptors on

Box 2 | Evidence for a dynamic GIRK–GPCR macromolecular complex from spectroscopic studies

High-resolution spectroscopic studies measuring fluorescence resonance energy transfer (FRET) have been used to investigate the spatial proximity of proteins within the macromolecular complex comprising G protein-gated inwardly rectifying K+ (GIRK) channels, G proteins, regulator of G protein signalling (RGS) proteins and G protein-coupled receptors (GPCRs). FRET occurs when two fluorophores with overlapping spectral properties are aligned and <100 Å apart. If there is movement in the protein such that the distance (or orientation) between the two fluorophores changes, then FRET will increase when they move closer or decrease when they move apart. In a macromolecular complex, FRET measurements can be used to determine whether two specific proteins are closer than 100 Å to each other. FRET studies have indicated that some GPCRs pre-couple with G proteins and channels94,96,101 (but see also ref. 170). Similarly, GABA

B

(γ-aminobutyric acid type B) receptors and RGS proteins also produce detectable FRET with GIRK channels26,102. Using a similar technique, a significant bioluminescence resonance energy transfer (BRET) signal has been detected between tagged β2 adrenergic receptors and GIRK channels96. A recent FRET study using total internal reflection fluorescence microscopy surprisingly found that FRET efficiency decreased upon receptor activation92; an increase in FRET would be expected as Gβγ and GIRK channels move closer together. These findings support the model in which the Gαβγ heterotrimer associates directly with the GIRK channel at rest and the Gβγ subunit moves within a restricted region around the channel during activation. Collectively, these studies have provided important evidence for the existence of a macromolecular signalling complex containing GIRK channels, GPCRs, G proteins and RGS proteins.

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a Autaptic transmission

b Synaptic transmission

c Volume transmission

αi/o βγ

GPCR GIRKK+K+

K+

DA

↓ Synaptic activity



GABA Glu



Collision couplingThe process by which proteins diffuse in a two-dimensional bilayer and randomly collide.

dendritic spines110. In the cerebellum, immunoelectron microscopy studies show that GIRK2–GIRK3 chan-nels are localized in the postsynaptic density, whereas GIRK1–GIRK3 channels are perisynaptic112. An interest-ing question is whether the subcellular localization of the GIRK channel isoforms in neurons correlates with their Gβγ affinity (GIRK2–GIRK3 has a lower EC50 for Gβγ activation than GIRK1–GIRK3 (ref. 20)).

Future immunoelectron or super-resolution micro-scopy studies may reveal additional details about the localization of GIRK channels, GABAB receptors, RGS and G proteins in neurons, which will provide further insights into their function. However, discussions con-tinue on whether a GIRK macromolecular signalling complex exists in vivo. Additional studies are needed to clarify the role of collision coupling and whether changes in GIRK signalling occur at the level of the GPCR–GIRK macromolecular complex or individual components of the signalling pathway.

Physiological roles for GIRK channelsThe physiological activation of GIRK channels can shape the neuronal network behaviour in many areas of the brain at different levels. The basal activity of GIRK channels contributes to the resting potential of neurons, shifting the membrane voltage by approximately –8 mV27. This hyperpolarization of the resting membrane poten-tial decreases electrical excitability. In addition, receptor activation of GIRK channels provides another level of inhibition, to which three different changes in signalling can be generally ascribed.

Neuronal self-inhibition. Some neurons release a neuro-transmitter that activates Gi-coupled and Go-coupled receptors and, in turn, GIRK channels on their own dendrites (autaptic transmission), causing self-inhibition (fIG. 3a). For example, a train of action potentials fired by low-threshold spiking (lTS) interneurons of the cortex results in long-lasting hyperpolarization. This change in excitability occurs in a cell-autonomous way through endocannabinoids released from dendrites and the activation of cannabinoid receptor 1 coupled to GIRK channels on the same dendrites113. This modu-lation of lTS neurons may lead to long-lasting changes in cortical networks owing to altered glutamate trans-mission in pyramidal neurons. Dopaminergic neurons may also exhibit a form of autaptic inhibition: dendro-dendritic release of dopamine activates dopamine D2 receptors coupled to GIRK channels, leading to suppression of firing114.

Neuron-to-neuron inhibition. Activation of post synaptic GABAB receptors115, D2 receptors116 and group II metabotropic glutamate receptors117 by transmitters released from neighbouring neurons (synaptic transmis-sion) may also activate GIRK channels. For GABAergic synapses, GABA released spontaneously or driven by a single action potential produces a fast inhibitory post-synaptic potential, mediated by GABAA channels (fIG. 1c). By contrast, strong or repetitive stimulation is required to elicit the slow inhibitory potential, suggesting that GABA released into the synaptic cleft diffuses and acti-vates perisynaptic GABAB receptors coupled to GIRK channels118–120 (fIGs 1c, 1d, 3b). Thus, GABAB receptors and GIRK channels could be positioned directly adjacent to synaptically localized GABAA receptors or in a neigh-bouring dendritic spine (for a review, see ref. 121). The slow time course of the outward current through GIRK channels therefore correlates with the time required for

Figure 3 | Three types of neuronal signalling pathways for GirK channels. a | In autaptic synapses, neurotransmitters such as dopamine (DA) bind to the same neuron from which they were released, stimulating G protein-coupled receptors (GPCRs) and activating G protein-gated inwardly rectifying K+ (GIRK) channels. The net effect of such an autaptic synapse is a reduction in the release of neurotransmitter (negative feedback). b | The slow inhibitory postsynaptic current is generated when high-frequency stimulation triggers release of GABA (γ-aminobutyric acid) from the presynaptic neuron that spills over to neighbouring GABA

B–GIRK complexes,

located on the dendritic spine and shaft. c | GIRK channels are involved in network modulation through the process of volume transmission. When a neurotransmitter is released from many neurons, the ambient concentration rises and the neurotransmitter diffuses to activate GIRK channels on target neurons. This leads to reduced network activity of neurons. Glu, glutamate.

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Epilepsy Cerebral cortex

Hipppcampus


Spinal cord

SNc

VTA PAG

DRG

LC

Cerebellum

Ataxia

Down’s syndrome

Learning and memory

Drug addictionParkinson’s disease Pain and analgesia

Apoptosis

diffusion of the neurotransmitter and is not limited by the speed of G protein activation. The reuptake or degra-dation of the neurotransmitter and the limited space for diffusion may affect the amplitude of the slow IPSC, thus regulating the degree of inhibition. In the hippocampus, generation of the slow IPSC is important for regulating the rhythmic activity of the network119. In midbrain dopaminergic neurons, slow activation of GIRKs through D2 receptors originates from local pooling of dendrodendritically released dopamine114,122. Future studies are needed to show a physiological role for the slow IPSC in vivo. In addition to postsynaptic activation,

recent characterizations of GABAB receptor-mediated presynaptic inhibition have revealed a component that is sensitive to a GIRK-specific channel inhibitor, ter-tiapin123, suggesting a presynaptic role for GIRK chan-nels in inhibiting neurotransmitter release2,124,125. Thus, GABA may inhibit presynaptic GABA release and con-currently elicit postsynaptic hyperpolarization.

Network-level inhibition. The regulation of ambient levels of several endogenous GPCR agonists (for exam-ple, adenosine and somatostatin) may have a modula-tory effect on large-scale neuronal networks through GIRK channel activation. This large-scale effect of neuromodulators is known as volume transmission. Moderate activation of GIRK channels in a population of neurons would be expected to reduce membrane excitability (fIG. 3c). For example, somatostatin, acting through somatostatin receptor subtype 5, might alter the oscillation behaviour of thalamic networks through postsynaptic activation of GIRK channels, together with presynaptic inhibition126. Similarly, endogenous adenosine may suppress gamma oscillations in the hippocampus127, possibly owing to the selective expres-sion of A1 receptors on pyramidal neurons, which also activate GIRK channels. Dopamine concentrations are difficult to measure after synaptic or dendritic release128 but can be modelled in the extracellular space based on known diffusion properties and the uptake efficiency of surrounding cells. The transmitter encounters pre-dominantly extrasynaptic dopamine autoreceptors on dopamine-containing axons and heteroreceptors on neighbouring cells. In this model, dopamine released from a single site can diffuse as far as ~8 μm, thereby activating many dopamine receptors on several neurons and affecting thousands of synapses simultaneously. Conditional-transgenic mouse lines or cell-specific knockouts of GIRK channel subunits are among the future studies that are required to specifically investi-gate the role of GIRK channels in signalling pathways beyond the direct hyperpolarization.

Moving channels to modulate GIRK currentsRecently, a plasticity of the slow IPSCs in response to GABAB and D2 receptor activation has been observed (fIGs 3b,4). For example, depolarization of the post-synaptic neurons to bring the membrane potential close to 0 mV increases the GABAB receptor-mediated IPSC several fold for the duration of the experiment129. This increased IPSC is independent of the activation of GABAB receptors but dependent on NMDAR activa-tion, similar to hippocampal NMDAR-dependent long-term potentiation (lTP) of AMPAR-mediated EPSCs. It has been suggested that GIRK currents become larger because additional channels are inserted130. Indeed, the activation of NMDARs in cultured dissociated hippo-campal neurons increases the surface expression of GIRK1 and GIRK2 subunit-containing channels in the soma and dendrites within minutes130. This insertion requires dephosphorylation mediated by protein phos-phatase 1 of a GIRK2 serine residue (Ser9) that pro-motes channel recycling. Increased expression of GIRK

Figure 4 | GirK channels are implicated in various disease states. The schematic shows a sagittal view of a rat brain, highlighting regions in which G protein-gated inwardly rectifying K+ (GIRK) channels have been implicated in neuronal disorders or diseases owing to changes in excitability (labelled in yellow) or cell death (labelled in grey). GIRK2-knockout mice develop spontaneous convulsions and show a propensity for generalized seizures145. Similar pro-convulsive effects are observed following intrathecal administration of the GIRK channel inhibitor tertiapin148. Therefore, changes in GIRK activity throughout the brain may contribute to epilepsy. Two mouse models of Down’s syndrome, the Ts65Dn mouse149 and the Ts1Cje DS mouse150, contain a gene duplication for Girk2 and show larger slow inhibitory postsynaptic currents mediated by GABA

B

(γ-aminobutyric acid type B) receptors than wild-type mice151, impairments in long-term potentiation and enhancement of long-term depression151. Although all neurons are affected, this phenotype may involve mostly hippocampal functions. GIRK function may therefore be important in Down’s syndrome and for learning and memory. GIRK- knockout mice show impairment in self-administration of cocaine35, an altered response to γ-hydroxybutyrate26, less severe withdrawal from sedatives34 and reduced conditioned place taste aversion for ethanol than wild-type mice158. These findings implicate GIRK channels in drug addiction through changes in the neuronal responses of the mesolimbic system, including the ventral tegmental area (VTA). GIRK channels may also have a role in pain: knocking out GIRK2–GIRK3 reduces the potency (coupling efficiency) of opioid-induced analgesia, but leaves efficacy (maximal response) intact30,137, effects that probably involve the periacqueductal grey (PAG), locus coeruleus (LC) and spinal cord. Chronic neuropathic pain can lead to tyrosine phosphorylation of GIRK1 subunits, which reduces basal GIRK activity171. The weaver mouse has been used to investigate the role of GIRK channels in ataxia and Parkinson’s disease. In these mice, GIRK2 channels contain a mutation that eliminates K+ selectivity and leads to degeneration of dopaminergic neurons in the midbrain substantia nigra pars compacta (SNc) and cerebellar granular neurons161. The gain-of-function phenotype in dopaminergic neurons is of clinical interest owing to its similarity to the degeneration in Parkinson’s disease. In dorsal root ganglion (DRG) cells, activation of the nerve growth factor receptor (also known as p75NTR) increases levels of phosphatidylinositol-4,5-bisphosphate, which activates GIRK2 channels and promotes programmed cell death through K+ efflux-induced apoptosis164.

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channels would reduce excitability, dampening signalling of hippocampal pyramidal neurons. For example, GIRK channel activation is implicated in the maintenance of lTP at glutamatergic synapses. In mice lacking GIRK2, the depotentiation of lTP in hippocampal neurons is impaired because adenosine A1 receptors fail to activate GIRK channels131.

By contrast, activation of dopamine D2 recep-tors causes long-term depression (lTD) of the dopamine-dependent slow IPSC116. This lTD is inhib-ited by the presence of the Ca2+ chelator BAPTA in the recording pipette and seems therefore to rely on a cell-autonomous postsynaptic effect (fIG. 3a). The D2-dependent reduction in inhibition would be expected to increase burst firing and dopamine release in termi-nal axon projections in vivo. less is known about the mechanism of this plasticity than the GABA-mediated plasticity discussed above, but it could involve desensiti-zation of D2 receptors, modulation of G protein function and/or GIRK channel availability (as GIRK channels can undergo rapid redistribution).

Taken together, these findings suggest that GIRK channels play a key part in many types of neuronal com-munication and, as outlined in the following sections, modification of GIRK channels can modulate CNS function in health and disease (fIG. 4).

GIRK channels in pain perception and analgesiaGIRK channels have been implicated in pain perception. This notion is based on studies in mice carrying muta-tions in GIRK channels, and the activation of GIRK chan-nels by analgesic drugs and endogenous pain modulators such as endorphins and endocannabinoids.

GIRK channels were first implicated in pain per-ception when opioid-induced analgesia was tested in weaver mice that carry a spontaneous mutation in the pore region of GIRK2 (GIRK2wv)132, leading to a loss of K+ selectivity and G protein insensitivity22,133,134. Upon receptor activation, GIRK2wv depolarizes rather than hyperpolarizes neurons. Morphine and a selec-tive κ-opioid agonist (U-50488), which are known to activate GIRK2-containing channels through opioid receptors, reduce pain perception in wild-type mice but not in the weaver mice135. However, the interpre-tation of the opioid-dependent behaviours is con-founded by the loss of neurons in the substantia nigra and cerebellum136.

Subsequent studies have taken advantage of GIRK channel-knockout mice (TABLe 1). In GIRK2–/– mice, for example, the dose–response curve for analgesia produced by morphine and clonidine (an α2 adren-ergic receptor agonist) is shifted to higher concen-trations, but the maximal effect is preserved137. This finding was confirmed in a recent study using GIRK2–GIRK3 double-knockout mice30; in these mice, the potency (coupling efficiency) of opioid-induced analgesia was reduced, but their efficacy (maximal response) was intact.

Knockout studies also suggest that GIRK channels may account for gender difference in nociception. The higher pain threshold in males, measured as a longer

latency for tail withdrawal to radiant heat, is abolished in GIRK2–/– mice137, indicating that GIRK channel acti-vation may account for some of the gender differences in normal pain perception. Further evidence for this notion came from a study that showed the analgesic effects of ethanol, oxotremorine (a muscarinic acetyl-choline receptor agonist), baclofen (a GABAB receptor agonist), clonidine and WIN 55,212-2 (a cannabinoid receptor agonist) were reduced or eliminated in male but not in female GIRK2–/– mice138. Studies using mice lack-ing GIRK1 or GIRK2 channels show a blunted response to μ-([d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin) (DAMGo) and ∂-(Tyr-d-Ala-Phe-Glu-Val-Val-Gly amide) but not the κ-opioid receptor agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulphonate hydrate when applied intrathecally139. Similarly, intra thecally admin-istered tertiapin, a selective GIRK channel inhibitor123, reduced the efficiency of DAMGo, increasing the latency for withdrawal in the immersion tail flick test. Therefore, although further studies are needed to clarify the role of GIRK channels in opioid-induced analgesia, there is general agreement that GIRK channels modu-late systemic opioid (or Gi and Go)-mediated analgesia through postsynaptic inhibition.

The periaqueductal grey (PAG), a small nucleus in the brainstem, has a crucial role in central analgesia. Direct application of opioids into the PAG inhibits GABAergic neurons through both GIRK-independent (presynap-tic) and GIRK-dependent (postsynaptic) mechanisms, which is sufficient to induce analgesia140,141. The disin-hibition of principal neurons of the PAG that project to nuclei of the caudal brainstem and modulate ascending pain pathways probably plays an important part in the analgesic effects of opioids.

GIRK channels have also been implicated in opioid tolerance and dependence, as the channels undergo adaptive changes during chronic exposure to morphine. However, desensitization of GIRK currents induced by opioid receptor agonists seems to involve additional mechanisms. In neurons of the locus coeruleus — an adrenergic nucleus in the pons that lowers pain thresh-old when activated — inhibiting signalling by both G protein-coupled receptor kinase 2 (GRK2; also known as ADRBK1) and extracellular signal-regulated kinase significantly reduces the met-enkephalin-induced GIRK channel desensitization that normally occurs within minutes of drug application142. However, GIRK channel desensitization may not occur when μ-opioid receptors are activated by morphine. Morphine causes less GIRK channel desensitization in vitro and in vivo143 than met-enkephalin. This response is surprising because met-enkephalin induces less drug tolerance and dependence than morphine. A recent report even suggests that the potency of morphine to reduce pain (that is, the effect at a submaximal concentration) may increase during the development of tolerance (that is, a reduction of maximal effect)144. This is paradoxical but could be explained if tolerance and dependence are mediated by effectors of opioid receptors other than GIRK channels.

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An alternate role of GIRK channels in opioid depend-ence is suggested by the following observations. Mice lacking GIRK2 and GIRK3 have strongly reduced with-drawal signs that are normally induced in wild-type con-trols by the opioid receptor antagonist naloxone after chronic exposure to morphine. In the mutant mice, the withdrawal syndrome can be rescued if the output from locus coeruleus neurons is inhibited during the expo-sure to morphine30. This suggests that GIRK channels ‘gate’ the induction of opioid dependence by silencing the neurons in the locus coeruleus every time opioids are injected.

Taken together, these data suggest that GIRKs have a role in pain perception under specific conditions, and that they contribute to opioid-induced analgesia. However, the literature remains divided on the role of the PAG and the locus coeruleus in the regulation of opioid- induced analgesia. Further insight may come from mouse models in which GIRK subunits are mutated in a cell type-specific manner (for example, only GIRK-expressing neurons in the locus coeruleus) or in which GIRK channels are coupled to light-activated GPCRs.

GIRK channels in diseaseGIRK channels are implicated in the pathophysiology of several diseases, including epilepsy, addiction, Down’s syndrome, ataxia and Parkinson’s disease (fIG. 4). Two broad principles have been distinguished. First, loss of GIRK function can lead to excessive neuronal excitability, such as in epilepsy, whereas a gain of GIRK function can considerably reduce neural activity, such as is postulated to occur in Down’s syndrome. Second, loss of selectiv-ity can cause aberrant ion fluxes across GIRK channels, such as aberrant Na+ influx that triggers cell death, which is exemplified in a model of Parkinson’s disease.

Changes in neuronal excitability. The involvement of GIRK channels in epilepsy has been inferred from the phenotype of mice in which a GIRK subunit has been deleted. GIRK2-knockout mice develop spontaneous convulsions and show a propensity for generalized seizures when injected with a pro-convulsive GABAA receptor antagonist145. Drugs such as desimipramine, fluoxetine, haloperidol, thioridazine, pimozide and clozapine146 (see Supplementary information S1 (table)), which are used for the treatment of various neurologi-cal disorders, may inhibit GIRK channels and hence cause seizures — a known side effect of these drugs. Conversely, electroconvulsive shock leads to increased expression of GIRK channels147, which may provide neuroprotection against inappropriate activity. In sup-port of this hypothesis, stimulation of galanin type 2 receptors that activate GIRK channels prevents kindled epileptogenesis in rats148, highlighting GIRK channels as a novel target for anticonvulsants.

Down’s syndrome is a genetic disorder caused by duplication of human chromosome 21, which contains the GIRK2 gene (KCNJ6). To investigate the contribu-tion of a third copy of KCNJ6 to the phenotype of Down’s syndrome, two mouse models have been generated that carry either a partial- or full-segment duplication of

the mouse chromosome 16, the orthologue of human chromosome 21 (refs 149,150). In both models, GIRK2 protein is upregulated, resulting in a larger slow IPSC mediated by GABAB receptors151. Moreover, hippocam-pal lTP is reduced and lTD is enhanced in both mod-els, and therefore synaptic plasticity of glutamatergic transmission is altered. As the duplicated chromosomal segment also contains other genes, additional studies are needed to specifically test the role of GIRK chan-nels in the malfunction of the nervous system in Down’s syndrome.

Addiction. GIRK channels are thought to have a role in the acute rewarding effects and/or the adaptation that occurs with chronic exposure to addictive drugs that work through GPCRs, such as opioids, the ‘club drug’ γ-hydroxybutyrate (GHB) and cannabinoids. Addictive drugs are known to strongly increase dopamine levels in the mesocorticolimbic system. Based on in vivo and in vitro experiments, distinct cellular mechanisms have been proposed for different classes of drugs152. opioids and GHB activate GIRK channels, leading to disinhi-bition of dopaminergic neurons. Morphine stimulates μ-opioid receptors that are selectively expressed on interneurons of the VTA and activate GIRK channels, reducing the firing of these cells and eventually lead-ing to disinhibition of dopaminergic neurons153. The effect of GHB is more complex, as GABAB receptors are expressed on both GABAergic and dopaminergic neurons. However, dopaminergic neurons in the VTA are an order of magnitude less sensitive (higher EC50) to GABAB receptor agonists than GABA interneurons43, probably owing to the selective expression of GIRK2c and GIRK3 and the lack of the GIRK1 subunit (discussed above). Consequently, low concentrations of GHB only activate GIRK currents in GABAergic neurons (which contain GIRK1 subunits), leading to disinhibition of dopaminergic neurons. In addition, dopaminergic neu-rons in the VTA also selectively express RGS2, which contributes to the low GABAB receptor–GIRK sensitiv-ity of these neurons26. Interestingly, chronic exposure to morphine or GHB enhances the coupling efficiency (EC50) of GABAB receptors and GIRK channels by reducing the levels of RGS2 in dopaminergic neurons26. Under these circumstances, the concentration window for disinhibition is narrowed and low concentrations of drug are sufficient to inhibit dopaminergic neurons and become behaviourally aversive26. Thus, the com-bination of GIRK subunits expressed by a neuron can determine the signalling properties of neuronal cir-cuits. In the case of cannabinoids, the disinhibition of dopaminergic neurons seems to occur primarily through presynaptic, GIRK-independent mechanisms154 (but see also refs 124,125).

GIRK channels may also be involved in the response to psychostimulants, such as cocaine and ampheta-mines. SNX27, a psychostimulant-inducible protein155, specifically regulates surface expression of GIRK2c and GIRK3 (ref. 40). GIRK2- and GIRK3-knockout mice exhibit dramatically reduced intravenous self-adminis-tration of cocaine relative to wild-type mice35 whereas,

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http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=190685



http://www.nature.com/nrn/journal/v11/n5/suppinfo/nrn2834.html

http://www.ncbi.nlm.nih.gov/gene/3763?ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum


surprisingly, GIRK2–GIRK3 double-knockout mice self-administer more cocaine than wild-type mice, sug-gesting compensation in the single-knockout mice that is no longer possible in the double-knockout mice. Elucidating the changes in GIRK signalling with psycho stimulants is an exciting area for future research and may lead to further insight into the role of GIRK channels in addiction.

Because ethanol directly binds and activates GIRK chan-nels46–48,156, GIRK channels may mediate some ethanol- related behaviours. Indeed, ethanol has been shown to enhance GABAB receptor-activated GIRK currents in neurons of the VTA156, which would decrease the firing frequency of dopaminergic VTA neurons in rodents. As discussed above, GIRK channels probably have a role in the analgesic effects of ethanol138,157, but whether they also contribute to the reinforcing effects of ethanol is less clear. GIRK2-knockout mice exhibit reduced con-ditioned taste aversion compared with wild-type mice, suggesting that GIRK activation could be involved in the motivational response of ethanol158. However, these differences were observed with moderate but not high doses of ethanol158. A different approach involved the identification of a quantitative trait locus (QTl) with large effects on the predisposition to physical depend-ence and on associated withdrawal from sedative hyp-notics such as ethanol. In these studies, the QTl was mapped to a 0.44 Mb region of mouse chromosome 1, which contains the gene encoding GIRK3 (Kcnj9)34. In a behavioural assay designed to measure withdrawal, GIRK3-knockout mice show less severe withdrawal symptoms from ethanol and barbiturate use34.

Based on these results, new approaches for addiction therapies have emerged and more may follow. For example, pharmacological activation of GIRK channels by administration of the GABAB receptor agonist baclofen reverses behavioural sensitization to morphine in rats159 and clinical Phase II and III trials are under way examin-ing the effectiveness of baclofen in treating ethanol and cocaine addiction.

Cell death owing to GIRK signalling. Parkinson’s disease is a debilitating motor coordination disorder caused by degeneration of dopaminergic neurons in the sub-stantia nigra. The involvement of GIRK channels in Parkinson’s disease was first inferred from the weaver mouse132, in which constitutively active GIRK2wv chan-nels produce chronic depolarization and cell death in a subset of neurons in the brain22,133,134, mimicking the neuronal degeneration observed in Parkinson’s dis-ease160. The gain-of-function phenotype in dopaminer-gic neurons of weaver mice is of clinical interest owing to the progressive degeneration of dopaminergic neu-rons in the substantia nigra. By contrast, dopaminergic neurons in the VTA are spared in the early stages of the disease161. Interestingly, dopaminergic neurons of the substantia nigra express only GIRK2, which results in a gain of function, whereas dopaminergic neu-rons in the VTA coexpress GIRK2–GIRK3, which may produce a loss of function owing to co-assembly with GIRK3 (refs 22,133). However, other mechanisms of

dopaminergic cell death in the weaver mouse have been proposed. one study has suggested that constitutively active GIRK2wv channels trigger activation of KATP (also known as Kir6.2) channels in response to mild mito-chondrial uncoupling in substantia nigra pars compacta dopaminergic neurons but not VTA dopaminergic neu-rons162. other studies suggest a general susceptibility to oxidative stress (owing to a reduced thiol redox state) in dopaminergic neurons163. Weaver mice have additional developmental abnormalities that result in a complex phenotype including learning deficits, epileptic seizures and motor disturbances such as ataxia.

Another line of evidence implicating GIRK channels in cell death comes from a recent study investigating nerve growth factor-mediated programmed cell death in dorsal root ganglion neurons, which is an important step in the development of the nervous system. This study shows that activation of nerve growth factor receptor (also known as p75NTR) increases plasma membrane levels of PtdIns(4,5)P2, activating GIRK2 channels and creating a sustained K+ efflux that stimulates pro-grammed cell death164.

Taken together, GIRK channels may contribute to the pathophysiology of various human diseases, either by altering the excitability of specific neurons, changing the slow IPSCs or promoting cell death. The suggested roles for GIRK channels in brain diseases are based on observations in animal models. Future genetic studies may provide direct evidence for altered GIRK function in human diseases.

ConclusionsResearch over the past 20 years has elucidated the molecular and structural determinants of GIRK chan-nel activation and modulation by G proteins (Gα and Gβγ), with the emerging concept of the existence of macromolecular signalling complexes. Knockout mice for all four mammalian subunits of GIRK channels have greatly contributed to the understanding of GIRK func-tions — namely, to regulate the excitability of neurons in a cell-autonomous fashion and to regulate synaptic transmission and the activity of large-scale networks. Although we are only beginning to understand the role of GIRK channels with different subunit compositions, their respective expression pattern in the brain will give further functional insight. For example, dopaminergic neurons of the VTA express a unique GIRK2–GIRK3 heteromeric channel that has a particularly low affin-ity for the Gβγ dimer. More research is needed to link such cellular observations to function and behav-iour. The development of novel tools — such as light- activated GPCRs (optoXRs)165 that couple to GIRK chan-nels, as well as conditional and cell-specific knockout mouse models — will be important to further advance our understanding of GIRK function. These tools will also be crucial for the investigation of GIRK channels in disease and may help to design specific drugs that selectively open or close GIRK channels with differ-ent subunit compositions. Such drugs may be useful for treating addiction, epilepsy, Down’s syndrome or Parkinson’s disease.

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AcknowledgementsWe thank P. Aryal for the three-dimensional structural figures and A. L. Lalive for compiling the Supplementary information table. Our work was support by McKnight Endowment Fund for Neuroscience (to P.A.S.), the Human Frontiers Science Foundation (to P.A.S. and C.L.), the NARSAD (to P.A.S.), the Swiss National Science Foundation (to C.L.), the National Institute of Neurological Disorders and Stroke (R01 NS37682; to P.A.S.) and the National Institute on Drug Abuse (R01 DA019022; to P.A.S. and C.L.). We apologize to our colleagues whose works could not be included owing to space limitations.

Competing interests statementThe authors declare no competing financial interests.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/geneKCNJ6OMIM: http://www.ncbi.nlm.nih.gov/omimDown’s syndrome | Parkinson’s diseaseUniProtKB: http://www.uniprot.orgGIRK1 | GIRK2 | GIRK3 | GIRK4 | GRK2 | RGS2 | SNX27

FURTHER INFORMATIONChristian Lüscher’s homepage: http://www.addictionscience.unige.ch/index.htmlPaul A. Slesinger’s homepage: http://www.salk.edu/faculty/slesinger.html

SUPPLEMENTARY INFORMATIONSee online article: S1 (table)

ALL LiNKs Are AcTiVe iN THe oNLiNe Pdf

R E V I E W S



http://www.ncbi.nlm.nih.gov/gene

http://www.ncbi.nlm.nih.gov/gene/3763?ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum

http://www.ncbi.nlm.nih.gov/omim



http://www.uniprot.org



http://www.uniprot.org/uniprot/Q92806




http://www.uniprot.org/uniprot/Q96L92

http://www.addictionscience.unige.ch/index.html

http://www.addictionscience.unige.ch/index.html

http://www.salk.edu/faculty/slesinger.html

http://www.salk.edu/faculty/slesinger.html

http://www.nature.com/nrn/journal/v11/n5/suppinfo/nrn2834.html

emerging roles for g protein-gated inwardly rectifying potassium (girk) channels in health and...

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