Summary. G protein-gated inwardly rectifying K+ 
(GIRK/Kir3) channels are mainly expressed in excitable 
cells such as neurons and atrial myocytes, where they 
can respond to a wide variety of neurotransmitters. Four 
GIRK subunits have been found in mammals (GIRK1-4) 
and act as downstream targets for various Gαi/o-linked 
G protein-coupled receptors (GPCRs). Activation of 
GIRK channels produces a postsynaptic efflux of 
potassium from the cell, responsible for hyper-
polarization/inhibition of the neuron. A growing body of 
evidence suggests that dysregulation of GIRK signalling 
can lead to excessive or deficient neuronal excitability, 
which contributes to neurological diseases and disorders. 
Therefore, GIRK channels are proposed as new 
pharmacological targets. The function of GIRK channels 
in neurons is not only determined by their biophysical 
properties but also by their cellular and subcellular 
localization patterns and densities on the neuronal 
surface. GIRK channels can be located within several 
subcellular compartments, where they have many 
different functional implications. This subcellular 
localization changes dynamically along the neuronal 
surface in response to drug intake and following 
plasticity processes. Ongoing research is focusing on 
determining the proteins that form macromolecular 
complexes with GIRK channels and are responsible for 
fast and precise signalling under physiological 
conditions, and how their alteration is implicated in 
pathological conditions. In this review, the distinct 
regional, cellular, and subcellular distribution of GIRK 
channel subunits in the brain will be discussed in view of 
their possible functional and pathological implications. 

Key words: GIRK, GPCR, G protein, Inhibition, 
Immunohistochemistry, Immunoelectron microscopy, 
Pathology 

 

 

Introduction 

 

 All neurons in the central nervous system (CNS) 
express an array of ion channels along their impermeable 
plasma membrane to directly affect the electrical 
excitability and/or to switch on intracellular signalling 
cascades. Ion channels differ in molecular structure, 
selectivity to ions, and how they operate. Broadly, ion 
channels can be selective for individual ions, i.e., Na+, 
K+, Cl-, and Ca2+ channels, or for groups of ions, i.e., 
anion and cation channels. The individual proteins that 
combine to form ion channels are diverse and underlie 
many different functions in addition to ion selectivity. 
One of the most important and broadest classes of ion 
channels are the potassium (K+) channels, which 
facilitate the movement of K+ ions across the membrane 
lipid bilayer (Gutman et al., 2005). In broad terms, K+ 
channels can be categorized into four different families: 
voltage-gated K+ (KV) channels, Ca2+-activated K+ 
(KCa) channels, two-pore K+ (K2P) channels, and 
inwardly-rectifying (Kir) channels (Gutman et al., 2005). 

 Unlike classical KV channels, which open and close 
upon changes in the membrane potential, the family of 
Kir channels conduct K+ ions generating currents that are 
predominantly inward rather than outward, and thus they 
contribute little to the formation of the action potential 
but instead have large effects on the resting membrane 
potential (Hibino et al., 2010). Thus, activation of Kir 
channels at resting membrane potential leads to 
hyperpolarization, decreasing cell excitability. These 
channels function in many tissues, including the brain, 
heart, kidney, endocrine, and sensory, where they play 
central roles in controlling neuronal signalling, 
membrane excitability, heart rate, vascular tone, and 

 

G protein-gated inwardly rectifying K+ (GIRK/Kir3) 
channels: Molecular, cellular, and subcellular diversity 

 

Alejandro Martín-Belmonte1,3,4, Carolina Aguado1,2, Rocio Alfaro-Ruíz1,2 and Rafael Luján1,2 

1Synaptic Structure Laboratory, Instituto de Biomedicina de la UCLM (IB-UCLM), Departamento de Ciencias Médicas, Facultad de 
Medicina, Universidad Castilla-La Mancha, Albacete, 2Laboratorio de Estructura Sináptica, Instituto de Investigación Sanitaria de 
Castilla-La Mancha (IDISCAM), 3Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine 
and Health Sciences, Institute of Neurosciences, University of Barcelona and 4Neuropharmacology and Pain Group, Neuroscience 
Program, Institut d’Investigació Biomèdica de Bellvitge, IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain

Histol Histopathol (2025) 40: 597-620

Corresponding Author: Rafael Luján, Synaptic Structure Laboratory, 
Instituto de Biomedicina de la UCLM (IB-UCLM), Departamento de 
Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La 
Mancha, Campus Biosanitario, C/ Almansa 14, 02008 Albacete, Spain. 
e-mail: Rafael.Lujan@uclm.es 

www.hh.um.es. DOI: 10.14670/HH-18-822

istology and 
istopathology

H

REVIEWOpen Access

©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.


From Cell Biology to Tissue Engineering



insulin release (Hille, 2001). There are seven Kir 
subfamilies (Kir1-Kir7), which are further subdivided 
into subtypes, based on their biophysical properties: 
classical inwardly rectifying K+ channels (Kir2), G 
protein-activated K+ channels (Kir3), ATP-sensitive K+ 
channels (Kir6), and ATP-dependent K+ channels (Kir1 
and Kir4) (Hibino et al., 2010). The Kir3 subfamily, also 
known as G protein-coupled inwardly rectifier potassium 
(GIRK) channels, is of special importance due to its 
direct coupling to heterotrimeric G proteins, thus 
mediating the inhibitory effect of a wide variety of 
GPCRs on neuronal excitability (Dascal, 1997; Kubo et 
al., 2005; Luján et al., 2009, 2014; Jeremic et al., 2021; 
Luo et al., 2022). 

 Research over the past two decades has largely 
contributed to our understanding of the structural 
determinants and functional roles of GIRK channel 
subunits in the brain. Parallel to this crucial information, 
the use of subunit-specific antibodies and the application 
of light and electron microscopic techniques allowed us 
to elucidate the cellular and subcellular distribution of 
the four GIRK channel subunits under physiological 
conditions. More recently, new information is available 
about how such distribution patterns are altered in 
pathological conditions. The structure and function of 
GIRK channels and their link to disease and drug 
addiction have been reviewed in several articles (Rifkin 
et al., 2017; Jeremic et al., 2021; Zhao et al., 2021; Luo 
et al., 2022). In this review article, we will only briefly 
describe some basic molecular, biochemical, and 
physiological features of these channels. The regional, 
cellular, and subcellular distribution of GIRK channels 
in the brain will be reviewed in greater detail. 

 

Structure and signalling of GIRK channels 

 

 To date, the mammalian GIRK channel family 
includes GIRK1 (Kir3.1), GIRK2 (Kir3.2), GIRK3 
(Kir3.3), and GIRK4 (Kir3.4) subunits that are encoded 
by the KCNJ3, KCNJ6, KCNJ9, and KCNJ5 genes, 
respectively. The four subunits have 60-80% amino acid 
sequence homology (Dascal, 1997). The GIRK subunits 
combine to form homo- and heterotetrameric complexes 
that are activated by direct interaction with Gβγ released 
from Gαi/o G proteins (Logothetis et al., 1987; 
Krapivinsky et al., 1995b). All GIRK subunits are 
expressed in the brain, where they show overlapping and 
distinct distribution patterns (Jelacic et al., 2000; Luján 
and Aguado, 2015). However, most neuronal GIRK 
channels are believed to be composed of GIRK1-3, due 
to the extremely limited distribution of GIRK4 in the 
brain (Wickman et al., 2000; Perry et al., 2008; Luján 
and Aguado, 2015). Topologically, each of the four 
subunits consists of two transmembrane domains, TM1 
and TM2, connected by a pore domain that acts as an 
ion-selective filter. Additionally, they possess N- and C-
terminal domains, both located intracellularly (Nishida 
and MacKinnon, 2002; Inanobe et al., 2007; Nishida et 
al., 2007). These cytoplasmic domains are responsible 
for mediating the interactions with different proteins that 
impact the trafficking and function of GIRK channels 
(Luo et al., 2022). The characteristics of the four 
subunits are discussed in the following sections. 

 

GIRK1 subunit 

 

 The cDNA for GIRK1 was the first GIRK channel 
subunit to be isolated from rat atrium by expression 
cloning in Xenopus oocytes (Dascal et al., 1993; Kubo et 
al., 1993). GIRK1 contains an Endoplasmic Reticulum 
(ER) retention signal that prevents it from being 
trafficked to the plasma membrane. This subunit cannot 
be trafficked to the cell membrane by itself to form 
functional homotetramers (Ma et al., 2002) but, when 
expressed alone, it localizes to internal cytoskeletal 
structures (Kennedy et al., 1996). However, GIRK1 does 
form functional heterotetramers with GIRK2, GIRK3, 
and GIRK4 in expression systems (Kofuji et al., 1995; 
Krapivinsky et al., 1995a; Velimirovic et al., 1996; 
Jelacic et al., 1999). When GIRK1 is co-expressed with 
GIRK2, GIRK3, or GIRK4, the resulting channel has an 
increased K+ conductance compared with any of the 
subunits alone (Kofuji et al., 1995); this unique 
characteristic is due to the 150 C-terminal amino acids 
of GIRK1 (Chan et al., 1997). Furthermore, when the N-
terminus of GIRK1 is deleted or substituted, the 
resulting channel has slower activation and deactivation 
kinetics (Slesinger et al., 1995; Mark and Herlitze, 
2000). GIRK1 C-terminal splice isoforms have been 
isolated from brain and heart tissue and are referred to as 
hGIRK1a,b,c,d,e (Nelson et al., 1997; Steinecker et al., 
2007; Wagner et al., 2010). 

 

GIRK2 subunit 

 

 GIRK2 was cloned from mouse brain based on its 
homology to GIRK1 (Lesage et al., 1994). GIRK2 
contains an ER export signal and post-Golgi surface-
promoting motifs (Ma et al., 2002) that favours its traffic 
to the neuronal surface to form functional homomeric 
GIRK channels (Kofuji et al., 1995; Lesage et al., 1995; 
Ma et al., 2002). Multiplicity in GIRK2 is further 
increased by the existence of four splice isoforms: 
GIRK2A-GIRK2D (Lesage et al., 1995; Isomoto et al., 
1996; Wei et al., 1998; Inanobe et al., 1999a). GIRK2A-
GIRK2C are expressed in the brain, and they differ in 
the length of their C-terminus. GIRK2A and GIRK2C 
are identical except for an extra 11 amino acids present 
in GIRK2C, and both contain a post-Golgi export motif 
in the C-terminus. GIRK2C contains a PDZ binding 
sequence within the extra 11 amino acids (Lesage et al., 
1995), as well as a postsynaptic density-95, discs large, 
zona occludens (PDZ) binding motif, which interacts 
with PDZ-containing trafficking motifs of sorting nexin 
27 (SNX27) (Ma et al., 2002). This interaction between 
GIRK2C and SNX27 could facilitate both forward 

598

GIRK channels in the CNS



trafficking and internalization (Lunn et al., 2007). In 
contrast, GIRK2B is 100 amino acids shorter than the 
other two isoforms and lacks a post-Golgi export motif 
(Isomoto et al., 1996). The fourth isoform GIRK2D was 
identified in the testis, where it plays a role in sperm 
function during fertilization (Inanobe et al., 1999a). 

 

GIRK3 subunit 

 

 GIRK3 was also cloned from mouse brain based on 
its homology to GIRK1 (Lesage et al., 1994). This 
subunit also contains a PDZ binding sequence (ESKV) 
within the C-terminus which is a binding domain for 
PDZ-containing proteins (Jelacic et al., 1999). GIRK3 is 
widely expressed in most regions of the brain during 
perinatal development and in the adult (Kobayashi et al., 
1995; Karschin et al., 1996; Chen et al., 1997; Dascal, 
1997; Karschin and Karschin, 1997; Jelacic et al., 1999; 
Fernández-Alacid et al., 2011), however, there is no 
clear consensus about its function. Some studies have 
failed to detect functional GIRK currents when GIRK3 
is expressed alone or with GIRK1 in expression systems 
(Kofuji et al., 1995; Dißmann et al., 1996; Ma et al., 
2002). GIRK3 homotetramers and GIRK1/GIRK3 
heterotetramers are not functional and are localized to 
the ER, due to the presence of a lysosomal targeting 
signal on the C-terminus of GIRK3 (Ma et al., 2002). In 
contrast, other studies have observed functional GIRK3 
channels when GIRK1 or GIRK2 is co-expressed 
(Dißmann et al., 1996; Wischmeyer et al., 1997; Jelacic 
et al., 1999, 2000). For example, co-expression of 
GIRK2 and GIRK3 has been observed to give rise to 
channels that show a five-fold decrease in Gβγ 
sensitivity (Jelacic et al., 2000). In the ventral tegmental 
area (VTA), γ-aminobutyric acid type B (GABAB)-
activated dopaminergic neurons, which express GIRK2 
and GIRK3, showed a higher EC50 for activation of 
GIRK channels than GABAergic neurons, which express 
GIRK1, GIRK2, and GIRK3 (Cruz et al., 2004). There 
are no reported splice variants for GIRK3. 

 

GIRK4 subunit 

 

 GIRK4 was identified in bovine atrial membrane by 
immunoprecipitation with GIRK1 (Krapivinsky et al., 
1995a). GIRK4 can form homotetrameric complexes in 
atrial myocytes (Corey and Clapham, 1998), however, 
biochemical and electrophysiological studies have also 
shown that this subunit can assemble with GIRK1 to 
form heterotetramers with 1:1 stoichiometry (Inanobe et 
al., 1995; Krapivinsky et al., 1995a; Corey and Clapham, 
1998). This GIRK1/GIRK4 heterotetrameric assembly 
elicits a current similar to the IKACh of the heart. 
Interestingly, GIRK4 knockout (KO) mice lack this 
IKACh in the heart (Wickman et al., 1998). GIRK4 
contains an ER export motif that allows for forward 
trafficking and the potential formation of functional 
homotetrameric channels (Ma et al., 2002). There are no 
reported splice isoforms for GIRK4. 

GIRK knockout mice: Insights into physiology and 
disease 

 

 The contribution of each subunit to GIRK channel 
function, primarily in the brain and heart, has been 
mostly unravelled by KO mice created using 
homologous recombination techniques (Signorini et al., 
1997; Bettahi et al., 2002; Torrecilla et al., 2002). 
Thanks to these transgenic animals, GIRK channels are 
thought to play a role in several disorders, including 
anxiety, neuropathic pain, epilepsy, addiction, obesity, 
and arrhythmias (Pravetoni and Wickman, 2008), and 
thus they became important targets for therapeutic 
intervention (Lujan and Ciruela, 2012; Zhao et al., 
2021). 

 

GIRK1 and GIRK2 KO mice 

 

 These animals display the following features; 
hyperalgesia and decreased analgesic response to 
morphine administration (Marker et al., 2002, 2004; 
Blednov et al., 2003; Mitrovic et al., 2003), decreased 
anxiety, decreased baclofen-induced ataxia, increased 
operant responding for food (Pravetoni and Wickman, 
2008), increased susceptibility to spontaneous and 
pharmacologically induced seizures (Signorini et al., 
1997), blunted behavioural responses to ethanol, 
including less severe withdrawal symptoms, handling-
induced convulsions, and lacking the anxiolytic effect of 
ethanol (Blednov et al., 2001), and reduced cocaine self-
administration (Morgan et al., 2003). GIRK1 KO mice 
often displayed similar phenotypes to those of GIRK2 
KO mice, including thermal hyperalgesia and decreased 
analgesic responses to high doses of intrathecal opioids 
(Marker et al., 2004), elevated open-field activity, 
decreased anxiety-like behaviour, decreased baclofen-
induced ataxia, and increased operant responding for 
food (Pravetoni and Wickman, 2008) albeit in a less 
pronounced manner than GIRK2 KO mice. 

 The use of GIRK1 KO mice has revealed that the 
GIRK1 subunit is essential for intact spatial learning and 
memory and synaptic plasticity in hippocampal brain 
slices (Mett et al., 2021). In addition, GIRK2 ablation in 
neuropeptide Y (NPY)/agouti-related peptide (AgRP) 
neurons results in increased body weight and adiposity, 
phenotypes that are attributed to decreased sympathetic 
activity and energy expenditure, rather than an increase 
in food intake (Oh et al., 2023). GIRK2 expressed by 
NPY/AgRP neurons also has a role in cold-induced 
thermogenesis (Oh et al., 2023). 

 The above behavioural data supports the idea of the 
immense importance of the GIRK2 subunit for normal 
physiology, arguing that GIRK2 contributes to most, if 
not all, neuronal GIRK channels (Lüscher et al., 1997; 
Slesinger et al., 1997; Torrecilla et al., 2002; Cruz et al., 
2004; Koyrakh et al., 2005; Labouèbe et al., 2007). 
Consistent with this idea, several studies have also 
demonstrated that GIRK currents were significantly 
reduced or absent in neurons from the GIRK2 KO 

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GIRK channels in the CNS



hippocampus (Lüscher et al., 1997; Koyrakh et al., 
2005), locus coeruleus (Torrecilla et al., 2002), 
substantia nigra (Koyrakh et al., 2005), cerebellum 
(Slesinger et al., 1997), and VTA (Cruz et al., 2004). In 
addition, GIRK2 KO mice show reduced levels of 
GIRK1 protein but normal levels of GIRK1 mRNA, 
suggesting that the GIRK2 subunit is necessary for post-
transcriptional modification of GIRK1 (Signorini et al., 
1997). Altogether, the behavioural alteration, the 
decrease in currents and GIRK1 expression indicated 
that the main functional GIRK channels in the brain are 
heterotetramers formed by GIRK1 and GIRK2. Recent 
ultrastructural data showing co-clustering of 
GIRK1/GIRK2 in CA1 pyramidal cells also favours this 
view (Martín-Belmonte et al., 2022). 

 

GIRK3 KO mice 

 

 These animals display reduced cocaine self-
administration (Morgan et al., 2003), hyperalgesia 
(Marker et al., 2002, 2004), and reduced analgesia from 
systemic morphine administration (Smith et al., 2008). 
Consistent with its role in analgesia, GIRK3 show 
reduced opioid inhibition in the locus coeruleus 
(Torrecilla et al., 2002). Behavioural experiments 
conducted to measure anxiety and motor activity and 
coordination revealed that GIRK3 KO mice are 
indistinguishable from wild-type (Pravetoni and 
Wickman, 2008). Moreover, GIRK3 ablation has little 
impact on GIRK currents in the hippocampus, substantia 
nigra pars compacta (SNc), or spinal cord (Koyrakh et 
al., 2005; Marker et al., 2006); furthermore, it results in 
increased potency of baclofen in dopamine neurons of 
the VTA (Labouèbe et al., 2007). 

 

GIRK4 KO mice 

 

 The most significant phenotypes detected in GIRK4 
KO mice relate to cardiac function and include a blunted 
heart rate decrease in response to vagal stimulation and 
resistance to pacing-induced arrhythmias (Wickman et 
al., 2000). The heart rate of GIRK4 KO mice shows 
reduced variability and diminished responsiveness to 
pharmacological stimulation of the baroreflex and 
adenosine receptors (Wickman et al., 1998). GIRK4 KO 
mice also fail to develop a training-induced sinus 
bradycardia (Bidaud et al., 2021). In phenotypes related 
to the CNS, GIRK4 KO mice are not majorly different 
from their wild-type counterparts with respect to 
locomotor activity, visual acuity, and pain perception, 
although they do exhibit impaired performance in the 
Morris water maze, a test of spatial learning and memory 
(Wickman et al., 2000). More recently, it has been 
shown that GIRK4 KO mice are predisposed to adult-
onset obesity and before this phenotype, they exhibited 
reduced overall net energy expenditure and a mild 
tendency toward elevated food intake (Perry et al., 
2008). 

Macromolecular complexes with GIRK channels 

 

 GIRK channels can respond to a variety of 
neurotransmitters, including opioids, acetylcholine, 
adenosine, dopamine, γ-aminobutyric acid (GABA), 
glutamate, serotonin, norepinephrine, and somatostatin 
(Lüscher et al., 1997; Inanobe et al., 1999b; Dutar et al., 
2000). GIRK channels are directly coupled to G proteins 
and mediate the effects of GPCRs in a membrane-
delimited manner (Fig. 1). In the absence of a ligand, the 
Gα subunit of the G protein binds guanosine 
diphosphate, which associates with the GPCR (Hibino et 
al., 2010). Following activation of the GPCR, Gβγ 
dissociates and activates GIRK channels (Lüscher and 
Slesinger, 2010). Certain GPCRs are preferentially 
bound to specific G proteins, and one of those is the Gi/o 
protein, unique in its sensitivity to pertussis toxin (PTX). 
This Gi/o protein pathway is the major regulator of 
GIRK channel activity (Fig. 1). 

 The GPCR signalling model is based on three main 
components: GPCRs, heterotrimeric G proteins, and the 
ion effector channel, i.e., the GIRK channel. There are 
two theories to explain how inactive G proteins 
encounter GPCRs in their active state: 1) The “collision-
coupling” theory, which postulates that inactive G 
proteins are free-floating in the membrane where they 
collide with and become activated by ligand-bound 
GPCRs (Brinkerhoff et al., 2008) and 2) the “pre-
coupling” theory, which suggests that inactive G proteins 
are associated with inactive GPCRs and become 
activated when GPCRs are activated (Challiss and Wess, 
2011). 

 Recently, a new concept in the organisation of 
macromolecular signalling complexes has emerged. This 
is known as GPCR-effector macromolecular membrane 
assembly (GEMMA), defined as a pre-assembled 
signalling complex composed of combinations of 
GPCRs, G proteins, and effectors proteins localised to 
the plasma membrane (Ferré et al., 2022). A specific 
GPCR relevant to the physiology of many central 
neurons is the GABAB receptor, which regulates the 
activity of inhibitory and excitatory neurons and 
functions as a safety brake to counteract neuronal 
overexcitation (Gassmann and Bettler, 2012). GABAB 
receptors are frequently identified as components of 
GEMMAs in a subcellular compartment-manner with 
GIRK channels, as well as with other regulatory 
proteins, to carry out fast and specific signal 
transduction (David et al., 2006; Kulik et al., 2006; 
Fowler et al., 2007; Ciruela et al., 2010; Fajardo-Serrano 
et al., 2013; Luján et al., 2018; Martín-Belmonte et al., 
2022). For instance, Co-IP experiments have suggested 
that GABAB receptors may form GEMMAs with GIRK 
channels and with voltage-gated calcium (CaV) channels 
in the cerebellum (Luján et al., 2018). In addition, 
immunoEM techniques have demonstrated a close 
spatial relationship between GABAB receptors and 
GIRK channels only in dendritic spines, and between 

600

GIRK channels in the CNS



GABAB receptors and CaV2.1 channels only in dendritic 
shafts (Luján et al., 2018). Altogether, the data show for 
the first time that GABAB receptors can be associated 
with different GEMMAs in different neuronal 
compartments, where they are expected to serve 
complementary functions. In the hippocampus, GABAB 
receptors are not only components of GEMMAs with 
GIRK channels composed of GIRK1 and GIRK2 (Kulik 
et al., 2006; Martín-Belmonte et al., 2022), but also can 
be associated with RGS7, a regulator of G protein 
signalling, and Gβ5 (Fajardo-Serrano et al., 2013). 
Moreover, increasing evidence indicates that not all 
GABAB receptors and GIRK channels form GEMMAs 
along the neuronal surface. Most GIRK channels and 
GABAB receptors in the hippocampus and cerebellum 
are closely associated, forming clusters, suggesting the 
existence of pre-coupled complexes, however, some 
GIRK channels can be found scattered, not forming 
clusters (Kulik et al., 2006; Luján et al., 2018; Martín-
Belmonte et al., 2022). This distribution pattern suggests 
a different signalling mode based on collision coupling, 
which is less efficient. Therefore, the two signalling 
models (GEMMA and collision-coupling) seem to co-
exist in pyramidal cells of the hippocampus and Purkinje 
cells of the cerebellum and are balanced according to 
plasticity requirements. 

 

Molecular diversity of GIRK channels 

 

 Native GIRK channels are homotetrameric and 
heterotetrameric complexes made up of various 
combinations of the GIRK1-4 subunits. Understanding 
the different channels that GIRK subunits can form and 
function in vivo, as well as the establishment of their 
roles in particular signalling pathways and biological 
processes, are key aspects. From a theoretical point of 
view, random tetrameric assembly of the four distinct 
GIRK subunits and/or the alternative splicing of GIRK2 
would generate a relatively large and heterogeneous 
population of neuronal GIRK channels. However, cell 
biological, biochemical, morphological, and electro-
physiological evidence has indicated that the molecular 
diversity of neuronal GIRK channels is more limited 
than we would expect. This is suggested by: 

 (1) The presence in GIRK1 of an ER retention 
signal, which requires co-expression with other GIRK 
subunits to achieve membrane localization (Krapivinsky 
et al., 1995a; Kennedy et al., 1996; Ma et al., 2002). 
Thus, when GIRK2 and GIRK3 are abolished, GIRK1 
accumulates in the rER (Koyrakh et al., 2005). 

 (2) The presence in GIRK2 of an ER export signal 
and an endosomal trafficking motif, which makes this 
subunit essential for membrane localization (Ma et al., 
2002). 

 (3) The proposed function of GIRK3 in trafficking 
functional GIRK channels towards lysosomal 
degradation (Ma et al., 2002) and in targeting the 
channels to the cell surface (Lunn et al., 2007). 

 (4) The existence of brain areas with functional 
GIRK channels but only expressing GIRK2, or GIRK2 
and GIRK3 (Jelacic et al., 2000; Cruz et al., 2004). A 
good example is in the locus coeruleus, where the 
complete loss of opioid-induced currents required the 
simultaneous ablation of both GIRK2 and GIRK3 
(Torrecilla et al., 2002). 

 (5) The ablation of GIRK2 which resulted in a near 
to complete loss of GIRK current in many brain regions, 
including the hippocampus (Lüscher et al., 1997; 

 601

GIRK channels in the CNS

Fig. 1. Scheme depicting some of the 
mechanisms regulating the macromolecular 
signalling complex of GIRK channels and 
GPCRs. GIRK channels are activated by 
pertussis toxin (PTX)-sensitive Gαi/o protein-
coupled receptors. After the activation of 
GPCRs, the trimeric Gαβγ dissociates giving 
rise to Gα and Gβγ. The Gβγ dimer induces 
the activation of the GIRK channel and outflux 
of K+. The endogenous GTPase activity of 
Gαi/o hydrolyses GTP, allowing Gαi/o and Gβγ 
to reassociate. This process can be hastened 
by the activity of RGS proteins with the help of 
the regulator of G protein signalling (RGS). 
Na+ and PIP2 are involved in the opening of 
the GIRK channel. On the other hand, Gαq 
protein-coupled receptors inactivate GIRK 
channels through the activation of different 
proteins, such as phospholipase C (PLC) and 
protein kinase C (PKC). PKC activates 
Phospholipase A2 (PLA2) which releases 
arachidonic acid and reduces the affinity 
between GIRK2 and PIP2. cAMP-dependent 
PKA phosphorylates GIRK channels and 
potentiates its effects. CaMKII and PP1 
potentiate GIRK channel effects, thus 
promoting its translocation to the plasmatic 
membrane.



Koyrakh et al., 2005), cerebellum (Slesinger et al., 
1997), substantia nigra (Koyrakh et al., 2005), locus 
coeruleus (Torrecilla et al., 2002; Cruz et al., 2004), 
ventral tegmental area (VTA) (Cruz et al., 2004), and 
spinal cord (Marker et al., 2006). 

 (6) The restricted expression of GIRK4 to a small 
number of neuron populations (Wickman et al., 2000; 
Perry et al., 2008). 

 All these observations indicate that GIRK1/GIRK2 
heteromultimers represent the dominant functional 
GIRK channel in the brain. Nevertheless, it is now well 
established that GIRK subunit composition can affect 
channel function. Several studies performed in 
expression systems found that GIRK channels of 
different subunit compositions differ in single-channel 
profiles, whole-cell current kinetics, and Gßγ sensitivity 
(Duprat et al., 1995; Lesage et al., 1995; Jelacic et al., 
2000). The GIRK1 subunit, although inactive by itself, 
enhances heteromeric channel activity when associated 
with either the GIRK4 or GIRK2 subunit (Kofuji et al., 
1995). In addition to affecting functions, GIRK subunit 
composition can affect subcellular localization. 
Increasing evidence shows that GIRK channels with 
different subunit compositions are localized to different 
compartments in the plasma membrane (Luján and 
Aguado, 2015). For instance, excitatory synapses of 
CA1 pyramidal cells contain GIRK2 and GIRK3 
subunits, while extrasynaptic plasma membranes contain 
GIRK1 and GIRK2 subunits (Fernández-Alacid et al., 
2011). 

 

Regional distribution of GIRK channels in the CNS 

 

 The regional distribution of GIRK1, GIRK2, and 
GIRK3 in the brain has been studied mostly using 
northern and western blots, RT-PCR, in situ 
hybridization, histoblots, and light microscopy 
immunohistochemistry (Luján and Aguado, 2015). The 
regional distribution of GIRK4 in the brain has been 
analysed using radioactive and non-radioactive in situ 
hybridization (Wickman et al., 2000) and transgenic 
mice expressing enhanced green fluorescent protein 
(EGFP) under the control of the Girk4 gene promoter 
(Aguado et al., 2008; Perry et al., 2008; Luján and 
Aguado, 2015). 

 The identification of the primary sequence of 
encoded polypeptides has enabled the generation of 
antibodies that target GIRK subunits. To date, several 
subunit-specific antibodies can be used to identify and 
map the cellular and subcellular localization patterns of 
the GIRK1-3 subunits in the brain. Some of these 
antibodies have been validated for different technical 
approaches using GIRK KO mice, confirming the 
specificity of the immunolabelling patterns (Koyrakh et 
al., 2005; Kulik et al., 2006; Aguado et al., 2008, 2013; 
Fernández-Alacid et al., 2009, 2011; Ciruela et al., 2010; 
Booker et al., 2013; Fajardo-Serrano et al., 2013; Kirizs 
et al., 2014; Brandalise et al., 2016; Luján et al., 2018). 
The use of these antibodies in histoblot and 
immunohistochemical techniques has provided 
invaluable information on the relative expression and 
distribution patterns of GIRK1-3 in different brain 
regions and in specific neuronal populations under 
physiological and pathological conditions. To date, no 
specific antibodies against the GIRK4 subunit have yet 
been developed. 

 

Distribution of GIRK1-3 subunits 

 

 The three neuronal GIRK subunits (GIRK1, GIRK2, 
and GIRK3) are broadly distributed in the CNS, where 
they exhibit overlapping but distinct distribution 
patterns, as shown using in situ hybridization, histoblot 
and immunohistochemical techniques (Fig. 2). GIRK1-3 
mRNAs and proteins show the highest expression levels 
in the olfactory bulb, neocortex, hippocampus, and the 

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GIRK channels in the CNS

Fig. 2. Expression of GIRK1, GIRK2, and GIRK3 in the brain. A-C. Localization of GIRK1 and GIRK2 subunits in mouse brain and GIRK3 subunits in 
rat brain, throughout the brain as detected by the histoblot technique. The GIRK1, GIRK2, and GIRK3 subunits are widely distributed in the brain 
showing overlapping distribution patterns. Strong immunoreactivity for the three subunits is detected in the cortex (Ctx), cerebellum (Cb), hippocampus 
(hp), and thalamus (Th), with the lowest intensity in the caudate putamen. Scale bars: 2 cm.



granule cell layer of the cerebellum, with strong 
expression in the piriform cortex, the thalamus, and the 
hypothalamus, and weak expression in the basal ganglia 
and globus pallidus (Kobayashi et al., 1995; Karschin et 
al., 1996; Liao et al., 1996; Ponce et al., 1996; Chen et 
al., 1997; Miyashita and Kubo, 1997; Inanobe et al., 
1999b; Koyrakh et al., 2005; Perry et al., 2008; Saenz 
Del Burgo et al., 2008; Fernández-Alacid et al., 2009, 
2011). 

 The following section provides a detailed description 
of the well-characterised expression of the GIRK1-3 
subunits. 

 

 Hippocampus 

 

 GIRK1, GIRK2, and GIRK3 mRNA and protein are 
strongly expressed in CA1-CA3 pyramidal neurons and 
dentate gyrus granule cells with overlapping distribution 
patterns in the same layers or subfields (Kobayashi et al., 
1995; Karschin et al., 1996; Chen et al., 1997; Karschin 
and Karschin, 1997; Koyrakh et al., 2005; Kulik et al., 
2006; Saenz Del Burgo et al., 2008; Fernández-Alacid et 
al., 2011; Kirizs et al., 2014; Alfaro-Ruiz et al., 2021; 
Martín-Belmonte et al., 2022) (Figs. 2, 3). Of the three 
subunits, GIRK2 exhibits the highest levels of 
expression, whereas GIRK3 exhibits the lowest 
(Signorini et al., 1997; Koyrakh et al., 2005; Fernández-
Alacid et al., 2011; Martín-Belmonte et al., 2022). In the 
CA1 region, immunoreactivity for GIRK1 and GIRK2 is 
strong in the stratum lacunosum-moleculare, the distal 
portion of the stratum radiatum, and the stratum oriens 
(Fig. 3). In the CA3 region, immunoreactivity for 
GIRK1 and GIRK2 is strong in the strata oriens, 
radiatum, and lacunosum-moleculare but moderate in 
the stratum lucidum (Fig. 3). In the dentate gyrus, 
immunoreactivity for GIRK1 and GIRK2 is strong in the 

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GIRK channels in the CNS

Fig. 3. Distribution of immunoreactivity for GIRK channel subunits in the hippocampus, cortex, and cerebellum. A-I. In the hippocampus, 
immunoreactivity for GIRK1 and GIRK2 was strong in all dendritic layers in the CA1 and CA3 fields, and dentate gyrus (DG), but weaker in the hilus (h). 
Immunoreactivity for GIRK3 was observed in the neuropil of all dendritic layers, with the strongest intensity occurring in the stratum lucidum (sl) in the 
CA3 field. In the cortex, immunoreactivity for GIRK1, GIRK2, and GIRK3 was strong in the neuropil of layers I-IV and was weaker in layers V-VI. In the 
cerebellum, immunoreactivity for GIRK1, GIRK2, and GIRK3 is very strong in the granule cell layer (gc), moderate in the molecular layer (ml), and 
absent in the white matter (wm). Scale bars: 100 μm.



molecular layer and moderate in the hilus (Koyrakh et 
al., 2005; Kulik et al., 2006; Fernández-Alacid et al., 
2011; Alfaro-Ruiz et al., 2021) (Fig. 3). Immuno-
reactivity for GIRK3 is moderate in the strata oriens, 
radiatum, and lacunosum-moleculare of the CA1 and 
CA3 regions but strong in the stratum lucidum. In the 
dentate gyrus, immunoreactivity for GIRK3 is moderate 
in the molecular layer and hilus (Fernández-Alacid et al., 
2011) (Fig. 3). 

 

 Basal ganglia and amygdala 

 

 The expression of GIRK1 and GIRK3 is weak in the 
basal ganglia and globus pallidus, and GIRK2 is mostly 
absent (Fig. 2). In contrast, the expression of GIRK1, 
GIRK2, and GIRK3 is high in different nuclei of the 
amygdala (Karschin et al., 1996; Saenz Del Burgo et al., 
2008). 

 

 Thalamus 

 

 GIRK1 and GIRK3 subunit mRNAs are abundant in 
all thalamic nuclei, and GIRK2 mRNA is present at high 
levels in the lateral and geniculate nuclei and at lower 
levels in the other thalamic nuclei (Karschin et al., 1996) 
(Fig. 2). GIRK1, GIRK2, and GIRK3 mRNAs are 
abundantly expressed in nuclei of the amygdala 
(Karschin et al., 1996). 

 

 Substantia nigra and ventral tegmental area 

 

 In the SNc and the adjacent VTA, GIRK2 mRNA 
and protein expression is high (Fig. 4), with significantly 
lower levels of GIRK1 and GIRK3 (Karschin et al., 
1996; Koyrakh et al., 2005; Labouèbe et al., 2007; Saenz 
Del Burgo et al., 2008). In the substantia nigra pars 
reticulata (SNr), the main GIRK expression is 
attributable to the GIRK3 subunit (Karschin et al., 1996; 
Saenz Del Burgo et al., 2008). 

 

 Cerebellum 

 

 GIRK1, GIRK2, and GIRK3 mRNA and protein are 
mainly expressed in the granule cell layer. In the 
molecular layer, GIRK2 is present at low levels, while 
GIRK1 and GIRK3 are more abundantly expressed 
(Karschin et al., 1996; Aguado et al., 2008; Saenz Del 
Burgo et al., 2008; Fernández-Alacid et al., 2011) (Figs. 
2, 3). The GIRK3 subunit shows the highest expression 
levels in the Purkinje cell layer (Karschin et al., 1996). 
Other anatomical parts of the cerebellum are the deep 
cerebellar nuclei, whose neurons express high levels of 
GIRK1 and GIRK3 mRNAs and very low levels of 
GIRK2 mRNA (Karschin et al., 1996; Saenz Del Burgo 
et al., 2008). 

 

 Midbrain, brainstem, and spinal cord 

 

 The inferior colliculus shows high expression of 
GIRK1 and GIRK3 mRNA, however, the expression of 
GIRK2 is mostly absent (Karschin et al., 1996; Saenz 
Del Burgo et al., 2008). GIRK1, GIRK2, and GIRK3 
transcripts and proteins are enriched throughout the 
brainstem, as well as in the superficial layers of the 
spinal cord dorsal horn (Karschin et al., 1996; Marker et 
al., 2005; Saenz Del Burgo et al., 2008; Luján and 
Aguado, 2015). 

 

Distribution of the GIRK4 subunit 

 

 GIRK4 expression has been detected in only a few 
regions and neuronal populations of the mouse brain 
(Karschin et al., 1996; Wickman et al., 2000). Strong 
GIRK4 expression is detected in the deep cortical 
pyramidal neurons, the endopiriform nucleus and 
claustrum of the insular cortex, and the central 
autonomic system, including the thalamic parafascicular 
and paraventricular nuclei. Lower expression levels of 
GIRK4 are observed in the laterodorsal and lateral 
posterior thalamic nuclei. In addition, small populations 
of neurons in the globus pallidus, the superior colliculus, 
and the medial vestibular and dorsal tegmental nuclei 
express GIRK4 (Karschin et al., 1996; Wickman et al., 
2000; Aguado et al., 2008; Perry et al., 2008). In the 
hippocampus, the expression of GIRK4 has not been 
observed in any of the principal cell layers and only in a 
few cells in the molecular layer of the dentate gyrus, 
often near the hippocampal fissure (Perry et al., 2008; 
Luján and Aguado, 2015) (Fig. 5). In the cerebellum, 
GIRK4 expression has not been observed in the Purkinje 
cell layer, molecular layer, and granule cells, but only in 
a subpopulation of Golgi cells in the granule cell layer 
(Aguado et al., 2008; Luján and Aguado, 2015) (Fig. 5). 
EGFP labelling in the substantia nigra is also found in a 
small population of neurons (Fig. 5) and is highest in the 
ventromedial hypothalamic nucleus, although robust 
expression is also seen in the paraventricular nucleus and 
posterior aspect of the arcuate nucleus (Perry et al., 
2008). 

 

Cellular distribution of GIRK channels in the CNS 

 

 A main characteristic of GIRK subunits is that they 
display cell-type specific distribution within brain 
regions (Luján and Aguado, 2015). The cellular 
distribution of GIRK1-3 subunits in different brain 
regions has been analysed using in situ hybridization 
(Koyrakh et al., 2005; Saenz Del Burgo et al., 2008), 
immunofluorescence (Aguado et al., 2008; Ciruela et al., 
2010; Booker et al., 2013), light microscopy immuno-
histochemistry (Koyrakh et al., 2005; Aguado et al., 
2008, 2013; Luján and Aguado, 2015), and immuno-
electron microscopy (iEM) (Koyrakh et al., 2005; 
Labouèbe et al., 2007; Aguado et al., 2008; Fernández-
Alacid et al., 2009, 2011; Ciruela et al., 2010; Arora et 
al., 2011; Padgett et al., 2012; Booker et al., 2013; Kirizs 
et al., 2014; Degro et al., 2015; Martín-Belmonte et al., 
2022). 

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GIRK channels in the CNS



 The cerebellum is the brain region where molecular 
heterogeneity is best illustrated. The cerebellar cortex is 
composed of at least five types of neurons; GABAergic 
(Purkinje, basket, stellate, Golgi, and Lugaro cells) and 
two types of glutamatergic neurons (granule and 
unipolar brush cells). The combination of different 
technical approaches has shown that Purkinje neurons 
express GIRK1/GIRK2/GIRK3 (although GIRK3 is the 
predominant subunit), basket cells express GIRK1/ 
GIRK3, stellate cells express GIRK3, Golgi cells 
express GIRK2/GIRK4, Lugaro cells express 
GIRK1/GIRK2/GIRK3 (although at very low levels), 

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GIRK channels in the CNS

Fig. 4. Immunoreactivity for the GIRK2 subunit in the mouse midbrain. A, B. In the substantia nigra, immunoreactivity for GIRK2 is more intense in pars 
compacta (SNc) than in pars reticulata (SNr), while in the ventral tegmental area (VTA), GIRK2 is intense throughout the nucleus. C-E. Using 
immunofluorescence, immunoreactivity for GIRK2 is present in cell bodies (arrows) and dendrites in the SNc, but mostly in SNr dendrites (arrowheads). 
F-H. Double-labelling confocal microscopy showing colocalization of the GIRK2 subunit and TH in cell bodies (arrows) and dendrites (arrowheads) of 
the SNc. This demonstrates the expression of GIRK2 in dopaminergic neurons. Scale bars: A,C, 500 μm; B, 200 μm; D-H, 50 μm.



606

GIRK channels in the CNS

Fig. 5. EGFP expression of GIRK4 in the brain of Tg(Kcnj5-EGFP)49Gsat mice. Representative images showing the restricted expression pattern of 
the GIRK4 subunit in the mouse brain. B. In the hippocampus, a small subpopulation of neurons found in the hippocampal fissure and molecular layer 
of the dentate gyrus show clear EGFP labelling (arrows). C. In the cerebellum, EGFP expression is found in small subsets of Golgi cells (arrows). D, E. 
EGFP labelling in the substantia nigra and ventral tegmental area (VTA) is found in small subsets of cells but does not colocalize with the GIRK2 
subunit. DG, dentate gyrus; gc, granule cell layer; gcl, granule cell layer of the cerebellum; h, hilus; ml, molecular layer; pc, Purkinje cell layer; SNc, 
substantia nigra pars compacta; SNr, substantia nigra pars reticulata. Scale bars: A,B, 150 μm; C, 55 μm; D-G, 300 μm.



granule cells express GIRK1/GIRK2/GIRK3 (at very 
high levels for all three), and unipolar brush cells 
express GIRK2/GIRK3 (Aguado et al., 2008; Luján and 
Aguado, 2015). In addition, GIRK2 is also expressed in 
different subpopulations of Golgi cells, identified using 
specific markers (Aguado et al., 2008) (Fig. 6). 

 In the hippocampus, both pyramidal cells and 
interneurons express GIRK1, GIRK2, and GIRK3 
(Koyrakh et al., 2005; Fernández-Alacid et al., 2011; 
Booker et al., 2013; Degro et al., 2015; Alfaro-Ruiz et 
al., 2021; Martín-Belmonte et al., 2022). However, the 
three subunits are not present in the same membrane 
microdomains. Thus, heteromeric complexes of 
GIRK1/GIRK2 are associated with GABAB receptors in 
pyramidal cells in dendritic spines (Koyrakh et al., 2005; 
Martín-Belmonte et al., 2022). GIRK3 is also present 
along the plasma membrane of pyramidal cells but never 
close to GIRK1/GIRK2, showing a different localization 
pattern in the neuronal surface (Fernández-Alacid et al., 
2011; Martín-Belmonte et al., 2022). This data suggests, 
therefore, that the GABAB-evoked currents in pyramidal 
cells are mediated by GIRK1/GIRK2. As regards 
interneurons, the two main neuronal populations are 
basket and chandelier cells; both play a critical role in 
shaping pyramidal cell activity (Klausberger and 
Somogyi, 2008). Using double labelling with 
parvalbumin (a marker of both basket and chandelier 
cells), it has been reported that GIRK1, GIRK2, and 
GIRK3 show the same localization with very similar 
densities (Booker et al., 2013), suggesting that the 
GABAB-evoked currents in interneurons are mediated 
by GIRK1, GIRK2, and GIRK3. 

 In the substantia nigra and VTA of the midbrain, the 
use of double-labelling immunohistochemistry with 
tyrosine hydroxylase (TH) as a marker revealed that the 
GIRK2 subunit is expressed in dopaminergic neurons 
(Koyrakh et al., 2005; Labouèbe et al., 2007; Arora et 
al., 2011; Padgett et al., 2012) (Figs. 4, 7), which do not 
express the GIRK1 subunit. Thus, in the SNc, 
dopaminergic neurons contain GIRK2 through the 

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GIRK channels in the CNS

Fig. 6. Neurochemical characterization of GIRK2-positive Golgi cells in the granule cell layer of the cerebellum. A1-A3. Triple labelling of GIRK2 (red), 
neurogranin (NG; blue), and GlyT2-GFP (green). GIRK2-expressing Golgi cells co-expressed neurogranin and GFP (arrows). The asterisk indicates a 
cell double-labelled for GIRK2 and neurogranin in the granule cell layer (GL) but negative for GlyT2-GFP. The arrowheads indicate a cell labelled for 
GIRK2 and GlyT2-GFP but immunonegative for NG. B1-B3. Triple labelling of GIRK2 (red), the metabotropic glutamate receptor subtype mGlu2/3 
(mGu2/3, blue) and GAD67-GFP (green) (arrows). GL, granule cell layer; ML, molecular layer; PC, Purkinje cell layer. Scale bar: A,B, 20 μm.



formation of heteromeric channels with the GIRK2A and 
GIRK2C isoforms (Inanobe et al., 1999b). However, 
dopaminergic neurons of the VTA express GIRK2 and 
GIRK3 (Cruz et al., 2004; Labouèbe et al., 2007), 
whereas GABAergic neurons express GIRK1, GIRK2, 
and GIRK3 (Labouèbe et al., 2007). 

 In addition to glutamatergic, GABAergic, and 
dopaminergic neurons, GIRK subunits have also been 
found in cholinergic neurons in the nucleus of the 
vertical limb of the diagonal band and the serotonergic 
nucleus of the raphe nucleus (Saenz Del Burgo et al., 
2008). Altogether, existing data demonstrate that GIRK 
subunits can be found in neurochemically different 
neurons throughout the brain, where they play a role in 
the regulation of neuronal excitability. The GIRK3 
subunit has also been described to be expressed in glial 
cells in the cerebellum (Fernández-Alacid et al., 2009). 

 

Cellular localization of GIRK channels in peripheral 
non-neural cells 

 

 In addition to the predominant expression of GIRK1, 
GIRK2, and GIRK3 in the nervous tissue, some studies 
have shown that GIRK subunits are also expressed in 
other organs and tissues, mainly in the heart, pancreas, 
adrenal gland, and testicles. One of the most important 
findings associated with the expression of GIRK 
channels in peripheral tissues has been the presence of 
GIRK1 and GIRK4 in the heart, both in atrial and 
ventricle cardiomyocytes. Indeed, GIRK1 and GIRK4 
are highly expressed in cardiac tissue, whereas GIRK2 
and GIRK3 are absent. The predominant GIRK channel 
subtype in cardiomyocytes is a heterotetrametric 
complex containing GIRK1 and GIRK4 in 1:1 
stoichiometry (Bettahi et al., 2002). Furthermore, 
GIRK4 can form functional GIRK4 homotetrameric 
complexes and there is evidence of their presence in 
atrial myocytes (Corey and Clapham, 1998). The PKC 
isoform, PKCε, strengthens the interactions of the 
cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP2 (Gada et al., 2023). Functionally, cardiac GIRK 
signalling plays a role in the parasympathetic regulation 
of heart rate. Parasympathetic modulation of cardiac 
output is mediated by acetylcholine release from the 
vagus nerve, which activates M2 muscarinic receptors 
(M2R). In sinoatrial nodal cells and atrial myocytes, the 
activation of M2R triggers the release of inhibitory G 
proteins (Gαi/o) releasing the Gβγ dimer, which then 
induces the activation of the GIRK channel IKACh. M2R-
IKACh signalling is negatively regulated by the 
regulator of G protein signalling 6 (RGS6) protein 
(Wydeven et al., 2014; Anderson et al., 2018, 2020; 

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GIRK channels in the CNS

Fig. 7. Cellular and subcellular localization of GIRK2 in the SNc. A-C. Colocalization of the GIRK2 subunit and TH in the SNc of mice as revealed using 
double-labelling pre-embedding methods. The peroxidise reaction product (TH immunoreactivity) filled somata and dendritic shafts, whereas 
immunoparticles (GIRK2 immunoreactivity) were located along the extrasynaptic plasma membrane (arrows). Scale bars: A, 1 μm; B,C, 0.2 μm.



Kulkarni et al., 2018). 

 GIRK channels also play a role in islet cells of the 
pancreas. GIRK1 and GIRK3 subunits are present in 
these cells and are responsible for most of the adrenaline 
action on K+ conductance and membrane potential 
changes in islet cells (Ferrer et al., 1995; Iwanir and 
Reuveny, 2008). GIRK1 is also moderately expressed in 
other peripheral tissues, such as aorta, lung, liver, 
stomach, small intestine, colon, urinary bladder, testis, 
uterus, and skin (Yamada et al., 1998). The expression of 
GIRK4 homomeric channels has also been reported in 
the adrenal cortex. Multiple mutations of GIRK4 
channels cause primary aldosteronism, a disease 
characterized by hypersecretion of aldosterone and the 
most common cause of secondary hypertension, 
accounting for 10% of patients with newly diagnosed 
hypertension (Fernandes-Rosa et al., 2017). 

 

Subcellular distribution of GIRK channels in the CNS 

 

 iEM is currently the method of choice for examining 
the subcellular localization of ion channels in general 
and in particular GIRK channels. The resolution offered 
by the technique is unparalleled, allowing the precise 
localization of ion channels and associated proteins in 
any neuron compartment. iEM is technically demanding, 
nevertheless, it is well worth the effort especially if 
quantitative analysis of channel localization is of 
importance. At the ultrastructural level, the use of high-
resolution immunohistochemical techniques, in 
particular the SDS-FRL, pre-embedding and post-
embedding immunogold methods, has revealed that 
GIRK channel subunits are localized with extraordinary 
precision in defined subcellular compartments (Koyrakh 
et al., 2005; Marker et al., 2005; Kulik et al., 2006; 
Labouèbe et al., 2007; Aguado et al., 2008; Fernández-
Alacid et al., 2009, 2011; Ciruela et al., 2010; Degro et 
al., 2015; Luján et al., 2018; Alfaro-Ruiz et al., 2021; 
Martín-Belmonte et al., 2022). These studies have shown 
that GIRK channels can be located within any 
subcellular compartment at the neuronal surface, both at 
synaptic and extrasynaptic sites, in somata, dendritic 
shafts, dendritic spines, and axons or axon terminals; 
however, each localization differs in subunit 
composition and density of channels or interacting 
proteins, as we will describe in the following section. 
This subcellular heterogeneity contributes significantly 
to GIRK specificity and the physiological diversity of 
GIRK signalling in the brain (Luján and Aguado, 2015; 
Luo et al., 2022). 

 

Distribution of GIRK channels in a neuronal compart-
ment-dependent manner 

 

 One way by which neurons control the assembly of 
GIRK1-3 involves differential localization of these 
subunits in specific neuronal compartments. Most GIRK 
channels are distributed along somatodendritic domains 
of central neurons. In CA1 pyramidal cells of the 
hippocampus, a high density of immunogold labelling 
for GIRK1 and GIRK2 along the extrasynaptic plasma 
membrane of dendritic shafts and spines has been 
described (Koyrakh et al., 2005; Kulik et al., 2006; 
Aguado et al., 2008; Fernández-Alacid et al., 2009, 
2011; Booker et al., 2013) (Fig. 8). This has been 
confirmed by quantitative iEM analysis, showing that 
around 75-85% of immunoparticles for GIRK1 and 
GIRK2 are present postsynaptically (Koyrakh et al., 
2005). Using double labelling SDS-FRL, we have 
recently demonstrated the co-clustering of GIRK2 and 
GIRK1, but not GIRK2 and GIRK3, in the extrasynaptic 
plasma membrane of dendritic spines and shafts (Martín-
Belmonte et al., 2022). In Purkinje and granule cells of 
the cerebellum, the subcellular localization of GIRK1, 
GIRK2, and GIRK3 was almost identical (Fig. 9), with 
80-90% of immunoparticles for GIRK2 and GIRK3 
present postsynaptically and 100% of immunoparticles 
for GIRK1 (Aguado et al., 2008; Fernández-Alacid et 
al., 2009; Ciruela et al., 2010; Luján et al., 2018). 

 Although at lower density compared with 
extrasynaptic sites, GIRK channel subunits are also 
present at synaptic sites. The post-embedding 
immunogold and SDS-FRL techniques have further 
revealed distinct patterns of GIRK distribution relative 
to neurotransmitter release sites. Thus, GIRK2 and 
GIRK3 but not GIRK1 have been localised to 
postsynaptic densities (PSDs) of glutamatergic synapses 
but not GABAergic synapses in the brain (Koyrakh et 
al., 2005; Marker et al., 2005; Fernández-Alacid et al., 
2009, 2011), demonstrating a segregation of GIRK 
channel subunits at synaptic sites. This localization is 
likely endowed by interactions between PDZ protein-
binding domains on the GIRK channels and PDZ 
domain-containing anchoring proteins and is consistent 
with the presence of GABAB receptors in the same 
compartment, as demonstrated using the SDS-FRL 
technique (Kulik et al., 2006). The demonstration of this 
subcellular compartmentalization at excitatory, but not 
inhibitory, synapses provide new insights into the role of 
GIRK channel subunits in information transfer and 
processing within neurons and neural networks, under 
both physiological and pathological conditions. 

 Within their extrasynaptic location, GIRK1 and 
GIRK2 subunits are found at a higher density at the 
periphery of asymmetrical synapses in many brain 
regions, including the hippocampus (Koyrakh et al., 
2005; Fernández-Alacid et al., 2011; Fajardo-Serrano et 
al., 2013), cerebellum (Aguado et al., 2008; Fernández-
Alacid et al., 2009; Ciruela et al., 2010), spinal cord 
(Marker et al., 2005), and VTA (Labouèbe et al., 2007). 
In addition, immunoreactivity for GIRK3 also follows 
the same distribution pattern as GIRK1 and GIRK2 in 
Purkinje and granule cells of the cerebellum (Ciruela et 
al., 2010; Fernández-Alacid et al., 2011) and in 
dopaminergic neurons of the VTA (Labouèbe et al., 
2007). This association of GIRK channels with 
asymmetrical (excitatory) synapses has been 
demonstrated using quantitative approaches. Thus, at the 

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GIRK channels in the CNS



head of CA1 pyramidal cell spines in the hippocampus 
(Koyrakh et al., 2005; Fajardo-Serrano et al., 2013) and 
Purkinje cell spines in the cerebellar cortex (Fernández-
Alacid et al., 2009), about 75% of the immunogold 
particles for GIRK1 and GIRK2 have been localized 
extrasynaptically, within a 300-nm annulus surrounding 
the edge of asymmetrical synapses, whereas the 
remaining particles are distributed at more distant 
positions along the extrasynaptic plasma membrane. 
This localization of GIRK channels relative to glutamate 
release sites in CA1 pyramidal and Purkinje cells is very 
similar to the localization of GABAB receptors (Koyrakh 
et al., 2005; Luján and Shigemoto, 2006; Fernández-
Alacid et al., 2009; Fajardo-Serrano et al., 2013). These 
data were the first morphological evidence of the 
association between GIRK channels and GABAB 

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GIRK channels in the CNS

Fig. 8. Postsynaptic and presynaptic localization of GIRK subunits in mice CA1 pyramidal cells. Electron micrographs showing postsynaptic and 
presynaptic immunoparticles for GIRK1, GIRK2, and GIRK3 in the stratum radiatum of the hippocampal CA1 field, detected using the SDS-FRL 
technique. A-D. Postsynaptically, GIRK1 and GIRK2 are mostly found along the extrasynaptic plasmatic membrane of dendritic spines (spine) and 
dendritic shafts. Both subunits can be found forming clusters (blue circles) or scattered (purple arrows). GIRK3 can be found in these same 
compartments but mainly as scattered immunoparticles (purple arrows). E-G. Presynaptically, GIRK1 and GIRK2 are found mainly in the extrasynaptic 
membrane of the axon terminal, although a few immunoparticles were also found in the active zone (az, red overlay) as opposed to postsynaptic 
densities (PSD, green overlay) of dendritic spines. Both subunits in these compartments can be found forming clusters (blue circles) or as scattered 
immunoparticles (purple arrows). GIRK3 can be found in the axon terminals and active zones, mainly as scattered immunoparticles. Scale bars: 0.2 
μm.



receptors forming GEMMAs. 

 In addition to their main postsynaptic localization, 
GIRK channel subunits can also be located at 
presynaptic sites (Fig. 8). Several studies have reported 
the presence of GIRK1, GIRK2, and GIRK3 subunits in 
axon terminals in the brain (Morishige et al., 1996; 
Ponce et al., 1996; Marker et al., 2005; Kulik et al., 
2006; Aguado et al., 2008; Ladera et al., 2008; 
Fernández-Alacid et al., 2009, 2011; Booker et al., 2013; 
Fajardo-Serrano et al., 2013; Luján et al., 2018; Alfaro-
Ruiz et al., 2021; Martín-Belmonte et al., 2022) (Fig. 8). 
Presynaptically, GIRK1, GIRK2, and GIRK3 are mainly 
distributed along the extrasynaptic membrane and the 
active zone of axon terminals (Koyrakh et al., 2005; 
Marker et al., 2005; Fernández-Alacid et al., 2009, 2011; 
Fajardo-Serrano et al., 2013; Luján et al., 2018; Alfaro-
Ruiz et al., 2021; Martín-Belmonte et al., 2022). 
Quantitative approaches have established that around 15-
25% of immunoparticles for GIRK1-3 were detected 
presynaptically in the hippocampus and cerebellum 
(Koyrakh et al., 2005; Fernández-Alacid et al., 2009). 
The association of GIRK1-3 subunits to the presynaptic 
active zone suggests that they likely form heteromeric 
channels and that they might be involved in the 
presynaptic modulation of neuronal activity. But what is 
the mechanism of GIRK presynaptic modulation? 
Compelling evidence so far shows that this mechanism 
might involve activation of GABAB receptors to inhibit 
neurotransmitter release (Ladera et al., 2008; Fernández-
Alacid et al., 2009; Luján et al., 2009, 2014). Whilst 
electrophysiological studies do not support this 
association of presynaptic GABAB receptors and GIRK 
channel subunits (Lüscher et al., 1997), however, the 
functional association of GIRK and GABAB has been 
demonstrated using functional assays and biochemical 
approaches on cortical and cerebellar synaptosomes 
(Ladera et al., 2008; Fernández-Alacid et al., 2009). 
Following the activation of GABAB receptors, the 
opening of GIRK channels hyperpolarizes the 
presynaptic plasma membrane of excitatory axon 
terminals and thus reduces excitability and glutamate 
release. Three sets of data support this functional 
coupling: (1) the up-regulation of the GIRK3 subunit in 
GABAB KO mice and of the GABAB1 subunit in GIRK3 
KO mice obtained using iEM (Fernández-Alacid et al., 
2009); (2) the co-clustering of GIRK channel subunits 
with GABAB receptors in axon terminals of the 
hippocampus and cerebellum (Luján and Aguado, 2015; 
Martín-Belmonte et al., 2022); and (3) the loss of this 
co-clustering in pathological conditions (Martín-
Belmonte et al., 2022). 

 

Distribution of GIRK channels in a plasticity- and drug-
dependent manner 

 

 GIRK channels are present in dopaminergic, 
glutamatergic, and GABAergic neurons of the 
mesocorticolimbic pathway, where they play important 
roles in the acute rewarding effects and/or the adaptation 
that occurs with chronic exposure to addictive drugs 
(Marron Fernandez de Velasco et al., 2015). Thus, 
signalling through GIRK channel subunits mediates the 
effect of opioids, cocaine, morphine, and ethanol. 
Distinct cellular mechanisms have been proposed for 

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GIRK channels in the CNS

Fig. 9. Subcellular localization of GIRK channel subunits in Purkinje cells of the cerebellum. Electron micrographs showing the distribution of 
immunoparticles for the GIRK3 and GIRK2 subunits using the SDS-FRL technique in the mouse cerebellum. A, B. Immunoparticles for the GIRK3 
subunit are detected in dendritic spines (s) and dendritic shafts of oblique dendrites (oDen) of Purkinje cells. C, D. Immunoparticles for the GIRK2 
subunit are also detected in (s) and dendritic shafts (Den) of Purkinje cells but at a lower frequency than GIRK3. Scale bars: 0.2 μm.



different classes of drugs, with the mesocorticolimbic 
dopaminergic system considered as a common anatomic 
substrate (Lüscher and Ungless, 2006). The 
mesocorticolimbic dopaminergic system is comprised of 
dopaminergic neurons originating from the VTA with 
axons projecting to limbic nuclei, mainly the nucleus 
accumbens (NAc) and cortical areas such as the 
prefrontal cortex (PFC). Opioids and γ-hydroxybutyrate 
activate GIRK channels, leading to the disinhibition of 
dopaminergic neurons. Morphine stimulates μ-opioid 
receptors that are selectively expressed on GABAergic 
interneurons of the VTA and likewise activate GIRK 
channels, leading to disinhibition of dopaminergic 
neurons (Marron Fernandez de Velasco et al., 2015). 
However, the γ-hydroxybutyrate effect is more complex 
because GABAB receptors are expressed on both 
interneurons and dopaminergic neurons in the VTA. 
GABAB receptors from dopaminergic neurons are less 
efficiently coupled to GIRK channels in dopaminergic as 
opposed to GABAergic neurons. This is likely due to the 
differential expression of GIRK subunits (Cruz et al., 
2004). 

 Psychostimulants such as cocaine alter GIRK 
channel activity in the dopaminergic neurons of the 
VTA. These neurons revealed decreased GABAB 
receptor and D2R signalling after the injection of 
cocaine (Arora et al., 2011). This adaptation appears to 
be mediated by the internalization of GIRK channels 
(Arora et al., 2011). This effect has not been detected in 
dopaminergic neurons in the substantia nigra (Arora et 
al., 2011). However, a similar study performed in 
dopaminergic neurons of the VTA showed no changes in 
GIRK channel activity after cocaine exposure (Padgett 
and Slesinger, 2010). Interestingly, a single injection of 
cocaine or methamphetamine decreases GIRK channel 
activity in GABAergic neurons in the VTA (Padgett and 
Slesinger, 2010). 

 Repeated cocaine exposure triggers adaptations in 
layer 5/6 of glutamatergic neurons in the medial 
prefrontal cortex (mPFC), where it affects not only 
GIRK channels but also their associated GPCRs. GIRK 
channels mediate most of the GABAB receptor-
dependent inhibition of layer 5/6 pyramidal neurons in 
the mPFC, and repeated cocaine suppresses this pathway 
(Hearing et al., 2013). GABAB receptor-GIRK signalling 
in these neurons is decreased following repeated 
exposure to cocaine (Hearing et al., 2013). A protein 
phosphatase inhibitor applied to these neurons of the 
mPFC, as well as to GABAergic neurons of the VTA, 
reversed the effects of psychostimulants on GIRK 
channel activity, suggesting that GIRK channel 
trafficking is likely to be mediated by a phosphorylation-
dependent mechanism (Padgett et al., 2012; Hearing et 
al., 2013). 

 GIRK channels are also involved in alcohol-induced 
behaviour and addiction, as alcohol directly binds and 
activates GIRK channels without the participation of 
GPCRs (Kobayashi et al., 1999; Lewohl et al., 1999). 
Indeed, alcohol directly activates GIRK2 through an 
interaction with a hydrophobic pocket in the cytoplasmic 
domain, inducing conformational changes and increasing 
the affinity of PIP2, which helps stabilize the open state 
of the GIRK channel (Aryal et al., 2009; Bodhinathan 
and Slesinger, 2013). Alcohol induces neuroadaptations 
in GIRK channel activity. For example, alcohol 
increases the GIRK current induced by GABAB receptors in the VTA, thereby decreasing the excitability 
of dopaminergic neurons (Federici et al., 2009). In 
addition, alcohol impacts somatodendritic GABAB 
receptor-dependent signalling in principal neurons of the 
basal amygdala, attributable to suppression of GIRK 
channel activity due to their internalization (Marron 
Fernandez de Velasco et al., 2023). 

 

Altered subcellular distribution of GIRK channels in 
pathological conditions 

 

 GIRK channels are expressed at high levels in the 
hippocampus, amygdala, and prefrontal cortex; three 
brain regions related to cognitive function (Karschin et 
al., 1996; Alfaro-Ruiz et al., 2021; Martín-Belmonte et 
al., 2022). It is not surprising that dysregulation of 
GIRK-dependent signalling is associated with cognitive 
deficits in humans (Luo et al., 2022). The activation of 
GIRK channels is required for some forms of synaptic 
plasticity such as LTP, LTD, and depotentiation of LTP 
(Chung et al., 2009). For example, constitutive and 
forebrain pyramidal neuron-specific GIRK2 KO mice 
are deficient in depotentiation of LTP (Chung et al., 
2009; Victoria et al., 2016). Furthermore, pharma-
cological disruption of GIRK channels causing loss or 
gain of function alters learning and memory processes 
through mechanisms that alter neuronal excitability and 
the maintenance of long-term processes of synaptic 
plasticity (Djebari et al., 2021). Together, these results 
show the importance of an optimal range of GIRK 
signalling for normal hippocampus-dependent cognition 
(Victoria et al., 2016; Sánchez-Rodríguez et al., 2020). 

 Alzheimer's disease (AD) is a neurodegenerative 
disorder that slowly and progressively destroys brain 
cells. It is the most common type of dementia and is 
characterized by a progressive cognitive impairment that 
affects memory, orientation, and language, as well as 
more complex features, such as personality and 
reasoning (Jucker et al., 2006). AD is one of the leading 
causes of death in the elderly and is expected to increase 
in the coming years (Weuve et al., 2014). The disease is 
characterized by the development of β-amyloid plaques, 
neurofibrillary tangles formed by hyperphosphorylated 
Tau protein, and loss of synapses; the latter shows the 
best correlation with disease progression (Selkoe, 2002; 
Walsh and Selkoe, 2004; Penzes et al., 2011; Spires-
Jones and Hyman, 2014). One of the most affected areas 
in AD is the hippocampus (Hyman et al., 1984), where 
GIRK channels are highly expressed. 

 GIRK channels play a key role in the processes of 
learning, memory, and synaptic plasticity in the 
hippocampus (Ostrovskaya et al., 2014; Marron 

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GIRK channels in the CNS



Fernandez de Velasco et al., 2015). Growing evidence 
has shown that GIRK channels are affected in different 
models of AD (Mayordomo-Cava et al., 2015; Sánchez-
Rodríguez et al., 2017, 2019, 2020; Alfaro-Ruiz et al., 
2021; Martín-Belmonte et al., 2022). In the APP/PS1 
model, which overexpresses the APP protein, a change 
in the subcellular localization of GIRK channels in 
hippocampal CA1 pyramidal cells has been reported 
(Alfaro-Ruiz et al., 2021). Similarly, in the P301S model 
of tauopathy, the quantity of GIRK2 was also decreased, 
and its subcellular localization was altered in CA1 
pyramidal cells (Alfaro-Ruiz et al., 2021). In addition, in 
models in which β-amyloid peptides are injected 
intracerebroventricularly, LTP, hippocampal oscillatory 
activity, and deficits in novel object recognition were 
altered. However, these phenotypes could be rescued by 
ML297, an activator of GIRK channels (Sánchez-
Rodríguez et al., 2017, 2019). On the other hand, there is 
also evidence that GABAB receptors, the most important 
GPCR activating GIRK channels in the hippocampus, 
are also downregulated in different regions of the 
hippocampus of APP/PS1 mice (Martín-Belmonte et al., 
2020a,b) as well as in humans (Iwakiri et al., 2005; 
Martín-Belmonte et al., 2020a). Consistent with this 
data, recent studies have established a loss of GABAB-
GIRK interaction in APP/PS1 mice, giving rise to a 
reduction in the co-clustering of these two proteins 
(Martín-Belmonte et al., 2022). Interestingly, there is a 
link between the GABAB receptor and β-amyloid 
peptides (Schwenk et al., 2016; Dinamarca et al., 2019), 
which could eventually affect GIRK channel function. 

 Interestingly, hyperexcitability has been described in 
the hippocampus of AD patients in the early stages of 
the disease (Dickerson et al., 2005; Bakker et al., 2012), 
and this could be related to a loss of function of GIRK 
channels. In addition, epileptic activity is frequently 
associated with AD in humans (Vossel et al., 2017). 
Similarly, these epileptic seizures have also been found 
in murine models of AD (Palop et al., 2007; 
Minkeviciene et al., 2009; García-Cabrero et al., 2013; 
Ittner et al., 2014). The fact that the alteration in GIRK 
channels is common to AD and epilepsy, which also 
share some symptoms, would reinforce the hypothesis of 
the involvement of GIRK channels in human diseases. 
Although many studies are still needed to unravel how 
GIRK channels are altered in other models of the disease 
and human patients, this channel is a promising 
therapeutic target for the development of new drugs 
against AD. 

 The relationship between GIRK channels and 
epilepsy has been inferred from the results obtained 
using GIRK KO mice (Signorini et al., 1997). GIRK2 
KO mice have reduced GIRK1 expression and 
spontaneous epileptic seizures (Signorini et al., 1997). In 
mice with prolonged seizures, altered processing of 
GIRK1 and GIRK2 has also been described (Baculis et 
al., 2017). Similarly, epileptogenesis is facilitated by the 
blockade of hippocampal GIRK channels with the 
agonist Tertiapin-Q or with pertussis toxin, an antagonist 
of the Gi subtype GPCR (Mazarati et al., 2006). 
Moreover, the use of ML297 is effective in reducing 
seizures in two models of epilepsy, in which seizures are 
provoked by electroshocks or using the GABAA 
antagonist, pentylenetetrazol (Kaufmann et al., 2013). 
Taken together, this set of data shows that GIRK 
channels play a key role in epileptic episodes and could 
be used as a therapeutic target for the treatment of this 
disease. Interestingly, the G-protein-independent GIRK 
channel activator 1 (GiGA1), a selective agonist of 
GIRK1 and GIRK2, can suppress the excitation of 
pyramidal cells in the CA1 region of the hippocampus 
(Zhao et al., 2020). Therefore, this compound is 
effective in induced models of epilepsy and is a 
promising compound as a treatment for epilepsy. 

 

Developmental expression and acquisition of GIRK 
channels 

 

 Changes in the expression of ion channels have been 
related to a variety of important developmental events, 
such as proliferation, neuronal migration, differentiation 
or survival processes, and the formation of synaptic 
circuitry (Luján et al., 2005). Given the role played by 
GIRK channels in physiological and pathological 
conditions, information regarding their onset of 
expression and precise localization in embryonic and 
postnatal development is critical to unravel the 
contribution of these channels to developmental 
processes. The application of an in situ hybridization 
technique has shown that the GIRK1, GIRK2, and 
GIRK3 subunits are highly expressed in the embryonic 
brain, where they display non-overlapping expression 
patterns (Karschin and Karschin, 1997). However, 
during postnatal development, the expression of GIRK 
channel subunits starts showing overlapping expression 
patterns in the different brain regions (Karschin and 
Karschin, 1997; Aguado et al., 2013). GIRK3 is the most 
widely expressed of all GIRK subunits in the developing 
and adult brain (Karschin and Karschin, 1997). The 
expression of GIRK1 and GIRK2 show higher levels 
during development than in the adult animal in most 
brain regions. Thus, during postnatal days P2 and P10, 
mRNA for both GIRK1 and GIRK2 are highly expressed 
in the cerebellum, pontine nucleus, and lateral reticular 
nucleus. In the cerebellum, the GIRK2 subunit is 
expressed in granule cells throughout their 
differentiation and migration and final positioning in the 
adult cerebellum (Aguado et al., 2013) (Fig.10). In other 
regions, such as the hippocampus and septum, high 
levels of the two subunits remain until adulthood 
(Karschin and Karschin, 1997). GIRK3 is expressed 
throughout all embryonic brain structures but, from P2 
to P10, the expression of this subunit is only present in 
the cortex, olfactory bulb, hippocampus, septum, 
thalamus, ventromedial hypothalamic nucleus, inferior 
olive, and cerebellum (Karschin and Karschin, 1997). 
Finally, the expression of GIRK4 is the most restricted 
of all GIRK subunits and is only detectable in the 

613

GIRK channels in the CNS



ventromedial hypothalamic nucleus prenatally (Karschin 
and Karschin, 1997). 

 An important question is how the distribution pattern 
of GIRK channel subunits observed in adult central 
neurons is acquired during development. During the first 
week of postnatal development, extrasynaptic GIRK1, 
GIRK2, and GIRK3 are mainly distributed intra-
cellularly in association with the rER or other 
cytoplasmic membranes of dendritic shafts and spine 
apparatus in CA1 pyramidal cells of the hippocampus 
(Fernández-Alacid et al., 2011). During the second and 
third weeks of postnatal development, the three GIRK 
subunits are distributed both at intracellular sites and 
along the plasma membrane, while in the adult they are 
mostly localised to the neuronal surface in dendrites and 
spines (Fernández-Alacid et al., 2011). At synaptic sites, 
significant changes in the expression and abundance of 
GIRK channel subunits are described during postnatal 
development. The expression of the GIRK2 subunit 
increases with age in CA1 spines, while the GIRK1 
subunit has never been detected at synaptic sites. GIRK3 
subunit levels are constant throughout postnatal 
development (Fernández-Alacid et al., 2011). These data 
support the idea that GIRK channels within the synaptic 
specialization of CA1 pyramidal cells are likely to be 
GIRK2⁄GIRK3 heterotetramers during development and 
adulthood. Altogether, this data shows progressive 
trafficking of GIRK channel subunits to dendritic 
compartments as a function of age, parallel to the 
establishment and maturation of excitatory synapses. 

 

Concluding remarks 

 

 During the last two decades, the work developed in 
many laboratories using multidisciplinary tools has 
contributed to the great progress in the knowledge and 
understanding of the molecular and structural 
determinants of GIRK channel activation and 
modulation. The generation of GIRK KO mice has also 
favoured our understanding of the role played by GIRK 
channel subunits in neurons. However, most studies have 
been performed in animals and as yet very little 
information is available in humans; a major 
disadvantage that significantly limits our understanding 
of the role played by GIRK channels in human 
physiology and disease. The development of novel tools, 
such as the highly sensitive SDS-FRL technique, is 
being used to unravel the precise subcellular localization 
of different GIRK channel subunits and their spatial 
relationship with associated proteins like GPCRs, G 
proteins, or enzymes, and it can be applied to human 
samples with theoretically similar efficiency as in 

614

GIRK channels in the CNS

Fig. 10. Distribution of GIRK2 during prenatal and postnatal development in the cerebellum. A-D. During embryonic development (E18.5) and postnatal 
development (P7 and P10), immunoreactivity for GIRK2 was strong in the external granule cell layer (EGL) and the internal granule cell layer (IGL) in 
all cerebellar lobules, and showed much weaker immunoreactivity in the molecular layer (ML) and no labelling at all in the Purkinje cell (PC) layer. E. At 
P20, a narrow external granule cell layer (EGL) with strong immunoreactivity for GIRK2 is still visible, as well as GIRK2-immunopositive granule cells 
(arrows) migrating through the molecular layer (ML) to the internal granule cell layer (IGL). Scale bar: 0.5 mm.



animals. The information obtained to date with this 
technology in animals is helping to discern the 
mechanisms underlying the nanoscale organization of 
GIRK channels. The application of other novel 
technologies has led to the design of new drugs that 
selectively modulate GIRK channels with different 
subunit compositions and that are more efficient and 
selective than those currently available. These and future 
drugs may be useful for the treatment of neurological 
diseases and disorders. 

 

Acknowledgements. We thank Ms Diane Latawiec for the English 
revision of the manuscript. Funding sources were the Spanish Ministerio 
de Economía y Competitividad and Junta de Comunidades de Castilla-
La Mancha (Spain). 

Funding. Grants RTI2018-095812-B-I00 and PID2021-125875OB-I00 
funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of 
making Europe” to RL. This work was also supported by a grant from 
Junta de Comunidades de Castilla-La Mancha (SBPLY/17/ 
180501/000229 and SBPLY/21/180501/000064) and Universidad de 
Castilla-La Mancha (2023-GRIN-34187) to RL. 

Availability of data and material. All data used and/or analysed during 
the current study are available from the corresponding author upon 
reasonable request. 

Author’s Contributions. All authors had full access to all data in the study 
and take responsibility for the integrity of the data and the accuracy of 
the data analysis. 

Consent for publication. All co-authors of the present manuscript can 
certify that it has not been submitted to more than one journal for 
simultaneous consideration and that the manuscript has not been 
published previously (partly or in full). The authors also can certify that 
our main study is not split into several parts to increase the number of 
submissions, that none of the data presented here has been fabricated 
or manipulated, and that we present our own data/text/theories/ideas. 
All authors and authorities have explicitly provided their consent to 
submit the present manuscript, and in general, we all agree with the 
ethical responsibilities of the authors of the journal. Finally, all authors 
give consent for publication in Histology and Histopathology. 

Competing interests. The authors of this manuscript declare that they 
have no competing interests. 

 

 

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Accepted September 26, 2024

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