1 - Sensory Functions for Degenerin/Epithelial Sodium Channels (DEG/ENaC)
Introduction
The rapid advancements in molecular and genomic biology have resulted in a wealth of information about how genes and their protein products affect cellular and organismal functions, and how such functions evolved. These data also led to the realization that multiple independent protein families have often evolved to serve similar physiological functions. The complex relationship between protein structure and physiological functions highlights the importance of studying such relationships with integrative and comparative approaches.
One of the most diverse groups of proteins in terms of the relationship between protein structure and function are ion channels. These membrane-targeted proteins are found in all cell types, including prokaryotes, and are critical for maintaining the appropriate ionic gradients across all cellular barriers, including the plasma membrane and intracellular compartments (Ashcroft and ScienceDirect (Online service), 2000). This review focuses on a relatively newly discovered and enigmatic family of ion channels; degenerin/epithelial Na+ channels (DEG/ENaC). DEG/ENaC proteins form nonvoltage gated, amiloride-sensitive cation channels (Bianchi and Driscoll, 2002, Garty and Palmer, 1997). DEG/ENaC channels comprise three to nine independent subunits, which can be either hetero- or homomultimers (Benson et al., 2002, Canessa et al., 1994b, Eskandari et al., 1999, Jasti et al., 2007, Kellenberger and Schild, 2002, Zha et al., 2009b). In cases where members of the family have been characterized electrophysiologically, subunit composition was found to have a significant effect on the pharmacological and electrical properties of the channel, suggesting that subunit composition is a critical regulatory mechanism in these channels (Askwith et al., 2004, Benson et al., 2002, Chu et al., 2004, Xie et al., 2003, Zha et al., 2009a, Zhang et al., 2008).
Despite of the high diversity in the primary sequence of individual subunits, several structural constituents indicated that all members of the family have a similar protein topology (Bianchi, 2007, Bianchi and Driscoll, 2002, Corey and Garcia-Anoveros, 1996, Tavernarakis and Driscoll, 2000, Tavernarakis and Driscoll, 2001a). The typical DEG/ENaC subunit has two transmembrane domains, two short intracellular domains and a large extracellular loop, which is a hallmark characteristic of the DEG/ENaC protein family topology (Fig. 1.1). The DEG/ENaC family seems to be animal specific and many different members have been identified in diverse species (Fig. 1.2).
In the few instances where the pharmacological, structural, and biophysical properties of specific DEG/ENaC subunits have been studied, the channels have been characterized as ligand-gated, voltage insensitive, depolarizing cation channels, which seem to be more selective for Na+ over Ca2+ and K+ (Garty and Palmer, 1997). The physical cloning of various DEG/ENaC subunits enabled the identification of selective agonists and antagonists for specific subunits. In addition, natural ligands and physical stimuli were found to activate or modulate channel functions. These include (1) peptides such as members of the invertebrate FMRFamide family (Askwith et al., 2000, Green et al., 1994, Lingueglia et al., 1995, Xie et al., 2003), mammalian FFamide and SFamide peptides (Deval et al., 2003, Sherwood and Askwith, 2008, Sherwood and Askwith, 2009), natural, and dynorphin-related opioid peptides (Sherwood and Askwith, 2009); (2) small increases in extracellular proton concentrations (Adams et al., 1998b, Benson et al., 2002, Price et al., 2001, Waldmann et al., 1997b, Xie et al., 2003, Xiong et al., 2004); (3) sulfhydryl compounds (Cho and Askwith, 2007); (4) small polyamines such as agmatine (Yu et al., 2010); and (5) mechanical stimuli (Bazopoulou et al., 2007, Lu et al., 2009, O'Hagan et al., 2005, Price et al., 2001, Simon et al., 2010, Tavernarakis and Driscoll, 2001a, Zhang et al., 2004, Zhong et al., 2010). Together, these data indicated that, like other ligand-gated ion channel families, DEG/ENaC channels have evolved to serve many different physiological functions, acting as ionotropic receptors to diverse extracellular stimuli.
Although amiloride-sensitive sodium currents from various epithelial tissues have been recorded since the early 1970s, the genes encoding for these channels, which were shown to be critical for regulating salt exchange in the kidney and blood pressure, were not identified until the early 1990s (Canessa et al., 1993, Canessa et al., 1994a, Lingueglia et al., 1993a, Lingueglia et al., 1993b). The successful cloning of ENaC-coding genes was achieved by using expression cloning in Xenopus oocytes, which demonstrated that the mature ENaC comprises proteins from three highly related but independent genes. These genes were subsequently termed ENaCα, ENaCβ, and ENaCγ (Canessa et al., 1994b). The existence of homologous channels in invertebrates was originally debated. Nevertheless, several studies suggested the existence of amiloride-sensitive sodium currents in the leech, Xenopus, and the pond snail, suggesting these channels were not mammalian specific (Green et al., 1994, Weber et al., 1992, Weber et al., 1993). Later, cloning of several DEG/ENaC-like proteins from the worm Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster showed that the DEG/ENaC family is likely to be ubiquitously present in animal genomes (for a comprehensive review of the early studies, see Garty and Palmer, 1997).
Subsequently, several additional members of the DEG/ENaC superfamily have been cloned from mammalian models, including several acid-sensitive ion channels (ASIC/Accn) (Price et al., 2000, Price et al., 2001, Price et al., 1996, Waldmann et al., 1997b, Waldmann et al., 1996, Xie et al., 2002). In contrast to ENaC-coding genes, which are transcriptionally enriched in epithelial tissues, members of the ASIC subfamily seem to be highly enriched in neuronal tissues, both centrally and peripherally (Lu et al., 2009, Xie et al., 2002). The completion of the sequencing of the human and other animal genomes revealed that mammals encode for eight to nine independent members of the DEG/ENaC protein superfamily. Surprisingly, the release of the completed genomes of the worm and the fruit fly revealed that the genomes of these invertebrates harbored a significantly larger number of independent DEG/ENaC-like genes (31 in the fruit fly and 30 in the worm), which also included several genes that can produce multiple variants due to alternative splicing and multiple transcriptional initiation sites (Bazopoulou et al., 2007, Liu et al., 2003a, Liu et al., 2003b). Hence, DEG/ENaC genes represent one of the largest ion channel families in invertebrate genomes. The expansion of the DEG/ENaC protein family in these animals suggests the hypothesis that DEG/ENaC ion channels have evolved to serve a much wider range of physiological functions in invertebrates relative to their roles in mammals. Alternatively, it may suggest that DEG/ENaC subunits in invertebrates are highly specialized; each subunit is performing a narrow slice of the physiological tasks performed by mammalian family members. Our group is focused on understanding the role of DEG/ENaC channels in invertebrate physiology, which we hope will help us to resolve these two alternative hypotheses.
Although members of the DEG/ENaC superfamily are easily recognized by their unique protein topology (Fig. 1.1), identifying the relationships between family members across distant species based on protein sequence alone is hampered by the poor overall sequence conservation of the extracellular loop domain. Hence, protein alignment analyses alone were not powerful enough to draw physiological homology conclusions (Fig. 1.2). Consequently, newly identified family members typically require physiological analyses de novo.
The best physiologically characterized members of the family are the three mammalian ENaC genes (Garty and Palmer, 1997, Horisberger and Chraibi, 2004). The mammalian ENaC channels are typically found at the apical membrane of epithelial cells where they play an essential role in regulating sodium gradients across epithelial barriers in a variety of tissues (Snyder, 2005, Snyder et al., 1995, Voilley et al., 1994). Mutations in ENaC subunits can lead to disorders such as Liddle's syndrome, which is a rare form of genetically inherited hypertension syndrome (Snyder et al., 1995). ASIC represent the other major mammalian branch of the DEG/ENaC family (Waldmann et al., 1999). These channels are enriched in peripheral and central neurons and are highly sensitive to changes in extracellular proton concentrations (Bassilana et al., 1997, Waldmann et al., 1997a, Wemmie et al., 2002). ASIC channels seem to play a major role in several pH-dependent physiological processes in the brain that include seizure termination (Ziemann et al., 2008), learning and memory (Askwith et al., 2004, Wemmie et al., 2002), and fear conditioning (Coryell et al., 2009, Coryell et al., 2007, Wemmie et al., 2003, Ziemann et al., 2009). Similar central neuronal roles have also been recently identified for DEG/ENaC channels in the worm model (Voglis and Tavernarakis, 2008). How these pH-sensitive channels affect neuronal functions is still a mystery. At least some of the functions might be mediated by direct, short-term effects on synaptic plasticity, possibly by sensing microchanges in pH that are associated with the low pH environment of the lumen of synaptic vesicles. In contrast to our understanding of the role of ASIC channels in the CNS, their roles in sensory functions are still controversial, which will be discussed in details below.
Despite the advances in understanding the role of DEG/ENaC signaling in the brain, its role in peripheral neuronal functions is still poorly understood. Nevertheless, recent work in invertebrate and mammalian models indicated that members of the DEG/EnaC superfamily are playing a major role in chemosensation and mechanosensation, although the capacity in which they exert their sensory functions is still not well understood in most systems. This review focuses on the current state of research on the sensory roles of DEG/ENaC channels in diverse animal models.
The emerging interest in DEG/ENaC-dependent signaling has resulted in many studies of their functions in diverse species. As more and more individual subunits are being characterized, a complex picture is emerging in terms of the physiological roles of DEG/ENaC and their diverse gating mechanisms. Although the first DEG/ENaC channels cloned were characterized as a constitutively open sodium channels, later studies of the ENaC channel and the majority of other family members suggested that members of the family are likely acting as either classic ligand-gated ion channels (Horisberger and Chraibi, 2004) or mechanically gated channels (Bazopoulou et al., 2007, Bianchi, 2007).
Section snippets
Salt taste
Maintaining appropriate ionic homeostasis is critical for all organisms, especially in regard to sodium, which is kept in relatively high extracellular concentrations in most animal tissues. Animals actively regulate their sodium intake via food consumption (Geerling and Loewy, 2008). In agreement with the importance of sodium, studies in rodent models indicated that some taste cells are specialized for responding to NaCl while others are less specialized and can be activated by many different
C. elegans
All organisms seem to have evolved on mechanisms to sense mechanical stimuli, and in most cases, physiological studies indicated that the mechanosensory complex acts as a cation channel (for a recent review, see Arnadottir and Chalfie, 2010). Yet, the molecular identities of the proteins responsible for sensing mechanical stimuli are still mostly unknown (Christensen and Corey, 2007, Corey, 2006). The difficulty in identifying the mechanosensory conducting channels is likely the result of
Peripheral Pain
Although the general role of DEG/ENaC signaling in eukaryotic mechanosensation is still controversial, the data discussed above indicated that, at least in invertebrates, DEG/ENaC subunits are playing an important role in the function of mechanically activated sensory neurons, often in the context of mechanical and thermal nociceptive stimuli (Albeg et al., 2010, Bounoutas and Chalfie, 2007, Chatzigeorgiou et al., 2010, Chelur et al., 2002, Goodman et al., 2002, Roza et al., 2004, Suzuki et
Conclusions
Degenerin/epithelial sodium channels are emerging as important molecular players in animal sensory biology. Their possible role in mediating nociceptive behaviors in both invertebrates and vertebrates suggest that these channels evolved to serve such functions early in the metazoan radiation. One puzzling aspect of DEG/ENaC diversification is the large number of independent subunits present in invertebrate genomes relative to mammalian genomes. To my knowledge, no other ligand-gated ion channel
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