In the last few years, the knowledge about the molecular and structural determinants of a special class of potassium channels regulated by G proteins, the G protein-activated inwardly rectifying K+ channels (GIRK channels) has increased (Luscher et al. 1997). GIRK channels are members of a large family of inwardly rectifying K+ channels (referred also with the symbols Kir1–Kir7), which stabilize the potential (by changing the current) to maintain the equilibrium potential for K+ (EK), corresponding to the zero current level. The term “inwardly rectifying K+ channels rectification” refers to the ability of these channels to pass current into the cells most efficiently when the cell is hyperpolarized, thus allowing a large influx of potassium ions. The mechanism behind this rectification, yet not fully understood, involves the blockade by high-affinity endogenous polyamines and Mg2+, which block the pore in a voltage-dependent manner, at depolarized potentials, thus resulting in a reduction of outward current (Yamada et al. 1998).
GIRKs channels are activated by G proteins through a mechanism mediated by inhibitory G protein–coupled receptors (GPCRs). The GPCRs activation opens GIRKs channels via Gβγ subunits released from G proteins. This can suppress neuronal excitability and inhibit transmitter release (Fernandez-Alacid et al. 2009). Numerous GPCRs, including opioid, adrenergic, dopaminergic, GABAB, muscarinic cholinergic, cannabinoid, endothelin (B), somatostatin, and galanin receptors, are functionally coupled with GIRK channels in the nervous system (Blednov et al. 2003; Ciruela et al. 2010; Nakatsuka et al. 2008). GIRK activation generally decreases neuronal firing, leading to neuronal self-inhibition, neuron-to-neuron inhibition, and inhibition at the network level (Lacey et al. 1987; Luscher and Keller 2004). In fact, GPCRs-GIRK signaling pathways contribute to many physiological and pathophysiological conditions, such as pain, reward, learning/memory, anxiety, schizophrenia, and addiction (Lujan et al. 2014).
KCNJ6 (GIRK2): Structure and Function
The GIRK2 channel is encoded by the gene KCNJ6 (potassium channel, inwardly rectifying subfamily J, member 6) localized on the chromosome 21. Mutations in this gene highlight the role of the GIRK2 channel in CNS (central nervous systems) function and pathological states (Luscher et al. 1997).
Mammals express four potassium channels: 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).
Knockout mice of all the mammalian GIRK channels have greatly contributed to the understanding of GIRK functions, namely, to regulate the excitability of neurons in a cell-autonomous fashion and to regulate synaptic transmission and the activity of large-scale networks. This chapter is focused on GIRK2 (KCNJ6), which is mainly expressed in heart and brain tissue.
GIRK2 is a component of different channels: in most brain regions it forms heterotetrameric GIRK channel with GIRK1 or GIRK3 (Jelacic et al. 2000) although GIRK2 may also form homotetramers (Duprat et al. 1995; Liao et al. 1996). GIRK 2 is formed by three variants expressed in the brain that differ in the length of the carboxy-terminal domain: GIRK2c contains a PDZ-binding motif that is absent in GIRK2a and GIRK2b (Isomoto et al. 1996; Lesage et al. 1994; Lesage et al. 1995).
The GIRK2 channel share the same basic topology and structure of the other GIRK channels. This protein crystallizes as a homotetramer in the presence of βγ G protein subunits, phosphatidylinositol-4,5-bisphosphate (PIP2), and intracellular sodium. GIRK2 assembles in four subunits to form the K+ pore forming transmembrane domain (TMD) and a large cytoplasmic domain (CTD). The TMD-CTD interface in GIRK2 is constituted by both hydrophilic and hydrophobic interactions between the interfacial helices of the TMD, the TM-CTD linker, and the bC-bD loop of the CTD (Whorton and MacKinnon 2011). This interface regulates the ion conduction through two gates: the first gate – the inner helix gate – is formed by the inner helices of the TMD, just inside the membrane, above the level of the interfacial helix (Doyle et al. 1998); and the second gate is the G loop gate – formed by the G loop at the apex of the CTD, just outside the membrane, below the level of the interfacial helix (Nishida et al. 2007).
Mutations in conserved positions within the TMD-CTD interface yielded nonfunctional channels that commonly generate channelopathies, such as the Andersen syndrome (Decher et al. 2007; Donaldson et al. 2003; Plaster et al. 2001).
KCNJ6 (GIRK2) in Rare Genetic Diseases
GIRK channels and in particular GIRK2 are implicated in the pathology of Down syndrome (DS), a congenital condition, caused by the trisomy of human chromosome 21, and characterized by learning disabilities, craniofacial abnormalities, and hypotonia and appearance of Alzheimer disease neuropathology (Wiseman et al. 2009).
The Down syndrome critical region (DSCR) of chromosome 21 contains a gene or genes whose dosage imbalance contributes to a number of phenotypes associated with DS. This region includes KCNJ6, which encodes the GIRK2 channel (Toyoda et al. 2002). The overexpression (trisomy) of Kcnj6 in mice evidenced deficits in hippocampal-dependent recall of contextual fear conditioning, modified reward mechanisms, hampered postsynaptic depotentiation (DP), and accentuated long-term depression (LTD). This suggested that an increased expression of GIRK2 channels may have significant functional consequences that can lead to some of the neurological abnormalities found in DS patients (Cooper et al. 2012).
Fetal heart disease is the most common human genetic anomaly associated with Down syndrome. GIRK2 channel is also present in fetal heart. The overexpression of GIRK2 gene in transgenic mice led to altered cardiac responses to drugs which activate GIRK2 channel in the sinoatrial node and the atria (Lignon et al. 2008).
Recently, a very rare disease was associated with mutations in KCNJ6 (GIRK2), the Keppen-Lubinsky syndrome (KPLBS) (Masotti et al. 2015). KPLBS is a very rare disorder affecting only three people in the world. KPLBS is characterized by a severely delayed psychomotor development, hypertonia, hyperreflexia, generalized lipodystrophy giving an aged appearance, and characteristic dysmorphic features, including microcephaly, prominent eyes, narrow nasal bridge, and open mouth. From the exome sequencing analysis of these affected individuals, common variants have been found in the exon 3 of KCNJ6. In particular, two individuals shared the same mutation, an inframe heterozygous deletion of three nucleotides (c.455_457del) leading to loss of one amino acid (p. Thr152del). The third individuals was heterozygous for a missense mutation (c.460G>A) that introduces an amino acid change from glycine to serine (p. Gly154Ser). These mutations are localized in the selectivity filter domain of KCNJ6. The substitution could also abolish the selectivity for K+, which could in turn cause an aberrant Na+ influx across the channel and trigger cell death (Masotti et al. 2015).
KCNJ6 (GIRK2): Pain and Depression
Variants in KCNJ6 (GIRK2) gene revealed the relationship between single-nucleotide polymorphisms (SNPs), especially within the exonic and 5′-flanking regions, and individual differences in opioid analgesic sensitivity (Nishizawa et al. 2009). Two SNPs, G1250A and A1032G, are representative SNPs for association studies of GIRK2 and pain. The homozygous carriers of the A allele (A1032G) required more frequent pain medications and higher opioid doses (methadone) for pain control compared to individuals with the G allele (Bruehl et al. 2013; Nockemann et al. 2013).
Interestingly, the GIRK2 variant rs2835859 has been demonstrated not only to be associated with postoperative analgesia and pain sensitivity but also to nicotine dependence and successful smoking cessation (Nishizawa et al. 2014).
The alteration of noradrenergic neurotransmission underlies the etiopathology of multiple neurologic diseases, such as attention deficit/hyperactivity disorder, anxiety, depression, and drug addiction (Itoi and Sugimoto 2010). The behavioral response to antidepressants has been linked to increased neurogenesis, which is mediated by stimulation of 5-HT receptors (Santarelli et al. 2003). The G protein-coupled inwardly rectifying potassium (GIRK) channels are the main inhibitory effectors of 5-HT receptors (Williams et al. 1988), and therefore they could be potential candidates for the study of depression and antidepressant responses involving the 5-HT receptor-mediated signaling. Mice lacking GIRK2 subunits of GIRK channels display a depression-resistant phenotype combined with a reduced behavioral response to citalopram (an antidepressive drug), an increase in the firing rate of dorsal raphe (DR) neurons, and a reduction of 5-HT1A receptor mediated responses. Furthermore, the deletion of the GIRK2 subunit does not affect basal adult neurogenesis (Llamosas et al. 2015).
KCNJ6 (GIRK2): Alcohol Addiction
Only a few papers deal with the role of KCNJ6 (GIRK2) mutations and two major addiction societal problems: alcohol and nicotine addiction.
It has been demonstrated that polymorphisms in KCNJ6 gene are associated with an increased risk of drinking in adolescents especially when early psychosocial stress conditions are present (Clarke et al. 2011). Furthermore, from the study of these polymorphisms, the authors suggested that the interaction between early psychosocial stress conditions and the KCNJ6 genotype may confer a risk to alcoholism that persists until adulthood. Mice lacking GIRK2 channels are more prone to self-administer themselves more ethanol compared to wild-type (WT) littermates (Blednov et al. 2001). Finally, the KCNJ6 polymorphism rs2836016 has been associated with alcoholism in an adult alcohol-dependent sample and correlated with the initiation of alcohol abuse (Clarke et al. 2011).
In the last few years, the interest on potassium channels has increased in consideration of the implications of these proteins in several diseases. G protein-coupled inwardly rectifying potassium (GIRK) channels are widely expressed in heart and brain and mediate the inhibitory effects of many neurotransmitters. As a result, these channels are important for normal CNS function and have also been implicated in Down syndrome, psychiatric disorders, dependence syndrome, and pain control. Our group has recently reported that mutations in KCNJ6 (GIRK2) gene cause the ultrarare syndrome referred as the Keppen Lubinsky syndrome (Masotti et al. 2015). Also single nucleotide polymorphisms within and around the coding regions of KCNJ6 gene have been demonstrated to be associated with other diseases, not necessarily of primary genetic origin. Therefore, further studies on KCNJ6 (GIRK2) and its function will surely lead to an indepth understanding of the physiological role of this channel, and the alterations that cause diseases. We believe that the knowledge of the structural features and precise functions of this channel will help to envisage novel drugs and suggest new pharmacological treatments.