Chapter Five - Localization and Targeting of GIRK Channels in Mammalian Central Neurons

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Abstract

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels are critical to brain function. They hyperpolarize neurons in response to activation of different G protein-coupled receptors, reducing cell excitability. Molecular cloning has revealed four distinct mammalian genes (GIRK1–4), which, with the exception of GIRK4, are broadly expressed in the central nervous system (CNS) and have been implicated in a variety of neurological disorders. Although the molecular structure and composition of GIRK channels are key determinants of their biophysical properties, their cellular and subcellular localization patterns and densities on the neuronal surface are just as important to nerve function. Current data obtained with high-resolution quantitative localization techniques reveal complex, subcellular compartment-specific distribution patterns of GIRK channel subunits. Recent efforts have focused on determining the associated proteins that form macromolecular complexes with GIRK channels. Demonstration of the precise subcellular compartmentalization of GIRK channels and their associated proteins represents a crucial step in understanding the contribution of these channels to specific aspects of neuronal function under both physiological and pathological conditions. Here, we present an overview of studies aimed at determining the cellular and subcellular localization of GIRK channel subunits in mammalian brain neurons and discuss implications for neuronal physiology.

Introduction

Ion channels are classified by their gating properties and ion selectivity for Na+, Ca2 +, Cl, and K+ (Hille, 2001). Potassium (K+)-selective channels are key determinants of membrane excitability and regulate a variety of cellular processes including membrane potential, signal transduction, hormone release, vascular tone, cell volume, and immune responses (Curran, 1998). Four different subfamilies of K+ channels have been proposed based on their structural and phylogenetic relationship: voltage-gated K+ (Kv) channels, Ca2 +-activated K+ (KCa) channels, two-pore K+ (K2P) channels, and inwardly rectifying (Kir) channels (Gutman et al., 2005).

Kir channels are formed by 16 subunits falling into 7 subfamilies (Kir1–Kir7), including the Kir3 subfamily, which is directly coupled to heterotrimeric G proteins and mediates the inhibitory effect of activated G protein-coupled receptors (GPCRs) on neuronal excitability (Dascal, 1997, Kubo et al., 2005, Luján et al., 2014, Luján et al., 2009, Yamada et al., 1998). This subfamily of Kir channels, also known as G protein-gated inwardly rectifying K+ (GIRK) channels, is composed of four subunits, designated GIRK1–4 (Dascal, 1997, Luján et al., 2014, Lüscher and Slesinger, 2010).

The past decade has witnessed great progress in our understanding of the diverse functional roles that GIRK channels fulfill in different neuron populations and brain regions. Molecular cloning of GIRK channel genes has led to the design and synthesis of antibodies that, combined with several immunohistochemical techniques, facilitated studies on the distribution of GIRK channel subunits in the CNS. Indeed, immunohistochemical approaches at both the light and electron microscopic levels have yielded important insights into the distribution and precise subcellular localization of GIRK channels in neurons. In particular, the use of high-resolution immunohistochemical techniques at the electron microscopic level has allowed unparalleled precision, showing a highly regulated subcellular distribution patterns for GIRK channel subunits that are also region- and cell type-dependent (Luján et al., 2014, Luján et al., 2009). The demonstration of this precise subcellular compartmentalization provides a new understanding of the role of GIRK channels in information transfer and processing within neurons and neural networks under physiological and pathological conditions. In this chapter, we summarize current anatomical knowledge describing the regional and cellular distribution of the four GIRK channel subunits in the mammalian CNS. The precise subcellular distribution of GIRK channels at the surface of central neurons will be discussed in view of their relationship to neurotransmitter release sites and of possible functional implications.

Section snippets

Molecular Organization and Heterogeneity of GIRK Channels

The structure and function of GIRK channels are reviewed in other chapters of this volume, and we refer the reader to those sections for further information. Here, we briefly describe basic molecular, biochemical, and physiological features of GIRK channels.

GIRK channels are composed of four different, but homologous, subunits (GIRK1–4) that are conserved in mouse, rat, and human (Hibino et al., 2010, Yamada et al., 1998). Each subunit contains hydrophilic amino- and carboxy-terminal domains

Neuroanatomical Approaches to the Study of GIRK Channel Distribution

The molecular definition of the mammalian GIRK family has led to the generation of subunit-specific molecular tools for investigating expression levels and cellular patterns of GIRK mRNAs in brain tissue with different techniques. Thus, northern blots, western blots, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, histoblots, and immunohistochemical studies have been used to observe the regional, cellular, and subcellular distribution of GIRK channels in the CNS

Regional Distribution of GIRK Channel Subunits in the CNS

In situ hybridization, histoblot, and immunohistochemical studies have revealed that GIRK1, GIRK2, and GIRK3 channel subunits are widely expressed throughout the CNS of rodents, showing distinct although partially overlapping patterns of mRNA and protein distribution (Aguado et al., 2008, Chen et al., 1997, Fernández-Alacid et al., 2009, Fernández-Alacid et al., 2011, Inanobe et al., 1999, Karschin et al., 1996, Kobayashi et al., 1995, Koyrakh et al., 2005, Liao et al., 1996, Miyashita and

Cellular Distribution of GIRK Channel Subunits

Cell biological, biochemical, and electrophysiological approaches indicate that the dominant functional GIRK channel in the CNS contains GIRK1 and GIRK2 (Liao et al., 1996). This view is supported by data obtained using genetic studies, which show that ablation of GIRK2 results in loss of GIRK current in many brain regions, including the hippocampus (Koyrakh et al., 2005, Lüscher et al., 1997), cerebellum (Slesinger, Stoffel, Jan, & Jan, 1997), substantia nigra (Koyrakh et al., 2005), VTA (

Subcellular Distribution of GIRK Channel Subunits

The classical notion that the subcellular localization of ion channels in a particular cell type readily applies to all brain neurons is not well supported by available data. The emerging view is that each ion channel possesses its own unique subcellular distribution pattern in each cell type (Luján, 2010), suggesting that there are no simple rules concerning their subcellular organization. This is particularly true for GIRK channels. Indeed, available evidence suggests that GIRK channels can

Developmental Aspects of GIRK Channel Distribution

Brain development results from a temporospatial pattern of events, beginning with neuronal proliferation, followed by migration and differentiation and ending with synapse formation and circuit refinements. A growing body of evidence suggests that each step in the developmental sequence of the CNS involves the appropriate expression and function of neurotransmitters, their receptors, and ion channels (Luján, 2010, Luján et al., 2005). For example, Kv channels are involved in the control of

Conclusions and Future Perspectives

The electrical signaling of neurons depends largely on the abundance and function of a large diversity of neurotransmitter receptors and ion channels located at specific sites in neuronal somata, dendrites, and axons. The selective placement of GIRK channel subunits at precise locations in mammalian neurons and their dynamic regulation through specific signaling pathways allows for a wide variety of neuronal function in the brain. However, the cellular diversity of the brain and the

Acknowledgments

The authors would like to thank Alexandra Salewski, M.Sc., for the English revision of the manuscript and Mercedes Gil for her excellent technical assistance. We also thank the Spanish Ministry of Education and Science (BFU-2012-38348; Consolider-Ingenio CSD2008-00005), the European Union (HBP—Project Ref. 604102), and the Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) for their generous support of our research. Additionally, our gratitude extends to all members of our laboratory

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